Insights into the structure and modulation of human TWIK-2

Domain: neuroscience
Published: 2026-01-31
Editorial outcome: major_revision

Abstract
The Tandem of pore domain in a Weak Inward Rectifying K+ channel 2 (TWIK-2; KCNK6) is a member of the Two-Pore Domain K+ (K2P) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. Despite its physiological relevance, the structure, regulatory mechanisms, and selective modulators of TWIK-2 remain largely unknown. Here, we present a 3.7 Å single particle cryo-electron microscopy structure of human TWIK-2 and highlight its conserved and distinctive features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing site-directed mutagenesis and provide insights into the possible lipid-mediated mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising anti-inflammatory therapeutics.

Introduction
Two-pore-domain K+ (K2P) channels are a diverse family of potassium channels from TREK (K+) selective ion channels that supervise background K+ currents. They are critical to maintaining membrane resting potential and regulating cellular excitability1–3. In mammals, the K2P family comprises 15 members, classified into six subgroups based on their functional diversity: TWIK, TREK, TRESK, TASK, TALK, and THIK4,5. Unlike Voltage-gated K+ (Kv) channels, K2P channels lack the canonical voltage-sensing domain. While the overall architecture of K2P channels is evolutionarily conserved, there are multiple structural variations and sites of modulation, which make these channels susceptible to regulation by voltage-independent factors such as temperature, pH, pressure, bioactive lipids, and volatile anesthetics5–7.

Among the 15 members of the K2P family, channels from TREK and TASK subfamilies have been extensively structurally characterized8–13, offering profound insights into their gating mechanisms, physiological functions, and pharmacological potential8,14–24. TREK channels are known for their mechano- and thermosensitivity and role in sensory response14,18,25,26, and TASK channels are well-characterized for their pH sensitivity and role in maintaining neuronal excitability12,13,27–29. Nevertheless, the gating mechanisms, physiological modulation, and pharmacological potential of the TWIK family remain underexplored primarily because of the weak currents30,31 and poor heterologous expression of these channels32–34.

TWIK-1 (KCNK1) is the only structurally characterized mammalian K2P channel member of the TWIK family30,35. The poor currents in TWIK-1 were initially believed to be due to intracellular localization resulting from SUMOylation36. However, mutations in the SUMOylation site and overexpression of a recombinant plasma-membrane-localized TWIK-1 demonstrated that the poor currents are characteristic of TWIK family members37,38. Recent studies involving a combination of electrophysiology, site-directed mutagenesis, molecular dynamics simulations, and structural analysis of TWIK-1 at low (pH 5.5) and neutral pH (pH 7.4) revealed the molecular basis for pH-mediated regulation of gating and conformational dynamics of the selectivity filter in TWIK-139,40.

TWIK-2 (KCNK6) shares ~54% sequence similarity with TWIK-1 and exhibits ubiquitous tissue distribution31,41. TWIK-2 is implicated in diverse pathological conditions such as pulmonary hypertension, acute lung injury, hearing loss, and NLRP3 inflammasome-induced inflammation42–46. A significant degree of variation in the current recordings has been reported for TWIK-1 and TWIK-2 by various groups, underscoring the challenges of robust electrophysiological studies for the TWIK subfamily31,33,41. The weak basal activity of TWIK-2 could be due to its subcellular partitioning between the plasma membrane and endolysosomes. Moreover, it has been reported that the Y308A mutation within the lysosomal-targeting YXXØ motif in TWIK-2 promotes plasma membrane expression of TWIK-234. Translocation of TWIK-2 from endosome to plasma membrane is implicated in NLRP3-mediated inflammasome activation in macrophages, with KCNK6 knockouts showing a suppressed inflammasome activation45,46. Despite the high sequence similarity with TWIK-1, TWIK-2 is believed to be insensitive to pH and indifferent to activation by phosphorylation30,31,38,40. The mechanism of regulation of TWIK-2 is poorly understood because of the lack of three-dimensional structures and due to inconsistent reports of its electrophysiological recordings31,34,41. Additionally, although substantial progress has been made in developing K2P channel modulators, particularly for the well-characterized TREK subfamily, there is a dearth of high-affinity, selective modulators of TWIK-2. The existing small molecule modulators of TWIK-2 activity, such as ML365 and NPBA, also cross-react with other K2P channels25,47–53.

To gain deeper insights into the molecular basis of channel regulation and to aid the pharmacological characterization of TWIK-2, we determine the structure of full-length human TWIK-2 by single particle cryo-electron microscopy (cryo-EM). We uncover conserved and distinctive features of this K2P channel subtype and, using a combination of structure-guided site-directed mutagenesis and automated high-throughput whole-cell patch clamp electrophysiology, we identify key amino acids that regulate TWIK-2 channel activity. Finally, using the existing K2P modulators, we develop an effective strategy for high-throughput screening of TWIK-2 modulators.

Results
Heterologous expression and functional characterization of recombinant human TWIK-2
We expressed full-length TWIK-2 (hereafter TWIK-2) in HEK293 GnTI− and verified that it was functional by whole-cell patch-clamp electrophysiology (top panel of Fig. 1A, B, Supplementary Fig. 1, and Supplementary Table 1). When recorded in high potassium (100 mM) at pH 7.4, the TWIK-2 channels evoked larger outward potassium currents than the non-transfected (non-T) cells (Supplementary Fig. 1 and Supplementary Table 2). TWIK-1 is sensitive to pH39,40. To assess whether TWIK-2 behaves similarly, we measured conductance at two pH levels (7.4 and 5.5) in high potassium (top panels of Fig. 1A, B, and Supplementary Fig. 1B, C). We did not observe significant changes in current densities or reversal voltage potential (Vrev) of TWIK-2 at pH 7.4 and pH 5.5 under these conditions (p=0.9003; p=0.1017), indicating that TWIK-2 is insensitive to changes in extracellular pH. To determine whether pH independence of channel activity is an inherent property of TWIK-2, we introduced a mutation at residue-equivalent position 111, known to affect pH dependence of TWIK-1 channels. In this position TWIK-2 has a tyrosine (Y) instead of a histidine (H) seen in TWIK-1 (Fig. 1C and Supplementary Fig. 2B). Functional analysis of the Y111H mutant at pH 7.4 and pH 5.5 showed no changes in current densities (p=0.5142) or voltage reversal potential (Vrev) (p=0.1394) related to pH dependence (bottom panels of Fig. 1A, B, and Supplementary Table 1). This result shows that TWIK-2 channels are pH-insensitive, indicating that the structural elements responsible for pH sensitivity in TWIK-1 differ from those in TWIK-2. To further explore structural differences within the TWIK family, we prepared a full-length human TWIK-2 construct for overexpression and structural analysis. We purified TWIK-2 using Decyl maltose neopentyl glycol (DMNG) at pH 7.5 and 150 mM KCl. We confirmed the protein homogeneity and purity by size-exclusion chromatography and SDS-PAGE analysis (Supplementary Fig. 3B). The peak fractions, corresponding to TWIK-2 dimer, that eluted at ~12.4 ml on a Superdex 200 Increase size-exclusion chromatography column were used for subsequent structural analysis (Supplementary Fig. 3B–F).

Molecular architecture of TWIK-2
We determined the structure of TWIK-2 at ~3.7 Å (Fig. 1D, E; Supplementary Fig. 3C–F, and Supplementary Table 3). We were able to reliably model all the secondary structure elements of TWIK-2 except the following disordered-loop regions: M1-G4 (N-terminus), V75-P89, T150-W171, and L262-R313 (C-terminus) (Fig. 1C–E, and Supplementary Fig. 3F–H). Notably, the V75-P89 loop region harbors two predicted N-linked glycosylation sites, N79 and N85 (Supplementary Fig. 2A). For the overexpression of homogenous TWIK-2, we used HEK293 GnTI− cells that lack N-acetylglucosaminyltransferase I activity, resulting in a minimally glycosylated recombinant protein. Perhaps complex native glycosylation at these sites is necessary for stabilization of this loop region. Each TWIK-2 protomer contains four transmembrane helices (M1–M4), two pore-forming helices (PH1 and PH2), two selectivity filter loops (SF1 and SF2), and two extracellular cap-forming helices (CH1 and CH2) (Fig. 1C). TWIK-2 assembles as a canonical domain-swapped homodimer with a pseudo-tetrameric central pore observed in all other K2P channels. The cap-forming helices within a protomer are arranged in an inverted “V” shaped cap domain over the central pore of the channel. The cap domains from both protomers form an “arched dome,” creating a bifurcated extracellular ion pathway (EIP, Fig. 1C–E). As observed in other known K2P structures, we notice that the cap domain is also responsible for pairing the M1 helix of one protomer in a three-dimensional module with the rest of the protein from the other protomer. Thus, the domain-swapping of the M1 helix leads to the formation of four pore-forming domains comprised of two PD1s that are non-identical to the two PD2s. PD1s are composed of the M1 helix from one protomer and PH1, SF1, and M2 from the other protomer. PD2s, on the other hand, are assembled by M3, PH2, SF2, and M4 of the same protomer (Fig. 1D, E). Three-dimensionally, each pore-forming domain consists of one pore-forming helix (PH) and one selectivity filter (SF) loop flanked by two transmembrane helices (Fig. 1C). The PH and SF regions from PD1 (PH1/SF1) and PD2 (PH2/SF2) of both protomers form the central ion-conducting pore. Our cryo-EM density map allows placement of three K+ ions in the central selectivity filter (Fig. 1D). M2 and M4 helices are positioned adjacent to the core, while M1 and M3 are located on the periphery. The domain-swapped M1 helices, the non-identical sequences of the SFs, and the unequal lengths of the SF1-M2 and SF2-M4 linkers, impart a pseudo-hetero-tetrameric symmetry to the elements surrounding the pore of the channel (Fig. 1E).

Ion conduction pore of TWIK-2
The ion conduction pathway of TWIK-2 extends from the hydrophobic cuff marked by a kink in the M2 helix within the vestibule, designed to funnel ions toward the central pore, to the bifurcated EIP on the extracellular side through a narrow selectivity filter (Fig. 2A, B). While the sequence within the selectivity filters of TWIK-2 is conserved mainly across different K2P channels, the entry and exit of the ion conductance pathway are flanked by non-conserved M135 and Y111, respectively (Fig. 2A and Supplementary Fig. 2). We found that the narrowest constriction in the ion conductance pathway has a radius of 4.1 Å, which is sufficiently wide for K⁺ ions to pass through (Fig. 2C, D). Except for the two conserved threonines at the bottom of the SF, the backbone carbonyl oxygens regulate the flow of K+ ions within the SF, as opposed to side chains. While the SF sequences are primarily conserved across the K2P family, TWIK subfamily members show some divergence. The canonical isoleucine (TIGY/FG) in SF1 is replaced by threonine in TWIK-1 and valine in TWIK-2. Similarly, the canonical phenylalanine (TI/VGFG) in SF2 is replaced by leucine in both TWIK-1 and TWIK-2 (Supplementary Fig. 2A). These variations could potentially govern the differences in the currents of the TWIK subfamily compared to other highly modulated K2P channels, such as TREK and TASK.

Unidentified non-protein molecules in TWIK-2
In our cryo-EM sample preparation, we did not include any exogenous lipids or small-molecule ligands that bind to TWIK-2, with the intention of determining the structure of apo-TWIK-2. However, we observed tubular lipid-liked densities below the SF in the canonical K2P vestibule, the modulatory lipid site, and fenestration sites, which could be relevant to the mechanism of TWIK-2 action (Fig. 2E). Endogenous lipids are known to co-purify with the protein in the detergent micelle54–56. While these tubular densities could not be assigned to the protein, the resolution of our cryo-EM map precludes their unambiguous identification. Alternatively, these densities could also be DMNG detergent. But we do not observe densities that can accommodate the DMNG head group. Hence, we modeled hydrocarbon tails in the tubular densities (Fig. 2E). We note that the tubular lipid-liked densities occupy the groove created by the PH2 and M4 helices of one protomer with the M2 helix of the other. The lower end of this groove is believed to be a “classic” modulatory phospholipid binding site, and the site that opens to the bottom of SF has been termed a “fenestration” site5. It is surmised that the binding of lipids in this groove pushes the M4 helix down, allowing better access to SF. In a “down” conformation, the M4 helix traverses the lipid bilayer at a ~45° angle, creating a hydrophobic “fenestration site” between the bottom of the PH2 helix and M2 helix of the opposite protomer that enables binding of hydrophobic compounds at this pocket that inhibit the channel5,57 (Supplementary Fig. 4A). In TWIK-2, we observe that M4 adopts a canonical “down” conformation, perhaps due to the presence of tubular densities in the “fenestration site” (Supplementary Fig. 4A). In addition, we also noticed three small tetrahedral blobs within the central cavity below the permeation pore that could accommodate an ion or water (blue, Fig. 2E). Molecular dynamics (MD) simulations of K2P channels treated with negatively charged activator have suggested the presence of ions or water within the “cavity binding sites,” stabilized by the negative moiety of the small molecule or lipids58. We have left these small tetrahedral densities unmodeled in our TWIK-2 structure because the precise identity of these densities remains unclear.

Automated patch-clamp recordings of variants of recombinant human TWIK-2
We observed that TWIK-2 channel activity was dependent on the applied voltage but not on the extracellular pH (Fig. 1A, B). Although TWIK-2 and TWIK-1 exhibit high structural similarity, including conserved residues in the SF and M2 domains, subtle differences in residue interactions may lead to distinct gating behaviors in the two channels. To identify and assess molecular determinants critical to K⁺ conductance in TWIK-2, we performed site-directed mutagenesis along the ion permeation pathway, near the hydrophobic cuff, and near the canonical K2P modulator pocket (Fig. 3A). Specifically, we generated alanine substitutions of F98 (PH1), T106 (SF1), Y111 (SF1-M2 loop), M135 (hydrophobic cuff), S213 (PH2), and T214 (SF2). We also introduced a charge into the hydrophobic cuff using the M135N mutation. Lastly, we generated the Y111H substitution in TWIK-2 to make it “TWIK-1-like,” as H122 in TWIK-1 (H98 in TASK-3) is believed to be a major pH-sensing amino acid39,40.

We conducted whole-cell recordings on cells expressing either wild-type or mutant TWIK-2 channels (Fig. 3B, C, and Supplementary Table 4). Overall, there were no significant differences in current densities between the mutant channels and wild-type TWIK-2, except for the double mutant T106A-T214A, which showed an increase in the current density (Fig. 3B, Supplementary Fig. 5A, and Supplementary Table 5). However, notable differences in the activation profiles of the macroscopic currents were observed among the mutants, as evident by varying activation and deactivation time constants (τ) (Fig. 3C and Supplementary Table 4). Mutations in amino acids at the extracellular and intracellular openings of the ion-permeation pore, specifically Y111 and M135, delayed TWIK-2 activation. The Y111H mutant, located at the extracellular exit of the ion-permeation pathway, exhibited the most significant increase in activation time, indicating slower channel activation. While the Y111A mutant did not show a noticeable change in activation time, it did exhibit faster deactivation. Both M135A and M135N mutations, that lie at the entrance of the ion permeation pathway within the central cavity, caused only a slight delay in activation, compared to wild-type TWIK-2 (Fig. 3C). The F98A mutation, in the K2P modulator binding pocket, did not affect the activation but led to a substantial increase in the deactivation time compared to wild-type TWIK-2 (Fig. 3C). Conversely, mutations at the bottom of the selectivity filter—T106A, S213A, T214A, and the double mutant T106A-T214A—all showed faster activation than the wild-type channel (Fig. 3B, C and Supplementary Table 4).

Measurements of activation rates at +60 mV showed that single and double (T106A and T214A) mutants have similar activation profiles (Fig. 3C). When examining the raw traces, the double mutant produced larger currents. However, the currents at higher potentials appeared to activate much more slowly. These results suggest that the double mutation enables a greater flow of potassium ions through the pore. Our findings indicate that alanine mutations of the conserved threonines in the selectivity filter (T106A and T214A), which coordinate the canonical site-4 K+ ion, directly affect the pore domain and enhance gating efficiency, favoring open states over closed ones. Mapping these variants onto the structural framework of TWIK-2 channels suggests that mutations near the GYG selectivity filter and adjacent pore helices affect the function of the channel by disrupting K⁺ coordination. Most mutants impacted gating, often changing the voltage-dependence of activation (V50) by interfering with the coupling between regulatory sites and the pore. This led to hyperpolarizing shifts, indicating a gain-of-function due to easier opening (Supplementary Fig. 5B and Supplementary Table 6). These effects highlight that channel opening and closing are sensitive to disruptions in the selectivity filter and pore helices, which block K⁺ permeation, as well as disturbances of the M2/M4 helices at the bundle crossing that alter voltage dependence, and impaired coupling between the cytoplasmic regulatory domains and the pore, which slow or destabilize gating transitions.

Modulation of TWIK-2 by known K2P modulators
The distinct pseudo-tetrameric architecture of the transmembrane region and the sequence asymmetry between the pore-forming domains of the K2P channels are believed to render them insensitive to existing K+ channel blockers. Moreover, the cross-reactivity of known modulators between different subfamilies of K2P channels has made the efficient targeting of selective K2P channels a bottleneck47. To establish a high-throughput, automated approach for screening modulators of TWIK-2, we investigated the modulation of TWIK-2 using four known K2P modulators: ML335, BL1249, ML365, and NPBA (Fig. 4A). In agreement with previous reports, we observed that ML365 and NPBA inhibit TWIK-2 with IC50 values of 1.5 and 5.7 µM, respectively (Fig. 4B, C)48,49. However, we did not observe any modulation of TWIK-2 by ML335 and BL1249, which have been shown to modulate TREK and TRAAK family channels (Fig. 4B)8,48,59,60. To better understand how ML365 and NPBA inhibit TWIK-2, we compared their effects on inhibiting TWIK-2 and its mutants (Fig. 4D, E, and Supplementary Table 7). We compared the IC50 values of wild-type TWIK-2 against four mutants: one each at the entry (Y111H) and exit (M135A) of the ion permeation pore, as well as two at the bottom of selectivity filters SF1 (T106A) and SF2 (T214A). In all four mutants, NPBA showed negligible changes in IC50 compared to wild-type TWIK-2. The IC50 of ML365 remained unchanged for the M135A and T214A mutants. However, the Y111H (0.16 µM) and T106A (46 µM) variants of TWIK-2 exhibited increased and decreased susceptibility to ML365, respectively. Based on these findings, we used 50 µM of either ML365 or NPBA to assess maximum inhibition across all variants. We observed reduced maximum inhibition at the highest concentration of NPBA tested in Y111A, Y111H, M135A, S213A, and T106A-T214A double mutants (Fig. 4D, E, Supplementary Fig. 5C, and Supplementary Table 8). In the case of ML365, we noticed that the maximum inhibition for all the variants remained similar to wild-type TWIK-2, except the T106A-T214A double mutant, where we saw a significant reduction (Supplementary Fig. 5C). While the T106A-T214A double mutation caused a decrease in maximum inhibition for both ML365 and NPBA, the differences seen in IC50 values and maximum inhibition observed in other TWIK-2 mutants underscore potential differences in the inhibition mechanisms of ML365 and NPBA.

Comparison of the TWIK family of K2P channels
Given the notable sequence and structural similarities between TWIK-1 and TWIK-2, we compared their channel activity and modulation using our automated whole-cell patch-clamp setup. Our results indicated that TWIK-1 and TWIK-2 have similar current densities (Fig. 5A, Supplementary Fig. 5A, Supplementary Tables 5 and 9). However, TWIK-1 shows a significantly faster activation rate compared to TWIK-2 and exhibits much weaker inhibition by compounds such as ML365 and NPBA (Fig. 3C, Supplementary Fig. 5C, and Supplementary Tables 4 and 7). Furthermore, we observed a strong pH-dependent change in the voltage reversal potential for TWIK-1, aligning with previous reports (Fig. 5B and Supplementary Table 9). In contrast, this phenomenon was not seen in TWIK-2, even after introducing a TWIK-1-like Y111H mutation into its structure (Fig. 1A, B).

TWIK-1 is a pH-sensitive K2P channel whose structures at pH 7.5 and 5.5 have been previously reported39,40. Upon comparing the TWIK-2 structure (this study) with that of TWIK-1, both at pH 7.5, we find that the differences in the structure are localized to the cap region (Fig. 5C, D, and Supplementary Fig. 6A). When compared with TWIK-1 at pH 5.5, the differences in TWIK-2 structure lie only in the SF region, specifically SF1-M2 linker. H122 in SF1-M2 linker of TWIK-1 (equivalent to Y111 in TWIK-2) is held towards the transmembrane region inside a hydrophobic pocket between SF1 and PH1 (Fig. 5E and Supplementary Fig. 6B). At low pH, protonation causes release of H122 from this hydrophobic pocket, flipping it into a “up” conformation over the ion permeation pore blocking the K+ ion exit (TWIK-1) (Fig. 5F, G). In addition, low pH displaces the cap domain upwards by ~4 Å to accommodate the “up” conformation of H122 (Fig. 5D). Despite the residue Y111 in TWIK-2 (H122 in TWIK-1) adopting this distinct “up” conformation, the SF1-M2 linkers themselves stays splayed apart and they do not close over the SF obstructing the ion permeation pathway (Fig. 5F, G). Coinciding with the up conformation of Y111, we notice that the cap domain in TWIK-2 superposes better with the acid-inhibited TWIK-1 structure than the TWIK-1 at pH 7.5 (Fig. 5D). Hence, TWIK-2, at pH 7.5, has the cap region that is similar to acid-inhibited TWIK-1 (pH 5.5) but has the SF loops positioned in a similar way as that of the active TWIK-1 (pH 7.5) (Fig. 5C–G and Supplementary Fig. 6A, B). As TWIK-2 is not pH-sensitive, it is likely that displacement of helical cap is not used to regulate K2P channel activity, as is also the case in other lipid-regulated TREK and TRAAK family of K2P channels. These findings emphasize the distinct functional and structural characteristics of TWIK-1 and TWIK-2.

Methods
Cloning and overexpression of recombinant TWIK-2
The codon-optimized full-length wild-type human TWIK-2 (KCNK6) was synthesized by GenScript and was subcloned into the pEGBacMam expression vector (Addgene plasmid #160683) to be expressed with an N-terminal Strep-tag-II, followed by a GFP tag and an HRV-3C protease site. TWIK-2 mutations (F98A, T106A, Y111A, Y111H, M135A, M135N, S213A, T214A, T106A/T214A) were also made in this background by GenScript. Wild-type (TWIK-2) and variants of TWIK-2 were expressed in HEK293 GnTI− cells (ATCC #CRL-3022). Large-scale protein production was done using a baculoviral expression system as per the protocol described by Goehring et al.74. Briefly, the TWIK-2 expression vector was transformed into chemically competent DH10Bac cells (Thermo Fisher Scientific), and white colonies were selected on gentamicin-kanamycin-tetracycline LB agar plates. Isolated Bacmid DNA was used to transfect sf9 cells (ATCC #12659017) with Cellfectin-II reagent (Gibco) per the manufacturer’s protocol to make baculovirus P1. P1 was then amplified to make P2 by adding 200 μl of P1 to 200 ml culture of sf9 cells at a 1×106 cells/ml density. Cells were grown at 27°C for 96 h and harvested by spinning at 4000×g for 20 min. The supernatant was filtered with a 0.22 μm filter and stored at 4°C in the dark as a P2 virus. This was subsequently used for large-scale transduction of HEK293 GnTI− suspension adapted cells grown in FreeStyle 293 media supplemented with 2% heat-inactivated fetal bovine serum (FBS, Gibco) at density of 2.5–3.0×106 cells/ml. Transduced cells were incubated on an orbital shaker at 37°C and 8% CO2 at 120 rpm. Eight hours after transduction, 10 mM Sodium butyrate was added, and cells were grown for an additional 16–24 h at 37°C, 8% CO2. Cells were harvested by centrifugation at 4000×g for 20 min, flash-frozen in liquid nitrogen, and stored at −80°C.

Purification of recombinant human TWIK-2
Cell pellets from 2.4 L culture were thawed and lysed in 120 ml lysis buffer (25 mM Tris-HCl, 150 mM KCl, 0.8 μM aprotinin, 2 μg/ml leupeptin, 2 μM pepstatin A, 1 mM PMSF, pH 7.5) by sonication. Lysed cells were centrifuged at 2400×g for 5 min to remove cell debris. The supernatant was then ultracentrifuged at 185,000×g for 45 min. The pellet from ultracentrifugation was resuspended using a Dounce homogenizer in 60 ml of lysis buffer. To solubilize the membrane, 60 ml 2X solubilization buffer (25 mM Tris-HCl, 150 mM KCl, 0.8 μM aprotinin, 2 μg/ml leupeptin, 2 μM pepstatin A, 1 mM PMSF, 2% DMNG, pH 7.5) was added, and the solubilization was carried out for 90 min at 4°C. The solubilized membrane was then centrifuged at 185,000×g for 1 h. The supernatant was filtered through a 0.22 μm filter and was loaded onto a column containing 3 ml Strep-Tactin resin (IBA Life Sciences) at a flow rate of 0.5 ml/min. The column was then washed with 15 ml of wash buffer (25 mM Tris-HCl, 150 mM KCl, 0.1% DMNG, pH 7.5) and eluted with 12 ml elution buffer (25 mM Tris-HCl, 150 mM KCl, 0.1% DMNG, 5 mM Desthiobiotin, pH 7.5) in 1.5 ml fractions. The homogeneity of eluted fractions was analyzed by SDS-PAGE and subsequently pooled and concentrated to 7 mg/ml. In-house purified HRV3C protease was added to purified TWIK-2 at a molar ratio of 1:20 and incubated overnight at 4°C to remove the GFP tag. The digested protein was further purified by size-exclusion chromatography using Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with SEC buffer (0.1% DMNG, 25 mM Tris-HCl, 150 mM KCl, pH 7.5). The fractions corresponding to GFP-tag-free TWIK-2 dimer were pooled and concentrated to 2.3 mg/ml for cryo-EM sample preparation.

Cryo-EM sample preparation and data collection
UltraAuFoil 300 mesh 1.2/1.3 grids (Quantifoil) were glow discharged (PELCO) for 60 s at 15 mA before use. 3.5 μl purified TWIK-2 was applied to the glow-discharged grids and blotted for 2.5–3 s at 4°C and 100% humidity using a Vitrobot (Mark IV, Thermo Fisher Scientific). Grids were immediately plunged-frozen in liquid ethane. Grids were screened, and movies were collected at the University of Michigan cryo-EM facility on 300 kV Titan Krios G4i cryo-EM (Thermo Fisher Scientific) equipped with K3 direct detector camera (Gatan), and a post-BioQuantum GIF energy filter (Gatan), slit width set to 20 eV. 15,006 movies were collected using SerialEM 4.0 software in counting mode at a magnification of 105,000× (nominal pixel size of 0.83 Å/pixel). The total dose was 60 e− Å−2 with 60 frames with a defocus range between 0.8 and 2.5 μm.

Cryo-EM data processing
Data processing was performed in cryoSPARC (version 4.5.3)75 Briefly, 15,006 movie stacks were motion corrected and patch-CTF estimated, followed by a manual curation to remove micrographs with relatively thick ice and CTF fit >7 Å. 10,629 micrographs were subsequently selected and used for reference-free particle picking by blob picker with minimum particle diameter of 80 Å and maximum particle diameter of 100 Å followed by extraction at a box size of 256 pixels at binning of 2. 5,447,554 particles were picked and subjected to two rounds of 2D classification that resulted in 1,387,475 relative particles. A subset of ~380,000 particles from 2D classes with clear secondary structure features were used to generate four ab-initio 3D classes with C1 symmetry. One “good” class with clear transmembrane features and one “bad” class representing empty micelle were selected as reference input for downstream heterogeneous refinement with the total relatively clean 1,387,475 particles. Multiple rounds of heterogeneous refinement were performed until no further improvement of the resolution. 301,000 selected particles from the best class after the last heterogeneous refinement were re-extracted with 256 pixels and subjected to one round of Non-uniform refinement with C1 symmetry. A 3D mask was generated using volume of the best ab-initio class in Chimera 1.17.1 by manually removing the micelle density and imported into cryoSPARC with a threshold of 0.064 and dilation radius of 3 pixels. This mask was used for a focused 3D classification of the clean re-extracted particles into 4 classes in cryoSPARC. 104,997 particles from the class with clear secondary structure features were selected for reference-based motion correction, and refined by non-uniform refinement with C1 symmetry to obtain a final map at ~3.7 Å.

Model building, refinement, and structure analysis
The sharpened map from the final non-uniform refinement in cryoSPARC was used to build a preliminary model in Model Angelo 1.076. The preliminary model was analyzed, and improperly built regions were rebuilt manually in Coot 0.9.8.8377. Final structure refinement was performed in Phenix 1.2178. Figures were prepared using UCSF ChimeraX 1.1779. The radii along the channel pore were calculated using HOLE80. FoldX was used to generate mutant models of TWIK-281.

Automated patch clamp recordings
The wild-type and mutant TWIK-2 expression constructs were used to transfect adherent HEK293 GnTI− cells for automated whole-cell patch-clamp recordings. In brief, 1–7 million cells, grown in DMEM medium with 10% FBS, were transfected at a density of approximately 0.6–0.7 million cells/mL using 1–4 µg of DNA and TurboFect, following the manufacturer’s instructions (Thermo Fisher Scientific). After an 8-h incubation, the media was replaced with fresh DMEM containing 10 mM sodium butyrate, and the cells incubated for another 16–24 h before use. The cells were maintained at 37°C, 8% CO2, and 100% humidity. Before experiments, the cells were centrifuged at 200×g for 5 min, and the resulting pellet was resuspended in an external recording solution (see below) to achieve a final density of 500,000 cells/mL. This cell suspension was transferred to the instrument cell hotel, kept at 10°C, and shaken at 200 rpm until recordings began.

Automated patch clamp recordings were carried out using the Syncropatch 384 PE platform (Nanion Technologies, Munich, Germany). We used four-hole, 384-well recording chips with medium resistance between 2 and 4 MΩ. Pulse generation and data collection were managed with the PatchController384 V.1.3.0 and DataController384 V1.2.1 software (Nanion Technologies). Whole-cell currents were filtered at 3 kHz and sampled at 10 kHz. Access resistance and apparent membrane capacitance were estimated using the built-in protocols. Recordings were obtained from cells expressing both wild-type and mutant K2P channels at room temperature, with an internal solution containing 110 mM KF, 10 mM KCl, 10 mM NaCl, 10 mM HEPES, 10 mM EGTA, with pH adjusted to 7.2 using KOH. The external solution included 44 mM NaCl, 100 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 5 mM glucose, buffered with 10 mM HEPES (pH 7.4, adjusted with NaOH) or 10 mM MES (pH 5.5, adjusted with NaOH). Recordings were only accepted from cells that showed a seal resistance greater than 100 MΩ, a series resistance below 10 MΩ, and stable cell capacitance throughout the session. To assess the biophysical properties of wild-type and mutant TWIK channels, we measured both current density and potassium ion reversal potential (Vrev). Whole-cell currents were recorded from a holding potential of −90 mV and elicited with depolarizing steps (200 ms) from −90 to +100 mV in 10 mV increments, followed by a return step to −90 mV (tail currents, 100 ms), and current–voltage (I–V) relationships were plotted for each condition. The voltage reversal potential (Vrev) was identified from the I–V curve as the membrane potential where no net current was observed. Current density was calculated by normalizing the peak current amplitude at the highest voltage step to the cell’s membrane capacitance, which accounts for differences in cell size. Tail currents measured 1 ms after returning to −90 mV were normalized to their maximum amplitude and graphed against the test potential (−90 to +100 mV). To find the voltage at half-maximal activation (V50) and the slope factor (k), normalized G–V curves were fitted with the Boltzmann equation:

G=1=(1+exp[(V-V50)=k]) (1)

The properties of each TWIK-2 mutant were compared to those of the wild-type (WT) channel measured in parallel, focusing on the difference (ΔV50) in voltage-dependent activation V50.

To evaluate the effect of small molecules on TWIK-2 channel activity, the test compound was applied to the extracellular face (cap domain) of the channel using an external solution. A depolarization step to +60 mV was then performed for 350 ms, starting from a holding potential of -90 mV, and this procedure was repeated every 5 s. During each depolarization, the peak outward current amplitude was carefully measured and normalized to the currents recorded before the addition of the compounds. For the concentration-response curves (CRCs), various concentrations of the compounds were applied across the four-hole, 384-well recording chip, or a single high concentration of 50 µM was used to achieve maximum blocking. Electrophysiological responses were analyzed in detail by calculating the ratios of peak currents in the presence (Icompound) and absence (I0) of the test compounds. Data analysis and graphing were performed using DataController384 V.3.2.0 (Nanion Technologies) and GraphPad Prism V.10.5.0, with concentration-response curves fitted to a three-parameter sigmoidal model. The rates of channel activation and deactivation were determined by fitting the outward currents elicited at +60 mV and the tail currents during -90 mV pulses to a single exponential function using DataController384 from Nanion Technologies. The number of cells used for each experimental condition is listed in the figure legends. Detailed statistical methods were also described in the figure legends.

Drug and reagents
K2P modulator ML365 (Sigma-Aldrich SML2643), NPBA (MedChemExpress), ML335 (MedChemExpress), and BL1249 (Tocris) were diluted to 20 mM in sterile DMSO (Sigma-Aldrich) and stored in -80°C before use.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study determines a TWIK2 cryoEM structure and uses a commercial automated patch clamp setup to assess the function of TWIK2 channel mutants and pharmacology. The structure of TWIK2 had not been previously determined and this study provides a first look at the TWIK2 channel architecture. To accompany the structure, the authors perform electrophysiological recordings of TWIK2, a channel that has been notoriously difficult (though not impossible) to record from. Overall, the methodology in the structural and functional work appears to be sound and the writing is clear. There are novel observations and interesting results in the manuscript but an informative connection between knowledge learned from the TWIK2 structure and the functional studies performed is largely lacking. The Y111H designed to mimic the TWIK1 pH sensing histidine and the S4 site threonine residues are all positions that did not require a cryoEM structure to be interesting candidates for mutation. While the M135A mutation is a candidate site based on the TWIK2 structure, it is unclear why the authors chose this position as it does not occlude the pore or play any clearly observable role in the TWIK2 structure. The experiments examining the effects of ML365 and NPBA are interesting but somewhat superficial, as the mutants show some effects on channel pharmacology but do not yield any substantive insight into where the drugs might bind or how they might act.\n\nMajor Points\n1) In the section entitled “Ion conduction pore of TWIK2”, the authors suggest that the carbonyl oxygens of selectivity filter residues V107 and Y109 face away from the central permeation axis in TWIK-2 and argue for the relevance of this SF orientation to TWIK2 function. Given the ~3.7 angstrom resolution of the overall structure, I cannot ascertain how the authors come to this conclusion. Using the map and model provided by the authors, I can easily model the selectivity filter within the map in a conventional orientation and find that I improve the number of residues in favored orientations when the SF is modeled more conventionally.\nIf there is a biophysical argument to be made that the positioning of the valine and tyrosine sidechains in the TWIK2 map somehow promote an unconventional SF backbone configuration, this should be provided. Simply based on the map provided by the authors, I find this section to be unsupported by experimental results. If the authors do feel their selectivity figure configuration is accurate, a supplemental figure showing how the experimental map is better fit by the outward rather than inward facing carbonyls at V107 and Y109 would be extremely helpful to make the authors case on this point.\n2) In Figure 2B, the pore radius profile shows a constriction of the pore at around 5A from the cytoplasmic face of the channel. This cytoplasmic constriction does not seem to be discussed in the manuscript. Is this constriction brought about by the lipids modeled in the structure? If so, is this at all relevant to the function of TWIK2? If the authors believe that these lipid densities are important to shaping the TWIK2 pore, then this pore radius plot is appropriate. If the relevance of these lipids is unclear and could simply be an artifact of the protein purification process, it would be better to omit the lipid densities when running HOLE (or include a second HOLE profile in the absence of the lipids) to show the profile of just the TWIK2 protein. It would also be appropriate to discuss the lipid densities present in the map in the results section of the manuscript, rather than the discussion. This would be a good spot to do so.\n3) For the experiments where G/GMAX–V curves are calculated, it does not appear that the recordings reach a steady state at 200 ms. The current is still rising for TWIK1 wt and this is even more pronounced in the mutants that slow activation (Figure 3B). Is it possible that the differences in V50 are artifactual, since the maximal steady state current is reached at 200ms in some of the mutants but not in all of them.\n4) I think the manuscript would be greatly improved by a more direct comparison of the TWIK2 structure with the previously published TWIK1 structures. Does this TWIK2 structure look more like the low pH or neutral pH TWIK2 structure? Are there clear differences in the positioning of the cap or TM helices? The entire section of the results devoted to an overview of the TWIK2 channel could be redone as a more direct comparison between TWIK1 and TWIK2 and would significantly increase interest.\n\nMinor Points\n- In the introduction, “Kcnk6 knockout studies have shown suppression of NLRP3 inflammasome activation in macrophages, implicating endolysosomal localization of TWIK-2 in inflammation 45,46.” This sentence is a bit misleading and should be clarified. From the references cited, it is movement of TWIK2 from the endolysosome to the plasma membrane that appears to be a critical step in the potassium efflux that mediates NLRP3 inflammasome activation.\n- In figure 1 (and for nearly all electrophysiological traces shown in the manuscript), recording traces are shown on a signal scale that reads “Normalized I”. It is unclear what the non-transfected cells traces are normalized to? For clarity throughout the manuscript, it would be helpful if the authors explicitly discuss whether normalization was applied using a universal metric (which could be described in the methods section of the manuscript) or ad hoc for each set of recordings (in which case this should be described in each figure legends)\n- Given that the C-terminally truncated TWIK construct was not used for structure of detailed functional studies, this data could be omitted or moved to the methods section.\n- Is it meaningful that the loop region between V75-P89 is unresolved in the cryo-EM structure? In all other K2P structures, this loop is well resolved. Does this mean that the V75-P89 is dynamic in TWIK2, suggestive of a functional role? Or is this just a limitation of the resolutions of this TWIK2 structure compared to other K2P structures? Does 3D variability analysis in cryosparc give any indication of what is going on in this loop region?\n- The panel in Figure 3 showing the relative locations of T106, T214, M135, and Y111 is critically important for understanding the orientation and relevance of these residues. I would suggest reworking this figure to make it clearer. The inset boxes are distracting, as each one has a different orientation relative to the main panel. Perhaps a clearly labeled “top view” showing the Y111 residues, “bottom view showing the M135 residues, and a side view like shown in the left side of Figure 3A would be sufficient for this.\n- “Notably, TWIK-2 channels elicited outward potassium currents that increased with depolarization (over +30 mV), suggesting voltage-dependent activation of TWIK-2” Isn’t this a surprising result, given that prior recordings in symmetrical potassium have shown inward rectification rather than outward rectification? Is there any methodologic explanation for this difference in observed functional behavior?\n- Reference 70 in the manuscript includes data that describe the effect of mutations at the S4 site threonines in K2Ps. For nearly all K2Ps, these S4 site mutations cause a loss of volage dependance. It is therefore interesting that in TWIK2, the T106A and T214A mutants maintain their voltage dependent gating. Is there any rationale for why this might be the case?\n- “Mutations T106A and T214A disturb these networks of hydrogen bonding and hydrophobic interactions that stabilize the base of the SF and make the channel less selective and more open (Figure 3B-3F).” The functional experiments presented in this section use only a single ionic composition and are not suited to adequately address ion selectivity. Please support with data or omit this claim.\n- Is the TWIK2 Y111H mutant pH sensitive like the TWIK1 channel? This seems like an obvious experiment to try and would be informative to understanding differences between TWIK1 and TWIK2 function.\n- More methodological detail of the automated patch clamp technique would be helpful to understand the experimental system. Are drugs applied to the intracellular face of the channel (via syncropatch pipette) or extracellular via bath?", "reviewer_2": "The manuscript of Ma et al, provides new insights into the modulation of the K2P channel TWIK-2 using structural and electrophysiological data. The study provides the first cryo-EM structure of the full-length TWIK-2 at 3.7Å resolution, which is accompanied by mutagenesis studies and the use of a whole-cell patch clamp electrophysiology workflow. The authors show that TWIK-2 is pH-insensitive but slightly voltage-gated, and suggest potential different binding modes or mechanisms of inhibition in TWIK-2 for two of the four tested K2P modulators (ML365 and NPBA).\nThe work is well executed and well written. However, overall the study fall a bit short. The new insights provided do not represent a big step forward in improving our understanding of how TWIK-2 (and K2P channels in general) function and are regulated. The substance and depth of the study could be/have been strengthened by more structure-guided mutagenesis studies, a more robust comparison of their new automated electrophysiology setup, a better literature comparison with (mutagenesis studies of) TWIK-1 and other K2P channels, or even more structural data or structural analysis. While I understand that addressing them all might be out of the scope of this study, the manuscript requires a bit more of depth.\nSpecific suggestion would be (major comments):\n1. In total ‘only’ four residues are identified as different/interesting and tested. Namely M135A, T106A, T214A and Y111H. What about other positions, e.g., around the cavity or along the ion conductance pathway? Is everything else conserved? What about testing mutating the same sites with other substitutes or using double and triple mutations to gain more insights? Compare in more depth with TWIK-1 and other K2P channels.\n2. Most striking in this endeavor is the mutant Y111H. The authors show that in contrast to TWIK-1, TWIK-2 is pH-insensitive, with the histidine in that position being the pH-sensing residue in TWIK-1. While the authors go on and attempt to describe some of the differences observed for TWIK-2-Y111H, they never actually tested if this mutant has become pH-sensitive. Considering the overall conservation between TWIK-1 and TWIK-2 this would be expected and highly interesting to test. Further, to test the importance of the tyrosine and decouple it from a pH sensor position, a mutation to another residue, e.g. Y111A and Y111L would be interesting. Finally, the fact that TWIK-2_Y111H mutant carries the pH sensor of TWIK-1 must be mentioned in all clarity also in the results and discussion section and taking into account during interpretation.\n3. I appreciate and highly welcome the high-throughput patch clamp workflow presented by the authors. However, considering i) the discrepancies mentioned by the authors on electrophysiology studies for TWIK-2 in the literature, and ii) the mentioned inconsistencies between some of their electrophysiology data with that published before, the authors should do a more thorough comparison. If possible, between methods conducted by themselves with the same non-glycosylated constructs (conventional patch clamp and high-throughput patch clamp). But perhaps more importantly, use TWIK-1 in their automated whole-cell patch clamp electrophysiology setup, for which more robust recordings have been reported. This would allow to asses and compare TWIK-1 and TWIK-2 and the different workflows better.\n4. The slightly different impact found for two of the tested inhibitors makes the discussion a bit ambiguous as to their potential different binding mode. I wonder if an attempt to solve an inhibitor-bound structure would be in the scope of this study as the authors seem to be able to obtain enough stable protein and the processing appears rather straightforward.\nOther major comments:\n5. Can the authors comment on the following: With regards to the processing of the cryo-EM data, I wonder if the authors also tested refining the other 3 classes obtained before proceeding with a final NU-refinement of one of the classes. Considering the authors interpret their structure as an intermediate conductive state, I wonder if there were some hints at other conformational states captured in the data.\n6. Could the authors comment why they mutated Y111H to mimic the corresponding TWIK-1 residue, but then mutated M135A instead of M135L (which would be the corresponding TWIK-1 residue)? What was the rational/strategy here? And here again, it would have been ideal to have TWIK-1 as a control experiment, and to compare Y111H with.\n7. Could the authors comment/expand on the voltage-dependence of TWIK-2 in the, and whether other K2P channels have also been found to be so.\n8. While I appreciate the modeling of potential lipids and co for the interpretation and figure rendering of non-protein densities, considering the high ambiguity, I expect that the authors have no intention of including it in the to be deposited model. (in the era of AlphaFold and machine learning, everything that is deposited can and will be use by AI or others)\nMinor comments:\n- Page 4: \"we identified key amino acids that differentiate TWIK-2 from TWIK-1 and other members of the K2P family\". From what I understood, only Y111 was a direct comparison with TWIK-1, so I suggest toning down this sentence.\n- Could the authors add details of this inhibition experiment in the Methods section (e.g. how was the small molecule screening set-up, quantity/volume of compound added, (pre-) incubation time, etc.). Could the authors show a blank/control measurement with just DMSO to account for the buffer?\n- Methods section: Cloning and overexpression of recombinant TWIK-2: The text should say for better clarity: \"with an N-terminal Strep-tag-II, followed by a GFP tag and a HRV-3C protease site\" instead of \"followed by an HRV-3C protease site and a GFP tag\" as it is then later stated that 3C cleavage results in removal of GFP tag.\n- As the authors mention in the main text the poor heterologous expression of TWIK channels, could they indicate the yield they obtained from their experiments?\n- Correct typo in: \"Mutations T106A and T214A\"\nFigures:\n- Please update figure numbers so that they are cited in correct order as mentioned in the text. E.g S2 should be called S1; swap order of Figure 4D and 4E\n- Figure S2F: could the authors explain the uV units on the y-axis of the FSEC curve\n- Specify in main text and S2 figure legend which detergent was used for solubilisation of ΔC-TWIK-2\n- For better clarity, keep one naming for each construct between figure and figure legends (e.g. in figure S2: TWIK-2 is sometimes called WT or wild-type; or ΔC-TWIK-2 (C-ter 1-276) is called c-ter 1-276, c-ter and del1-276).\n- In general, could the authors fix all the inconsistencies in the figure legend of S2: missing periods, having a mixture of (n=15) and \",n=15\" etc.\n- Figure 2A: could the authors improve the illustration so that the ion conduction pathway is clearer to the reader?\n- Figure S1: indicate the non-conserved M135 and Y111 residues on the sequence alignement\n- Figure S3: FSC curves indicates the mask during refinement is too tight and could be optimised\n- Could the authors mention/indicate the possible glycosylation sites that TWIK-2 possess? This could be added to S1.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank all the reviewers for their critical assessment of our manuscript. Our point-by-point response is below. For purposes of clarity, we have reviewer comments in red italics.\n\nReviewer #1 (Remarks to the Author):\n“This study determines a TWIK2 cryoEM structure and uses a commercial automated patch clamp setup to assess the function of TWIK2 channel mutants and pharmacology. The structure of TWIK2 had not been previously determined and this study provides a first look at the TWIK2 channel architecture. To accompany the structure, the authors perform electrophysiological recordings of TWIK2, a channel that has been notoriously difficult (though not impossible) to record from. Overall, the methodology in the structural and functional work appears to be sound and the writing is clear. “\nWe sincerely thank the reviewer for the overall positive feedback.\n“There are novel observations and interesting results in the manuscript but an informative connection between knowledge learned from the TWIK2 structure and the functional studies performed is largely lacking. The Y111H designed to mimic the TWIK1 pH sensing histidine and the S4 site threonine residues are all positions that did not require a cryoEM structure to be interesting candidates for mutation. While the M135A mutation is a candidate site based on the TWIK2 structure, it is unclear why the authors chose this position as it does not occlude the pore or play any clearly observable role in the TWIK2 structure. The experiments examining the effects of ML365 and NPBA are interesting but somewhat superficial, as the mutants show some effects on channel pharmacology but do not yield any substantive insight into where the drugs might bind or how they might act.”\nWe thank the reviewer for pointing out weaknesses in this manuscript that could be improved. We agree with the reviewer that the mutant selection was sparse. To address this, the analysis in the revised version of the manuscript includes the following single and double mutants: F98A, Y111H, Y111A, T106A, T214A, S213A, M135N, M135A, and T106A-T214A, focusing on the regions near the SF, the K2P modulator pocket (F98A), and the hydrophobic cuff (M135A and M135N). The alanine substitution of M135 (M135A) drastically reduces the size of the side chain, affecting not only the size of the pocket near M135 but also the relative hydrophobic surface area. As we observed lipid-like densities around the M135 amino acid, suggesting their role in regulating channel activity, we anticipated that the M135A mutation would have a drastic effect on TWIK-2 activity. In the revised manuscript, we also tested M135N to explore the role of introducing a polar amino acid in this region on TWIK-2 activity. We observe that both M135A and M135N mutants exhibit slightly slower channel activation, with no drastic effect on channel activity. However, M135A shows a greater loss of NPBA inhibition compared to M135N, suggesting that removing the bulky amino acid at this position affects the efficacy of NPBA. Similarly, we also tested the effect of changing the pocket around Y111 on TWIK-2 channel activity using a Y111A mutant. We found that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition, highlighting the role of Y111 in maintaining SF1-M2 loop integrity.\nTo study the binding modes of ML365 and NPBA, we collected and analyzed cryo-EM data using TWIK-2 complexed with ML365 and NPBA. We observed that in the presence of these ligands, the sample appeared to be more heterogeneous, resulting in maps of lower quality compared to the apo TWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound TWIK-2 maps are fairly similar (~ 4 Å), with one common feature being the loss of strong tubular lipid-like densities that we observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is possible that loss of tubular lipid-like densities is a factor of poor resolution of the map. To make reasonable comparisons, we filtered the apo-TWIK-2 map to 4Å (Response Fig. 1).\nResponse Figure 1: cryo-EM map comparison of TWIK-2 Apo, TWIK-2 ML365 and TWIK-2 NPBA. Two bottom views on the right indicate the occupancy of the cavity close to the selectivity filter (top) and cytoplasmic site (bottom).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe some non-protein density immediately below the SF in the case of the NPBA dataset. The non-protein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor resolution of the map, and the size and shape of the density, preclude us from unambiguously modeling NPBA at this site. Hence, we have not included these efforts in the manuscript and are presenting them here in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound structures were not successful, we employed site-directed mutagenesis to identify a potential NPBA binding site by performing more mutations in the SF, K2P modulator pocket, and around the hydrophobic cuff. We have discussed these in the main manuscript and our findings suggest that the potential binding site for NPBA, as hinted by additional density in the map, could be below the SF. The mutagenesis exploration still does not identify the site for ML365 unambiguously. However, loss of strong tubular lipid-like densities around the hydrophobic cuff compared to apo TWIK-2 makes us suspect that ML365, like NPBA could bind through the cavity, albeit interacting with different residues compared to NPBA.\n“1) In the section entitled “Ion conduction pore of TWIK2”, the authors suggest that the carbonyl oxygens of selectivity filter residues V107 and Y109 face away from the central permeation axis in TWIK-2 and argue for the relevance of this SF orientation to TWIK2 function. Given the ~3.7 angstrom resolution of the overall structure, I cannot ascertain how the authors come to this conclusion. Using the map and model provided by the authors, I can easily model the selectivity filter within the map in a conventional orientation and find that I improve the number of residues in favored orientations when the SF is modeled more conventionally.\nIf there is a biophysical argument to be made that the positioning of the valine and tyrosine sidechains in the TWIK2 map somehow promote an unconventional SF backbone configuration, this should be provided. Simply based on the map provided by the authors, I find this section to be unsupported by experimental results. If the authors do feel their selectivity figure configuration is accurate, a supplemental figure showing how the experimental map is better fit by the outward rather than inward-facing carbonyls at V107 and Y109 would be extremely helpful to make the authors' case on this point.”\nWe thank the reviewer for a thorough review of our structure. In our TWIK-2 structure, the SF has been modeled with carbonyl oxygens of three amino acids – V107, Y109, and L217 – facing away from the ion-conductance pore. While we agree that at ~ 3.7 Å resolution it could be challenging to resolve the orientation of carbonyl oxygens in the dynamic loops of the SF region unambiguously, we would like to highlight that the local resolution for this region is much better than 3.5 Å (Supplementary Fig. S3F in the manuscript). As per the reviewer’s critique, we attempted to model these three carbonyls in various combinations of them facing away or towards the SF. However, just based on visually comparing the fits of these various models to density, it is not straight forward to identity one particular combination of the carbonyl oxygens that fits better to the map (see figures & table below). Hence, based on a comparison of the model statistics such as bond RMSDs, Ramachandran outliers, Rama-Z score, & CaBLAM outliers for the SF region, and the overall model quality statistics for the whole protein, we believe that the model as it has been presented in the manuscript is the best possible fit (Response Table 1 in this rebuttal).\nIn K2P channels, the two conserved threonines at the bottom of the selectivity filter (SF) are the only amino acids within the SF that coordinate K+ ions through their side chains. All other amino acids at the SF coordinate K+ ions through carbonyl oxygens. It has been shown by multiple groups previously that reorientation of the carbonyl oxygens away from the SF is a means to regulate K+ ion flow, including channel inhibition, in potassium channels. Using X-ray crystallography and cryo-EM, several groups have shown that reorientation of carbonyl oxygens away from the SF pore affects the K+ ion coordination at the SF as part of C-type or pH-mediated inactivation of the channel in KcsA, Kv, and TWIK-11–4. The flipped carbonyls often co-existed with reduced occupancy at the canonical K+ ion sites (S0-S4) in the SF. As noted in the manuscript, our cryo-EM map allowed us to confidently model K+ ions in only three out of these five sites. Similarly, MD simulations on the prototypical K+ channels like MthK and KcsA have shown that reorientation of the carbonyl oxygen of the conserved valine at the SF is one of the initial steps that promotes dissociation of K+ from the SF and allows the SF to adopt nonconducting or inactivated states5,6. As pointed out by the reviewer, we had provided only a brief biophysical explanation for non-canonical carbonyl positioning in the discussion in the original manuscript. To address that, we have expanded on the explanation associated with flipped carbonyl observation, by including examples of other eukaryotic K+ channels (TWIK-1, Kv, and Kir) and their prototypic prokaryotic homologs (MthK and KcsA) where similar observations have been made, as a separate section in the discussion, & added more references to this section.\nResponse Figure 2: Various TWIK2 selectivity filter models fit to the cryo-EM density. The model quality statistics for the variations 1-7, and a comparison of the statistics are provided in table 1. Cryo-EM density is highlighted as blue mesh and the three K+ ions as blue spheres. The two protomers, TWIK-2 chain A and B, are colored light magenta and light cyan. All the amino acids at the SF are labeled in variation 7 where none of the carbonyls are flipped away from the SF, and in the remaining panels only the carbonyls that are flipped away from the SF have been labeled.\nResponse Table1: TWIK2 selectivity filter model quality statistics. Selectivity filter model quality statistics were obtained by running PDB validation on the amino acids 104-111 and 213-220, and K+ ions at the SF using Phenix. The values in the parentheses represent the whole PDB. As the resolution of the structure is ~3.7 Å, CaBLAM outliers, Rama-Z score, Bond RMSDs for the SF region, and the overall statistics of the whole PDB were compared to decide on the carbonyl orientations of the amino acids V107, Y109, and L217, which surround S0/S1 and S2/S3 sites at the SF. Each variation (Var. 4-6) or combination of variations are show (Var. 1-3)\nDeposited Parameter Var. 1 Var. 2 Var. 3 Var. 4 Var. 5 Var. 6 Var. 7 9E94\nAmino acids with V107, carbonyl Y109 & V107 & V107 & oxygens flipped L217 Y109 V107 None away from SF L217 L217 Y109 L217\nBond length 0.007 0.014 0.014 0.007 0.017 0.018 0.016 0.017\nRMSD (Å) (0.009) (0.010) (0.009) (0.009) (0.010) (0.010) (0.010) (0.010)\nBond angle 1.617 2.103 2.161 1.724 2.292 2.188 2.265 2.441\nRMSD (o) (1.564) (1.651) (1.624) (1.595) (1.672) (1.662) (1.643) (1.693)\n1.97 1.49 2.01 2.53 2.05 1.97 2.48 2.45\nMolProbity score (1.78) (1.75) (1.78) (1.89) (1.78) (1.82) (1.86) (1.88)\nRamachandran 0.00 0.00 8.33 8.33 0.00 0.00 0.00\noutliers (%) (0.00) (0.00) (0.47) (0.47) (0.00) (0.00) (0.00) 0.00 (0.00)\nRamachandran 8.33 8.33 25.00 41.67 8.33 50.00 58.33 50.00\nallowed (%) (6.07) (6.54) (7.01) (7.94) (6.07) (8.88) (8.88) (8.88)\nRamachandran 91.67 91.67 75.00 50.00 83.33 50.00 41.67 50.00\nfavored (%) (93.93) (93.46) (92.99) (91.59) (93.46) (91.12) (91.12) (91.12)\n-5.32 -6.25 -7.54 -6.18 -7.92 -6.96 -8.91 -9.44\nRama-Z (whole) (-3.75) (-3.79) (-3.86) (-3.78) (-3.90) (-3.81) (-3.93) (-4.00)\nRama-Z (loop) -4.05 -4.76 -5.74 -4.70 -6.03 -5.30 -6.79 -7.19\n(-4.87) (-5.04) (-5.29) (-5.06) (-5.33) (-5.09) (-5.53) (-5.64)\nCaBLAM outliers 25.00 50.00 50.00 37.50 62.50 50.00 62.50 75.00\n(%) (2.88) (3.85) (4.33) (3.37) (4.33) (3.85) (4.81) (4.81)\nClash score 8.81 2.20 4.39 10.96 6.62 2.21 8.81 8.83\n(6.76) (5.91) (6.05) (6.34) (6.34) (5.62) (6.20) (6.63)\n“2) In Figure 2B, the pore radius profile shows a constriction of the pore at around 5A from the cytoplasmic face of the channel. This cytoplasmic constriction does not seem to be discussed in the manuscript. Is this constriction brought about by the lipids modeled in the structure? If so, is this at all relevant to the function of TWIK2? If the authors believe that these lipid densities are important to shaping the TWIK2 pore, then this pore radius plot is appropriate. If the relevance of these lipids is unclear and could simply be an artifact of the protein purification process, it would be better to omit the lipid densities when running HOLE (or include a second HOLE profile in the absence of the lipids) to show the profile of just the TWIK2 protein.\nIt would also be appropriate to discuss the lipid densities present in the map in the results section of the manuscript, rather than the discussion. This would be a good spot to do so.”\nThe pore radius plot in Fig. 2B was constructed using the TWIK-2 model with hydrocarbon tails modeled into the tubular lipid-like densities. We have now edited Fig. 2B to show the pore radius profile with and without the modeled lipid-like densities. We did not include any exogenous lipids during the purification. So, we believe these tubular densities arise from the molecules that co-purified with TWIK-2 from HEK293 GnTI- cells. Because we could not verify the exact nature of these molecules, we have only modeled hydrocarbon tails in these densities and called them lipid-like in the manuscript. The discussion on the lipid-like densities has now been moved to the results section in the revised version of the manuscript, as per the reviewer’s suggestion. While this manuscript was under revision, another group reported a preprint suggesting a potential lipid mediated regulation of TWIK-2 channel activity. They argued that lipid molecules “plug” the channel on the cytoplasmic side by occupying the central cavity and by physically obstructing the passage of ions, rendering TWIK-2 inactive. They demonstrate that R257, an amino acid towards the C-terminus that lies below the hydrophobic cuff, is crucial for this lipid binding in the central cavity and that the R257A mutation increases the channel activity, suggesting that channel activation is a result of lipid removal or repositioning in the central cavity. In our TWIK-2 structure, we observe lipid-like densities in similar positions (central cavity and along the groove formed by the PH2, M2, and M4 helices) as reported by Mondal et al7. However, these lipid-like densities do not entirely obstruct the access to the central pore (Fig. 2). The observation of lipid-like density, corroborated by a different group independently, suggests that these molecules are relevant to the function. We have added a few lines discussing this preprint and its relevance in the context of our structure to the discussion section of the revised manuscript.\n“3) For the experiments where G/GMAX–V curves are calculated, it does not appear that the recordings reach a steady state at 200 ms. The current is still rising for TWIK1 wt and this is even more pronounced in the mutants that slow activation (Figure 3B). Is it possible that the differences in V50 are artificial, since the maximal steady state current is reached at 200ms in some of the mutants but not in all of them.”\nWe appreciate the reviewer’s concerns regarding the possible underestimation of steady-state currents due to incomplete activation at the end of the 200-ms depolarizing pulse, especially in slow-activating TWIK-2 mutants. To reinforce the validity of our conclusions regarding mutant-induced shifts in voltage dependence, we performed a new analysis based on normalized tail currents, which better reflect the relative conductance by capturing the instantaneous current upon repolarization—thus minimizing the effects of kinetic differences in channel activation. Specifically, we constructed revised G/GMAX–V curves using tail currents, enabling a more accurate assessment of the voltage dependence of activation across all variants. From these curves, we calculated the ΔV50 values, which provide a more reliable measure of the voltage shift caused by each mutation. Importantly, this alternative approach produced similar V50 shifts as previously reported, confirming that the observed differences are genuine and not artifacts, reflecting fundamental changes in channel gating properties. These new results are now included in the revised Supplementary Fig. 5 and discussed in the Results section. To enable a better comparison of currents across TWIK-2 variants, we have also modified Fig. 3 to now display whole-cell current traces and activation time constants for all the mutants that we tested.\n“4) I think the manuscript would be greatly improved by a more direct comparison of the TWIK2 structure with the previously published TWIK1 structures. Does this TWIK2 structure look more like the low pH or neutral pH TWIK2 structure? Are there clear differences in the positioning of the cap or TM helices? The entire section of the results devoted to an overview of the TWIK2 channel could be redone as a more direct comparison between TWIK1 and TWIK2 and would significantly increase interest.”\nWe agree with the reviewer that a comprehensive comparison of TWIK-2 with TWIK-1 could be a beneficial addition to the manuscript. In our first version of the manuscript, we had presented a comparison of TWIK-2 and TWIK-1 in Figs. 5B, C and Supplementary Fig. S4, focusing only on the conformational changes in pore domains, SF, and M4 helix. As TWIK-2 is physiologically unlike TWIK-1, for the sake of completeness, we have also included comparisons with other pH-sensitive and lipid-regulated K2P channels. To make these comparisons more apparent, we have reformatted and expanded the figures and text to include a separate comparison section of the TWIK-2 and TWIK-1 structures in their entirety. In addition, we have also included the results from whole-cell recordings studying the pH dependency of channel activity in TWIK-1 and the TWIK-1-like Y111H mutant of TWIK-2. Interestingly, we find that the differences in the structure of TWIK-2 are localized to the cap region when compared with the TWIK-1 structure at pH 7.5. When compared with TWIK-1 at pH 5.5, the differences lie only in the SF region, specifically the SF1-M2 linker that houses the pH-sensing amino acid H122 in TWIK-1. TWIK-2 has the cap region that is similar to inactive TWIK-1, but has the SF loops positioned in a similar way as that of the active TWIK-1, despite the residue Y111 in TWIK-2 (H122 in TWIK-1) adopting a unique ‘up’ conformation. These new observations have been discussed in the revised version of the manuscript, alongside the TWIK-2 and TWIK-1 comparisons from the older version (Fig. 1, 5). The comparison of TWIK-2 with other K2P channels has been reformatted into a separate figure (Supplementary Figs. 4, 7).\nMinor Points\n1. - In the introduction, “Kcnk6 knockout studies have shown suppression of NLRP3 inflammasome activation in macrophages, implicating endolysosomal localization of TWIK-2 in inflammation 45,46.” This sentence is a bit misleading and should be clarified. From the references cited, it is movement of TWIK2 from the endolysosome to the plasma membrane that appears to be a critical step in the potassium efflux that mediates NLRP3 inflammasome activation.\nWe thank the reviewer for the correction. We have now replaced the sentence with “Translocation of TWIK-2 from endosome to plasma membrane is implicated in NLRP3-mediated inflammasome activation in macrophages, with Kcnk6 knockouts showing a suppressed inflammasome activation”.\n2. - In figure 1 (and for nearly all electrophysiological traces shown in the manuscript), recording traces are shown on a signal scale that reads “Normalized I”. It is unclear what the non-transfected cells traces are normalized to? For clarity throughout the manuscript, it would be helpful if the authors explicitly discuss whether normalization was applied using a universal metric (which could be described in the methods section of the manuscript) or ad hoc for each set of recordings (in which case this should be described in each figure legends)\nWe thank the reviewer for pointing out this important issue related to the normalization of electrophysiological traces. In the revised manuscript, all figures now display traces showing actual absolute current values instead of normalized signals. This update allows for direct comparison of current amplitudes across wild-type and mutant channels as well as non-transfected controls. Additionally, we now include current density for each experimental condition where appropriate, ensuring that differences in current amplitudes are not influenced by variations in cell size. We believe these revisions significantly improve the clarity and transparency of our data presentation and address the reviewer’s concern about normalization.\n3- Given that the C-terminally truncated TWIK construct was not used for structure of detailed functional studies, this data could be omitted or moved to the methods section.\nWe agree with this reviewer’s observation that the data on C-terminally truncated TWIK-2 is not informative, beyond suggesting that TWIK-2 devoid of C-terminus is unstable compared to WT. In accordance, we have now removed the C-term truncation data of TWIK-2 from the revised manuscript.\n4 - Is it meaningful that the loop region between V75-P89 is unresolved in the cryo-EM structure? In all other K2P structures, this loop is well resolved. Does this mean that the V75-P89 is dynamic in TWIK2, suggestive of a functional role? Or is this just a limitation of the resolutions of this TWIK2 structure compared to other K2P structures? Does 3D variability analysis in cryosparc give any indication of what is going on in this loop region?\nThe loop V75-P89 is a part of the CH2-PH1 region that harbors two predicted glycosylation sites, N79 and N85. We used HEK293 GnTI- cells, which lack N-acetylglucosaminyltransferase I activity, for overexpression of TWIK-2. So, it is likely that glycosylation at these sites is essential for stabilizing this loop, and without such glycosylation, this loop is flexible and hence poorly resolved in the cryo-EM density map. Interestingly, TWIK-2 structures reported by a different group recently also lacked this region 8. Despite using glycosylation-competent Expi293F cells and achieving an overall higher resolution structure (~2.9 Å). Perhaps, in TWIK-2 this loop is more dynamic than other K2Ps. Another group reported structures of TWIK-2 while this manuscript was under revision 7. They too use glycosylation-capable Expi293F cells as Khanra et. al., but were able to resolve the V75-P89 region in their cryo-EM map. Moreover, they find that amino acids 75-80 in this loop actually form a short helix called ‘ear helix’ that is unique to TWIK-2, proposed to stabilize the TWIK-2 cap domain. As this loop region can also potentially interact with the keystone inhibitor site and the K2P modulator pocket, we attempted to resolve this region using 3DVA and subsequent classification in cryoSPARC (Response Fig. 3). However, our attempts were not successful.\n5- The panel in Figure 3 showing the relative locations of T106, T214, M135, and Y111 is critically important for understanding the orientation and relevance of these residues. I would suggest reworking this figure to make it clearer. The inset boxes are distracting, as each one has a different orientation relative to the main panel. Perhaps a clearly labeled “top view” showing the Y111 residues, “bottom view\" showing the M135 residues, and a side view like shown in the left side of Figure 3A would be sufficient for this.\nWe have modified the figure as per the reviewer’s suggestions.\n- “Notably, TWIK-2 channels elicited outward potassium currents that increased with depolarization (over +30 mV), suggesting voltage-dependent activation of TWIK-2” Isn’t this a surprising result, given that prior recordings in symmetrical potassium have shown inward rectification rather than outward rectification? Is there any methodologic explanation for this difference in observed functional behavior?\nWhile it may seem surprising at first, the voltage-dependent activation of TWIK-2 seen in our recordings agrees with previous studies, especially those indicating that TWIK-2 is one of the least inwardly rectifying members of the K2P family, that inactivates and has a potential role in electrogenesis9–12. Compared to strongly rectifying K2P channels, such as TREK-1 or TASK-3, TWIK-2 exhibits more linear or even outwardly biased I–V relationships under certain recording conditions. Moreover, differences in rectification behavior—particularly outward currents at depolarized potentials—can depend on the expression system (e.g., Xenopus oocytes versus mammalian cells), ionic composition, and intracellular environment (e.g., Mg²⁺, polyamines, or pH). In our experiments, outward currents at voltages above +30 mV are probably caused by a combination of weak inherent rectification and the electrochemical driving force for K⁺ efflux under symmetrical or nearly symmetrical K⁺ conditions, rather than unusual voltage-dependent gating. To clarify this, we added a section in the discussion explaining how our findings align with the known electrophysiological properties of TWIK-2 and reflect system-dependent variability rather than an unexpected gating mechanism.\nResponse Figure 3: 3D variability analysis (3DVA) and subsequent classification to resolve V75-P89 loop. The region shaded in gray is a part of the supplementary figure 3 in the manuscript and has been included here only for reference. Particles from the best cluster displaying all the features of TWIK-2 from 3DVA step was used to generate an ab initio initial reference (cyan box). Next, particles from all clusters with robust cap domain features (red boxes) from the 3DVA step were pooled for clean up by heterogenous refinement and ab initio jobs. The final map obtained after NU-refinement, using the ab-initio reference generated from the best 3DVA cluster, resulted in a map with features and resolution that resembled the map already presented in the manuscript.\n- Reference 70 in the manuscript includes data that describe the effect of mutations at the S4 site threonines in K2Ps. For nearly all K2Ps, these S4 site mutations cause a loss of voltage dependance. It is therefore interesting that in TWIK2, the T106A and T214A mutants maintain their voltage dependent gating. Is there any rationale for why this might be the case?\nThe reference 70 reports that in channels without a dedicated voltage-sensing domain, the SF itself acts as a gate and sensor. They demonstrate that mutating the conserved threonines at the SF to cysteines causes loss of voltage-dependent behavior. However, they also show that in addition to just these mutations, voltage-dependent behavior is also regulated by other factors such as mechanical force, ligands, and lipids13. Regulation of voltage-dependency of K2P channels by these auxiliary factors, apart from the residues at SF, has been shown in several K2P channels, especially the TREK family14–18. These reports suggest that the same K2P channel can switch between voltage-dependent and voltage-independent current responses depending on their conditions, perhaps a regulatory mechanism of certain K2P channels. T106A and T214A mutants display a reduced-voltage dependency in our experiments, but not a complete loss of voltage-dependency. Because we see that the central cavity is occupied by lipid-like densities in our cryo-EM map, and because TWIK-2 has an ability to translocate between endosomes and plasma membrane and has been proposed to interact with other proteins via its C-terminus, we believe it is possible that voltage-dependency in TWIK-2 could be a context dependent phenomenon, in addition to being regulated by the residues at the SF. However, identification of all the factors in TWIK-2 that regulate its channel activity needs furthermore substantial efforts.\n- “Mutations T106A and T214A disturb these networks of hydrogen bonding and hydrophobic interactions that stabilize the base of the SF and make the channel less selective and more open (Figure 3B-3F).” The functional experiments presented in this section use only a single ionic composition and are not suited to adequately address ion selectivity. Please support with data or omit this claim.\nWe agree with the reviewer that with only a single ionic composition, it is hard to address the ion selectivity of TWIK-2. We have rephrased this statement and added the observations from T106A and T214A double mutants’ whole cell recordings to this section. We find that the double mutants display greater current density compared to single mutants and wild-type TWIK-2.\n- Is the TWIK2 Y111H mutant pH sensitive like the TWIK1 channel? This seems like an obvious experiment to try and would be informative to understanding differences between TWIK1 and TWIK2 functions.\nAs already discovered by a few groups, H122 in TWIK-1 and H98 in TASK-3 although regarded as major proton-sensing amino acids in the SF1-M2 loop, are not the only determinants of pH sensitivity in K2P channels4,19–22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation, we did not anticipate that it would single-handedly change the pH sensitivity of TWIK-2. In the revised version of the manuscript, we show that the pH dependency of channel activation and activity of Y111H TWIK-2 is the same as that of wild-type like TWIK-2. Furthermore, we observe that in our assay setup, TWIK-1 showed pH-dependent current changes, albeit mild. These observations have been included in the revised version of the manuscript in a separate figure, and the corresponding text has been modified accordingly.\n- More methodological detail of the automated patch clamp technique would be helpful to understand the experimental system. Are drugs applied to the intracellular face of the channel (via syncropatch pipette) or extracellular via bath?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the automated patch clamp system. To address this, we have expanded the Methods section to include more detailed information about the experimental setup. Unless otherwise noted, all compounds were applied extracellularly using an external addition protocol, following the standard procedures for the SyncroPatch system. The SyncroPatch features a high-speed, multi-channel pipetting robot that manages all liquid additions and removals during the experiments. This robot can simultaneously add various external compounds—such as drugs, ligands, and chemicals—to each of the 384 wells, enabling a large number of experiments to be conducted simultaneously. Additionally, the robot performs quick and efficient solution exchanges in the wells. Although the system permits compound delivery to the intracellular face via an intracellular exchange solution, this study only examined the external application of pharmacological agents to aid in easier application, better ability to perform cumulative concentration-response curves, and for clear pre- and post-drug comparisons. This clarification has been added to the ‘Automated patch clamp recordings’ section within the Materials and Methods, to improve reproducibility and transparency in our data interpretation.\nReviewer #2 (Remarks to the Author):\n“The manuscript of Ma et al, provides new insights into the modulation of the K2P channel TWIK-2 using structural and electrophysiological data. The study provides the first cryo-EM structure of the full-length TWIK-2 at 3.7Å resolution, which is accompanied by mutagenesis studies and the use of a whole-cell patch clamp electrophysiology workflow. The authors show that TWIK-2 is pH-insensitive but slightly voltage-gated and suggest potential different binding modes or mechanisms of inhibition in TWIK-2 for two of the four tested K2P modulators (ML365 and NPBA).\nThe work is well executed and well written. However, overall the study fall a bit short. The new insights provided do not represent a big step forward in improving our understanding of how TWIK-2 (and K2P channels in general) function and are regulated. The substance and depth of the study could be/have been strengthened by more structure-guided mutagenesis studies, a more robust comparison of their new automated electrophysiology setup, a better literature comparison with (mutagenesis studies of) TWIK-1 and other K2P channels, or even more structural data or structural analysis. While I understand that addressing them all might be out of the scope of this study, the manuscript requires a bit more of depth. Specific suggestion would be (major comments):”\nWe sincerely thank the reviewer for the constructive suggestions to increase the depth of our work. We have revised the manuscript accordingly and addressed the changes point-by-point in our response below.\n1. In total ‘only’ four residues are identified as different/interesting and tested. Namely M135A, T106A, T214A and Y111H. What about other positions, e.g., around the cavity or along the ion conductance pathway? Is everything else conserved? What about testing mutating the same sites with other substitutes or using double and triple mutations to gain more insights? Compare in more depth with TWIK-1 and other K2P channels.\nWe agree with the reviewer that the mutant selection was sparse. To address this, we have included 4 more single mutants in the revised version of the manuscript, focusing on the regions around the SF (S213A and Y111A), the K2P modulator pocket (F98A), and hydrophobic cuff (M135N). Additionally, we also tested the effect of the T106A/T214A double mutant on TWIK-2 channel activity. We have also included an extensive comparison of TWIK-2 and TWIK-1, especially the channel activity, inhibition, and differences in the structure. Lastly, we have reformatted the existing comparison with other K2P channels to make it more apparent. Please see the remaining rebuttal, the results, and the discussion sections for more details.\n2. Most striking in this endeavor is the mutant Y111H. The authors show that in contrast to TWIK-1, TWIK-2 is pH-insensitive, with the histidine in that position being the pH-sensing residue in TWIK-1. While the authors go on and attempt to describe some of the differences observed for TWIK-2-Y111H, they never actually tested if this mutant has become pH-sensitive. Considering the overall conservation between TWIK-1 and TWIK-2 this would be expected and highly interesting to test. Further, to test the importance of the tyrosine and decouple it from a pH sensor position, a mutation to another residue, e.g. Y111A and Y111L would be interesting. Finally, the fact that TWIK-2_Y111H mutant carries the pH sensor of TWIK-1 must be mentioned in all clarity also in the results and discussion section and taking into account during interpretation.\nWe thank the reviewer for highlighting the importance of Y111H. H122 in TWIK-1, although regarded as a major proton-sensing amino acid in the SF1-M2 loop, is not the only determinant of pH4,20,22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation, we did not anticipate that it would single-handedly change the pH sensitivity of TWIK-2. In the revised version of the manuscript, as per the reviewer’s suggestion, we also compare the pH-dependency of TWIK-1 in our assay setup and test the effect of the TWIK-1-like Y111H mutant on the pH-dependence of TWIK-2 channel activity. We find that TWIK-1 is pH-dependent while TWIK-2 is not, despite the introduction of TWIK-1 like Y111H mutation. As per the reviewer’s suggestion, we also tested the effect of changing the bulky Y111 to a smaller aliphatic amino acid as alanine, on TWIK-2 channel activity. We found that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition. Perhaps, to speculate, the role of Y111 in TWIK-2 is maintaining the SF1-M2 loop integrity to facilitate allosteric coupling between the bottom and top of the SF.\n3. I appreciate and highly welcome the high-throughput patch clamp workflow presented by the authors. However, considering i) the discrepancies mentioned by the authors on electrophysiology studies for TWIK-2 in the literature, and ii) the mentioned inconsistencies between some of their electrophysiology data with that published before, the authors should do a more thorough comparison. If possible, between methods conducted by themselves with the same non-glycosylated constructs (conventional patch clamp and high-throughput patch clamp). But perhaps more importantly, use TWIK-1 in their automated whole-cell patch clamp electrophysiology setup, for which more robust recordings have been reported. This would allow us to assess and compare TWIK-1 and TWIK-2 and the different workflows better.\nWe thank the reviewer for these constructive suggestions. While we agree that not all experiments performed using conventional patch clamp can be adopted to a high-throughput automated patch clamp approach, screening of inhibitors and testing the relative change in channel activity upon mutagenesis has been reliably demonstrated by several groups. As there are already reports that compare high-throughput APC with conventional patch clamp techniques23–25, we prioritized to compare between TWIK-1 and TWIK-2. Using the same expression construct background and Syncropatch assay set up, we found that the overall TWIK-1 currents are comparable to TWIK-2. However, we notice that TWIK-1, unlike TWIK-2, shows pH dependency, albeit only mild. We also find that TWIK-1 is faster activating than TWIK-2 and is more susceptible to inhibition by ML365 and NPBA compared to TWIK-2. In addition to the comparison of electrophysiological properties of TWIK-2 and TWIK-1, in the revised version of the manuscript, we also include a detailed structure comparison at the end of the results section.\n4. The slightly different impact found for two of the tested inhibitors makes the discussion a bit ambiguous as to their potential different binding mode. I wonder if an attempt to solve an inhibitor-bound structure would be in the scope of this study as the authors seem to be able to obtain enough stable protein and the processing appears rather straightforward.\nWe agree with the reviewer that the structures of TWIK-2 with ML365 and NPBA could provide more information about their mechanism of binding. We collected and analyzed cryo-EM datasets of TWIK-2 with ML365 or NPBA. We observed that in the presence of these ligands, the sample appeared to be more heterogeneous, resulting in maps of lower quality compared to the apo TWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound TWIK-2 maps are fairly similar, with one common feature being the loss of strong tubular lipid-like densities that we observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is possible that loss of tubular lipid-like densities is a factor of poor resolution of the map. To make reasonable comparisons, we filtered the apo-TWIK-2 map to ~4Å (Response Fig. 1 above in page 2 of this document).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe some non-protein density immediately below the SF in the case of the NPBA dataset. The non-protein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor resolution of the map, and the size and shape of the density, preclude us from modeling NPBA at this site. Hence, we have not included these efforts in the manuscript and are presenting them here in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound structures were not fruitful, we employed site-directed mutagenesis to identify a potential NPBA binding site by performing more mutations in the SF, K2P modulator pocket, and around the hydrophobic cuff. We have discussed these in the main manuscript, and our findings suggest that the potential binding site for NPBA, as hinted by additional density in the map, could be below the SF. The mutagenesis exploration still does not identify the site for ML365 unambiguously. However, loss of strong tubular lipid-like densities around the hydrophobic cuff compared to apo TWIK-2 makes us suspect that ML365, like NPBA could bind through the cavity, albeit interacting with different residues compared to NPBA.\nOther major comments:\n5. Can the authors comment on the following: With regards to the processing of the cryo-EM data, I wonder if the authors also tested refining the other 3 classes obtained before proceeding with a final NU-refinement of one of the classes. Considering the authors interpret their structure as an intermediate conductive state, I wonder if there were some hints at other conformational states captured in the data.\nWe tried refining the other 3 classes shown in the penultimate step of data processing pipeline, but the resultant refined maps from these classes were of poor quality and offered no additional new information. Hence, we went ahead with the map denoted as “class 0” for final refinement and model building (Response Fig. 4)\nResponse Figure 4: NU-refinements of all the classes post 3D classification.\n6. Could the authors comment why they mutated Y111H to mimic the corresponding TWIK-1 residue, but then mutated M135A instead of M135L (which would be the corresponding TWIK-1 residue)? What was the rationale/strategy here? And here again, it would have been ideal to have TWIK-1 as a control experiment, and to compare Y111H with.\nMethionine, leucine, and alanine are all hydrophobic nonpolar amino acids. Between M135L and M135A, the alanine substitution of methionine reduces the size of the side chain drastically affecting not only the size of the pocket near M135 but also affecting the relative hydrophobic surface area. As we saw lipid-like densities around M135 amino acid, suspecting their role in regulation of channel activity, we anticipated M135A mutation to have drastic effect on TWIK-2 activity compared to M135L. In the revised version of the manuscript, we also tested M135N to explore the role of introduction of polar amino acid in this region on TWIK-2 activity. We notice that both M135A and M135N mutants exhibit slightly slower channel activation and show no drastic effect on the channel activity. However, M135A shows a greater loss of NPBA inhibition compared to M135N suggesting that removing the bulky amino acid at this position affects the efficacy of NPBA. Similarly, we also tested the effect of changing the pocket around Y111 on TWIK-2 channel activity using a Y111A mutant. We found that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type, like TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition. In the revised version of the manuscript, as per the reviewer’s suggestion, we also compare pH-dependency of TWIK-1 in our assay set up and test the effect of TWIK-1 like Y111H mutant on the pH-dependence of TWIK-2 channel activity. We find that TWIK-1 is pH-dependent while TWIK-2 is not, despite the introduction of TWIK-1 like Y111H mutation.\n7. Could the authors comment/expand on the voltage-dependence of TWIK-2 in the, and whether other K2P channels have also been found to be so.\nIn a 2016 study Schewe et al. provided significant insights into the voltage-dependent gating mechanisms of K2P channel, challenging the traditional view that voltage-sensing channel requires a canonical voltage-sensing domain. Instead, they proposed the SF to have a “check valve” gating behavior. They also note that this type of voltage-dependency has been observed in most K2Ps except TWIK113. Apart from the residues at the SF, voltage-dependent behavior in K2Ps, especially the TREK family, is also regulated by other factors such as mechanical force, ligands, and lipids 14–18. These reports suggest that the same K2P channel can switch between voltage-dependent and voltage-independent current responses depending on their conditions, perhaps a regulatory mechanism of certain K2P channels. Because we see that the central cavity is occupied by lipid-like densities in our cryo-EM map, and because TWIK-2 has an ability to translocate between endosomes and plasma membrane and has been proposed to interact with other proteins via its C-terminus, we believe it is possible that voltage-dependency in TWIK-2 could be a context dependent phenomenon.\n8. While I appreciate the modeling of potential lipids and co for the interpretation and figure rendering of non-protein densities, considering the high ambiguity, I expect that the authors have no intention of including it in the to be deposited model. (in the era of AlphaFold and machine learning, everything that is deposited can and will be use by AI or others)\nCompared to other K2Ps, the extra non-protein densities of TWIK-2 are a unique feature and may be endogenous molecules involved in channel regulation. As noted in the manuscript, we have not verified the exact nature of these lipid-like molecules, and hence we have modeled hydrocarbon tails in these densities. While this manuscript was under revision, two other groups reported structures of TWIK-2, and both groups observed tubular “non-protein” densities, especially in the fenestration site and in the central cavity 7,8. The former modeled acyl chains in the fenestration sites, while in the latter manuscript, the authors modeled lipids in these densities and proposed a mechanism of lipid-mediated regulation of TWIK-2 channel activity. Encouraged by multiple groups, independently, discovering lipid-like tubular densities in TWIK-2, we have decided to deposit the model with hydrocarbon chains modeled into these densities.\nMinor comments:\n- Page 4: \"we identified key amino acids that differentiate TWIK-2 from TWIK-1 and other members of the K2P family\". From what I understood, only Y111 was a direct comparison with TWIK-1, so I suggest toning down this sentence.\nWe thank the reviewer for the correction. We have rephrased it to “We identified key amino acids that regulate TWIK2 channel activity”, as we have included functional analysis of more mutations in the revised version of the manuscript.\n- Could the authors add details of this inhibition experiment in the Methods section (e.g. how was the small molecule screening set-up, quantity/volume of compound added, (pre-) incubation time, etc.). Could the authors show a blank/control measurement with just DMSO to account for the buffer?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the automated patch clamp system and the experimental setup (see above comments to reviewer 1). The SyncroPatch uses 384-well chips with micro-apertures, where negative pressure captures individual cells and forms high-resistance seals. A robotic pipetting unit handles liquids automatically, enabling parallel compound addition, quick solution changes, and precise concentration control, which ensures reliable dose–response measurements. We have updated the Methods section to include more details about the inhibition experiments. Importantly, we clarify that the reference (control) buffer always contained the same DMSO concentration as the compound solutions, accounting for any potential nonspecific effects of the solvent. As a result, the vehicle condition was consistently included in all experiments, ensuring that the observed effects are due to the test compounds.\n- Methods section: Cloning and overexpression of recombinant TWIK-2: The text should say for better clarity: \"with an N-terminal Strep-tag-II, followed by a GFP tag and a HRV-3C protease site\" instead of \"followed by an HRV-3C protease site and a GFP tag\" as it is then later stated that 3C cleavage results in removal of GFP tag.\nWe thank the reviewer for pointing this out. We have corrected it.\n- As the authors mention in the main text the poor heterologous expression of TWIK channels, could they indicate the yield they obtained from their experiments?\nAs mentioned in the introduction, poor heterologous expression of TWIK channels has been reported by a few groups before us 9,12,26. We were able to obtain ~0.4 mg of protein from 1L of HEK293 GnTI- cells, after the affinity purification step.\n- Correct typo in: \"Mutations T106A and T214A\"\nWe have rephrased that sentence in the discussion.\nFigures:\n- Please update figure numbers so that they are cited in correct order as mentioned in the text. E.g S2 should be called S1; swap order of Figure 4D and 4E\nWe have made sure that the figures are in the correct order.\n- Figure S2F: could the authors explain the uV units on the y-axis of the FSEC curve\n- Specify in main text and S2 figure legend which detergent was used for solubilization of ΔC-TWIK-2\n- For better clarity, keep one naming for each construct between figure and figure legends (e.g. in figure S2: TWIK-2 is sometimes called WT or wild-type; or ΔC-TWIK-2 (C-ter 1-276) is called c-ter 1-276, c-ter and del1-276). In general, could the authors fix all the inconsistencies in the figure legend of S2: missing periods, having a mixture of (n=15) and \",n=15\" etc.\nAs per the other reviewer’s suggestion, we removed the ΔC-TWIK-2 data from Supplementary Fig. 2 and the legend associated with it in the revised version of the manuscript. We have fixed all other inconsistencies.\n- Figure 2A: could the authors improve the illustration so that the ion conduction pathway is clearer to the reader?\nWe have remade this figure in current version of our manuscript.\n- Figure S1: indicate the non-conserved M135 and Y111 residues on the sequence alignment\nAll the sites that have been mutated in this study, including M135 and Y111, have now been highlighted in the sequence alignment figure in the current version of our manuscript.\n- Figure S3: FSC curves indicates the mask during refinement is too tight and could be optimized\nWe thank the reviewer for this suggestion. However, the unique architecture of K2P channels allows detergent micelles to occupy grooves formed between various secondary structure elements in the transmembrane region. Moreover, the detergent that we used for purification is DMNG. Neopentyl glycol class of detergents has low CMCs and are known to form heterogeneous micelles with varying sizes depending on the effective concentration of the detergent 27. To avoid heterogeneity in micelle size and stochastic detergent densities from interfering with refinement, we had to use a relatively tight mask to reconstruct a map with interpretable features at the protein-micelle interface. We have tried to dilate the mask more, and the resulting structure could not reach as high a resolution as the current map. A relatively tight mask is needed to effectively remove the micelle density that is tightly bound to our protein.\n- Could the authors mention/indicate the possible glycosylation sites that TWIK-2 possesses? This could be added to S1.\nWe have now highlighted the glycosylation sites N79 and N85 on the sequence alignment (current Figure S2).\nReferences\n1. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Nature 414, 43–48 (2001).\n2. Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type inactivation in K(+) channels. Nature 466, 203–208 (2010).\n3. Pau, V., Zhou, Y., Ramu, Y., Xu, Y. & Lu, Z. Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel. Nat. Struct. Mol. Biol. 24, 857–865 (2017).\n4. Turney, T. S., Li, V. & Brohawn, S. G. Structural Basis for pH-gating of the K+ channel TWIK1 at the selectivity filter. Nat. Commun. 13, 3232 (2022).\n5. Thomson, A. S. et al. Initial steps of inactivation at the K+ channel selectivity filter. Proc. Natl. Acad. Sci. U. S. A. 111, E1713-22 (2014).\n6. Rohaim, A. et al. A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant E71V. Nat. Commun. 13, 1574 (2022).\n7. Mondal, A., Niranjan, S. & Minor, D. L., Jr. A lipid plug affects K2P6.1(TWIK-2) function. bioRxivorg (2025) doi:10.1101/2025.06.11.659167.\n8. Khanra, N. K., Wang, C., Delgado, B. D. & Long, S. B. Structure of the human TWIK-2 potassium channel and its inhibition by pimozide. Proc. Natl. Acad. Sci. U. S. A. 122, e2425709122 (2025).\n9. Lloyd, E. E., Marrelli, S. P., Namiranian, K. & Bryan, R. M., Jr. Characterization of TWIK-2, a two-pore domain K+ channel, cloned from the rat middle cerebral artery. Exp. Biol. Med. (Maywood) 234, 1493–1502 (2009).\n10. Patel, A. J. et al. TWIK-2, an inactivating 2P domain K+ channel. J. Biol. Chem. 275, 28722–28730 (2000).\n11. Chavez, R. A. et al. TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family. J. Biol. Chem. 274, 7887–7892 (1999).\n12. Bobak, N. et al. Recombinant tandem of pore-domains in a Weakly Inward rectifying K+ channel 2 (TWIK2) forms active lysosomal channels. Sci. Rep. 7, 649 (2017).\n13. Schewe, M. et al. A non-canonical voltage-sensing mechanism controls gating in K2P K(+) channels. Cell 164, 937–949 (2016).\n14. Brennecke, J. T. & de Groot, B. L. Mechanism of mechanosensitive gating of the TREK-2 potassium channel. Biophys. J. 114, 1336–1343 (2018).\n15. Harrigan, M. P., McKiernan, K. A., Shanmugasundaram, V., Denny, R. A. & Pande, V. S. Markov modeling reveals novel intracellular modulation of the human TREK-2 selectivity filter. Sci. Rep. 7, 632 (2017).\n16. Yang, F. A gating mechanism of K2P channels by their selectivity filter. J. Mol. Cell Biol. 14, (2022).\n17. Rinné, S. et al. The molecular basis for an allosteric inhibition of K+-flux gating in K2P channels. Elife 8, e39476 (2019).\n18. Conrad, L. J., Proks, P. & Tucker, S. J. Effects of ionic strength on gating and permeation of TREK-2 K2P channels. PLoS One 16, e0258275 (2021).\n19. Morton, M. J., O’Connell, A. D., Sivaprasadarao, A. & Hunter, M. Determinants of pH sensing in the two-pore domain K(+) channels TASK-1 and -2. Pflugers Arch. 445, 577–583 (2003).\n20. Niemeyer, M. I., Cid, L. P., Peña-Münzenmayer, G. & Sepúlveda, F. V. Separate gating mechanisms mediate the regulation of K2P potassium channel TASK-2 by intra- and extracellular pH. J. Biol. Chem. 285, 16467–16475 (2010).\n21. Hall, P. R. et al. Structures of TASK-1 and TASK-3 K2P channels provide insight into their gating and dysfunction in disease. Structure 33, 115-122.e4 (2025).\n22. Lesage, F. et al. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. EMBO J. 15, 1004–1011 (1996).\n23. Li, T. et al. High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE. PLoS One 12, e0180154 (2017).\n24. Seibertz, F. et al. A modern automated patch-clamp approach for high throughput electrophysiology recordings in native cardiomyocytes. Commun. Biol. 5, 969 (2022).\n25. Obergrussberger, A. et al. The suitability of high throughput automated patch clamp for physiological applications. J. Physiol. 600, 277–297 (2022).\n26. Pountney, D. J. et al. Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K+ channel principal subunit. FEBS Lett. 450, 191–196 (1999).\n27. Lee, H. J., Lee, H. S., Youn, T., Byrne, B. & Chae, P. S. Impact of novel detergents on membrane protein studies. Chem 8, 980–1013 (2022)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have now addressed all of my remaining concern and I recommend for publication", "reviewer_2": "We thank and congratulate the authors for the thorough revision and the inclusion of a significant bulk of new data. The work as gained in substance, providing a solid contribution to the field and merits publication.\nMinor comments to help the authors in the final revision of the manuscript (If taken care of, no need to be checked again by me).\n1. The authors have now included the mutant Y111A alongside the originally used Y111H, which shows some differences in the activation and inhibition profile. However, the differences are not always clearly stated. One example can be found on page 12: \"Moreover, we notice a reduction in maximum inhibition for NPBA in M135A, S213A, and the T106A-T214A double mutants of TWIK-2 (Fig. 4 and Supplementary Fig. 5C).\" However, according to the figures, Y111A/H should also be added in the sentence. In particular Fig S5 shows that Y111A goes from a significant 80% NPBA-inhibition to 40%. When Y111A is added to this statement the subsequent sentences will require revision too: \"These mutants outline a pocket underneath the SF in the central vestibule (Fig. 3A). Based on these observations, we hypothesize that NPBA diffuses through the fenestration site and binds underneath the SF in the central vestibule.”\n2. Adjust statement on top of page 5: \"As we used HEK293 GnTI- cells, which lack N-acetylglucosaminyltransferase I activity, for the overexpression of TWIK-2, glycosylation at these sites is prevented. Glycosylation at these sites may be necessary for stabilization of this loop region\". --> This is not entirely true. GntI- cells limit N-linked glycosylation of expressed glycoproteins to a pattern dominated by the Man GlcNAc N-glycan, and therefore result in a more \"homogenous\" glycosylation of the protein. Hence, better to rephrase that a more complex glycosylation than the shorter and more homogenous produced by GnTI- cells might be the cause.\n3. End of page 7, top of page 8: \"We compared the IC50 values of wild-type TWIK-2 against four mutants: one each at the entry (Y111) and exit (M135) of the ion permeation pore, as well as two at the bottom of selectivity filters SF1 (T106A) and SF2 (T214A).\" --> Please indicate to which residues Y111 and M135 have been mutated into. It is indicated later in the text, so I suspect this is just an oversight.\n4. Correct sentence on Page 8: \"For ML365, the maximum inhibition remained similar to that of wild-type TWIK-2 in all variants, except for the T106A-T214A double mutant, which showed. In the case of ML365, we noticed that the maximum inhibition for all the variants remained similar to wild-type TWIK-2, except the T106A-T214A double mutant, where we saw a significant reduction\"\n5. Page 13: The original conclusion paragraph is missing. Ignore if this was intentional.\n6. Methods section, data processing: Please indicate whether the last refinement was still done with C1symmetry.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to reviewer comments\nWe thank all the reviewers for their critical assessment of our manuscript. Our point-by-point\nresponse is below. For purposes of clarity, we have reviewer comments in red italics.\nReviewer #1 (Remarks to the Author):\n“This study determines a TWIK2 cryoEM structure and uses a commercial automated patch clamp\nsetup to assess the function of TWIK2 channel mutants and pharmacology. The structure of\nTWIK2 had not been previously determined and this study provides a first look at the TWIK2\nchannel architecture. To accompany the structure, the authors perform electrophysiological\nrecordings of TWIK2, a channel that has been notoriously difficult (though not impossible) to\nrecord from. Overall, the methodology in the structural and functional work appears to be sound\nand the writing is clear. “\nWe sincerely thank the reviewer for the overall positive feedback.\n“There are novel observations and interesting results in the manuscript but an informative\nconnection between knowledge learned from the TWIK2 structure and the functional studies\nperformed is largely lacking. The Y111H designed to mimic the TWIK1 pH sensing histidine and\nthe S4 site threonine residues are all positions that did not require a cryoEM structure to be\ninteresting candidates for mutation. While the M135A mutation is a candidate site based on the\nTWIK2 structure, it is unclear why the authors chose this position as it does not occlude the pore\nor play any clearly observable role in the TWIK2 structure. The experiments examining the effects\nof ML365 and NPBA are interesting but somewhat superficial, as the mutants show some effects\non channel pharmacology but do not yield any substantive insight into where the drugs might bind\nor how they might act.”\nWe thank the reviewer for pointing out weaknesses in this manuscript that could be improved. We\nagree with the reviewer that the mutant selection was sparse. To address this, the analysis in the\nrevised version of the manuscript includes the following single and double mutants: F98A, Y111H,\nY111A, T106A, T214A, S213A, M135N, M135A, and T106A-T214A, focusing on the regions near\nthe SF, the K2P modulator pocket (F98A), and the hydrophobic cuff (M135A and M135N). The\nalanine substitution of M135 (M135A) drastically reduces the size of the side chain, affecting not\nonly the size of the pocket near M135 but also the relative hydrophobic surface area. As we\nobserved lipid-like densities around the M135 amino acid, suggesting their role in regulating\nchannel activity, we anticipated that the M135A mutation would have a drastic effect on TWIK-2\nactivity. In the revised manuscript, we also tested M135N to explore the role of introducing a polar\namino acid in this region on TWIK-2 activity. We observe that both M135A and M135N mutants\nexhibit slightly slower channel activation, with no drastic effect on channel activity. However,\nM135A shows a greater loss of NPBA inhibition compared to M135N, suggesting that removing\nthe bulky amino acid at this position affects the efficacy of NPBA. Similarly, we also tested the\neffect of changing the pocket around Y111 on TWIK-2 channel activity using a Y111A mutant. We\nfound that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type\nTWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition, highlighting the role\nof Y111 in maintaining SF1-M2 loop integrity.\nTo study the binding modes of ML365 and NPBA, we collected and analyzed cryo-EM data using\nTWIK-2 complexed with ML365 and NPBA. We observed that in the presence of these ligands,\nthe sample appeared to be more heterogeneous, resulting in maps of lower quality compared to\nthe apo TWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound\nTWIK-2 maps are fairly similar (~ 4 Å), with one common feature being the loss of strong tubular\nlipid-like densities that we observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is\npossible that loss of tubular lipid-like densities is a factor of poor resolution of the map. To make\nreasonable comparisons, we filtered the apo-TWIK-2 map to 4Å (Response Fig. 1).\nResponse Figure 1: cryo-EM map comparison of TWIK-2 Apo, TWIK-2 ML365 and TWIK-2 NPBA.\nTwo bottom views on the right indicate the occupancy of the cavity close to the selectivity filter (top)\nand cytoplasmic site (bottom).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe\nsome non-protein density immediately below the SF in the case of the NPBA dataset. The non-\nprotein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor\nresolution of the map, and the size and shape of the density, preclude us from unambiguously\nmodeling NPBA at this site. Hence, we have not included these efforts in the manuscript and are\npresenting them here in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound\nstructures were not successful, we employed site-directed mutagenesis to identify a potential\nNPBA binding site by performing more mutations in the SF, K2P modulator pocket, and around\nthe hydrophobic cuff. We have discussed these in the main manuscript and our findings suggest\nthat the potential binding site for NPBA, as hinted by additional density in the map, could be below\nthe SF. The mutagenesis exploration still does not identify the site for ML365 unambiguously.\nHowever, loss of strong tubular lipid-like densities around the hydrophobic cuff compared to apo\nTWIK-2 makes us suspect that ML365, like NPBA could bind through the cavity, albeit interacting\nwith different residues compared to NPBA.\n“1) In the section entitled “Ion conduction pore of TWIK2”, the authors suggest that the carbonyl\noxygens of selectivity filter residues V107 and Y109 face away from the central permeation axis\nin TWIK-2 and argue for the relevance of this SF orientation to TWIK2 function. Given the ~3.7\nangstrom resolution of the overall structure, I cannot ascertain how the authors come to this\nconclusion. Using the map and model provided by the authors, I can easily model the selectivity\nfilter within the map in a conventional orientation and find that I improve the number of residues\nin favored orientations when the SF is modeled more conventionally.\nIf there is a biophysical argument to be made that the positioning of the valine and tyrosine\nsidechains in the TWIK2 map somehow promote an unconventional SF backbone configuration,\nthis should be provided. Simply based on the map provided by the authors, I find this section to\nbe unsupported by experimental results. If the authors do feel their selectivity figure configuration\nis accurate, a supplemental figure showing how the experimental map is better fit by the outward\nrather than inward-facing carbonyls at V107 and Y109 would be extremely helpful to make the\nauthors' case on this point.”\nWe thank the reviewer for a thorough review of our structure. In our TWIK-2 structure, the SF has\nbeen modeled with carbonyl oxygens of three amino acids – V107, Y109, and L217 – facing away\nfrom the ion-conductance pore. While we agree that at ~ 3.7 Å resolution it could be challenging\nto resolve the orientation of carbonyl oxygens in the dynamic loops of the SF region\nunambiguously, we would like to highlight that the local resolution for this region is much better\nthan 3.5 Å (Supplementary Fig. S3F in the manuscript). As per the reviewer’s critique, we\nattempted to model these three carbonyls in various combinations of them facing away or towards\nthe SF. However, just based on visually comparing the fits of these various models to density, it\nis not straight forward to identity one particular combination of the carbonyl oxygens that fits better\nto the map (see figures & table below). Hence, based on a comparison of the model statistics\nsuch as bond RMSDs, Ramachandran outliers, Rama-Z score, & CaBLAM outliers for the SF\nregion, and the overall model quality statistics for the whole protein, we believe that the model as\nit has been presented in the manuscript is the best possible fit (Response Table 1 in this rebuttal).\nIn K2P channels, the two conserved threonines at the bottom of the selectivity filter (SF) are the\nonly amino acids within the SF that coordinate K+ ions through their side chains. All other amino\nacids at the SF coordinate K+ ions through carbonyl oxygens. It has been shown by multiple\ngroups previously that reorientation of the carbonyl oxygens away from the SF is a means to\nregulate K+ ion flow, including channel inhibition, in potassium channels. Using X-ray\ncrystallography and cryo-EM, several groups have shown that reorientation of carbonyl oxygens\naway from the SF pore affects the K+ ion coordination at the SF as part of C-type or pH-mediated\ninactivation of the channel in KcsA, Kv, and TWIK-11–4. The flipped carbonyls often co-existed\nwith reduced occupancy at the canonical K+ ion sites (S0-S4) in the SF. As noted in the\nmanuscript, our cryo-EM map allowed us to confidently model K+ ions in only three out of these\nfive sites. Similarly, MD simulations on the prototypical K+ channels like MthK and KcsA have\nshown that reorientation of the carbonyl oxygen of the conserved valine at the SF is one of the\ninitial steps that promotes dissociation of K+ from the SF and allows the SF to adopt nonconducting\nor inactivated states5,6. As pointed out by the reviewer, we had provided only a brief biophysical\nexplanation for non-canonical carbonyl positioning in the discussion in the original manuscript. To\naddress that, we have expanded on the explanation associated with flipped carbonyl observation,\nby including examples of other eukaryotic K+ channels (TWIK-1, Kv, and Kir) and their prototypic\nprokaryotic homologs (MthK and KcsA) where similar observations have been made, as a\nseparate section in the discussion, & added more references to this section.\nResponse Figure 2: Various TWIK2 selectivity filter models fit to the cryo-EM density. The model\nquality statistics for the variations 1-7, and a comparison of the statistics are provided in table 1. Cryo-\nEM density is highlighted as blue mesh and the three K+ ions as blue spheres. The two protomers,\nTWIK-2 chain A and B, are colored light magenta and light cyan. All the amino acids at the SF are\nlabeled in variation 7 where none of the carbonyls are flipped away from the SF, and in the remaining\npanels only the carbonyls that are flipped away from the SF have been labeled.\nResponse Table1: TWIK2 selectivity filter model quality statistics. Selectivity filter model quality statistics were\nobtained by running PDB validation on the amino acids 104-111 and 213-220, and K+ ions at the SF using Phenix. The\nvalues in the parentheses represent the whole PDB. As the resolution of the structure is ~3.7 Å, CaBLAM outliers, Rama-\nZ score, Bond RMSDs for the SF region, and the overall statistics of the whole PDB were compared to decide on the\ncarbonyl orientations of the amino acids V107, Y109, and L217, which surround S0/S1 and S2/S3 sites at the SF. Each\nvariation (Var. 4-6) or combination of variations are show (Var. 1-3)\nDeposited\nParameter Var. 1 Var. 2 Var. 3 Var. 4 Var. 5 Var. 6 Var. 7\n9E94\nAmino acids with\nV107,\ncarbonyl Y109 & V107 & V107 &\nY109, & L217 Y109 V107 None\noxygens flipped L217 L217 Y109\nL217\naway from SF\nBond length 0.007 0.014 0.014 0.007 0.017 0.018 0.016 0.017\nRMSD (Å) (0.009) (0.010) (0.009) (0.009) (0.010) (0.010) (0.010) (0.010)\nBond angle 1.617 2.103 2.161 1.724 2.292 2.188 2.265 2.441\nRMSD (o) (1.564) (1.651) (1.624) (1.595) (1.672) (1.662) (1.643) (1.693)\n1.97 1.49 2.01 2.53 2.05 1.97 2.48 2.45\nMolProbity score\n(1.78) (1.75) (1.78) (1.89) (1.78) (1.82) (1.86) (1.88)\nRamachandran 0.00 0.00 8.33 8.33 0.00 0.00 0.00\n0.00 (0.00)\noutliers (%) (0.00) (0.00) (0.47) (0.47) (0.00) (0.00) (0.00)\nRamachandran 8.33 8.33 25.00 41.67 8.33 50.00 58.33 50.00\nallowed (%) (6.07) (6.54) (7.01) (7.94) (6.07) (8.88) (8.88) (8.88)\nRamachandran 91.67 91.67 75.00 50.00 83.33 50.00 41.67 50.00\nfavored (%) (93.93) (93.46) (92.99) (91.59) (93.46) (91.12) (91.12) (91.12)\n-5.32 -6.25 -7.54 -6.18 -7.92 -6.96 -8.91 -9.44\nRama-Z (whole)\n(-3.75) (-3.79) (-3.86) (-3.78) (-3.90) (-3.81) (-3.93) (-4.00)\nRama-Z (loop) -4.05 -4.76 -5.74 -4.70 -6.03 -5.30 -6.79 -7.19\n(-4.87) (-5.04) (-5.29) (-5.06) (-5.33) (-5.09) (-5.53) (-5.64)\nCaBLAM outliers 25.00 50.00 50.00 37.50 62.50 50.00 62.50 75.00\n(%) (2.88) (3.85) (4.33) (3.37) (4.33) (3.85) (4.81) (4.81)\nClash score 8.81 2.20 4.39 10.96 6.62 2.21 8.81 8.83\n(6.76) (5.91) (6.05) (6.34) (6.34) (5.62) (6.20) (6.63)\n“2) In Figure 2B, the pore radius profile shows a constriction of the pore at around 5A from the\ncytoplasmic face of the channel. This cytoplasmic constriction does not seem to be discussed in\nthe manuscript. Is this constriction brought about by the lipids modeled in the structure? If so, is\nthis at all relevant to the function of TWIK2? If the authors believe that these lipid densities are\nimportant to shaping the TWIK2 pore, then this pore radius plot is appropriate. If the relevance of\nthese lipids is unclear and could simply be an artifact of the protein purification process, it would\nbe better to omit the lipid densities when running HOLE (or include a second HOLE profile in the\nabsence of the lipids) to show the profile of just the TWIK2 protein.\nIt would also be appropriate to discuss the lipid densities present in the map in the results section\nof the manuscript, rather than the discussion. This would be a good spot to do so.”\nThe pore radius plot in Fig. 2B was constructed using the TWIK-2 model with hydrocarbon tails\nmodeled into the tubular lipid-like densities. We have now edited Fig. 2B to show the pore radius\nprofile with and without the modeled lipid-like densities. We did not include any exogenous lipids\nduring the purification. So, we believe these tubular densities arise from the molecules that co-\npurified with TWIK-2 from HEK293 GnTI- cells. Because we could not verify the exact nature of\nthese molecules, we have only modeled hydrocarbon tails in these densities and called them lipid-\nlike in the manuscript. The discussion on the lipid-like densities has now been moved to the results\nsection in the revised version of the manuscript, as per the reviewer’s suggestion. While this\nmanuscript was under revision, another group reported a preprint suggesting a potential lipid\nmediated regulation of TWIK-2 channel activity. They argued that lipid molecules “plug” the\nchannel on the cytoplasmic side by occupying the central cavity and by physically obstructing the\npassage of ions, rendering TWIK-2 inactive. They demonstrate that R257, an amino acid towards\nthe C-terminus that lies below the hydrophobic cuff, is crucial for this lipid binding in the central\ncavity and that the R257A mutation increases the channel activity, suggesting that channel\nactivation is a result of lipid removal or repositioning in the central cavity. In our TWIK-2 structure,\nwe observe lipid-like densities in similar positions (central cavity and along the groove formed by\nthe PH2, M2, and M4 helices) as reported by Mondal et al7. However, these lipid-like densities do\nnot entirely obstruct the access to the central pore (Fig. 2). The observation of lipid-like density,\ncorroborated by a different group independently, suggests that these molecules are relevant to\nthe function. We have added a few lines discussing this preprint and its relevance in the context\nof our structure to the discussion section of the revised manuscript.\n“3) For the experiments where G/GMAX–V curves are calculated, it does not appear that the\nrecordings reach a steady state at 200 ms. The current is still rising for TWIK1 wt and this is even\nmore pronounced in the mutants that slow activation (Figure 3B). Is it possible that the differences\nin V50 are artificial, since the maximal steady state current is reached at 200ms in some of the\nmutants but not in all of them.”\nWe appreciate the reviewer’s concerns regarding the possible underestimation of steady-state\ncurrents due to incomplete activation at the end of the 200-ms depolarizing pulse, especially in\nslow-activating TWIK-2 mutants. To reinforce the validity of our conclusions regarding mutant-\ninduced shifts in voltage dependence, we performed a new analysis based on normalized tail\ncurrents, which better reflect the relative conductance by capturing the instantaneous current\nupon repolarization—thus minimizing the effects of kinetic differences in channel activation.\nSpecifically, we constructed revised G/GMAX–V curves using tail currents, enabling a more\naccurate assessment of the voltage dependence of activation across all variants. From these\ncurves, we calculated the ΔV50 values, which provide a more reliable measure of the voltage shift\ncaused by each mutation. Importantly, this alternative approach produced similar V50 shifts as\npreviously reported, confirming that the observed differences are genuine and not artifacts,\nreflecting fundamental changes in channel gating properties. These new results are now included\nin the revised Supplementary Fig. 5 and discussed in the Results section. To enable a better\ncomparison of currents across TWIK-2 variants, we have also modified Fig. 3 to now display\nwhole-cell current traces and activation time constants for all the mutants that we tested.\n“4) I think the manuscript would be greatly improved by a more direct comparison of the TWIK2\nstructure with the previously published TWIK1 structures. Does this TWIK2 structure look more\nlike the low pH or neutral pH TWIK2 structure? Are there clear differences in the positioning of\nthe cap or TM helices? The entire section of the results devoted to an overview of the TWIK2\nchannel could be redone as a more direct comparison between TWIK1 and TWIK2 and would\nsignificantly increase interest.”\nWe agree with the reviewer that a comprehensive comparison of TWIK-2 with TWIK-1 could be a\nbeneficial addition to the manuscript. In our first version of the manuscript, we had presented a\ncomparison of TWIK-2 and TWIK-1 in Figs. 5B, C and Supplementary Fig. S4, focusing only on\nthe conformational changes in pore domains, SF, and M4 helix. As TWIK-2 is physiologically\nunlike TWIK-1, for the sake of completeness, we have also included comparisons with other pH-\nsensitive and lipid-regulated K2P channels. To make these comparisons more apparent, we have\nreformatted and expanded the figures and text to include a separate comparison section of the\nTWIK-2 and TWIK-1 structures in their entirety. In addition, we have also included the results from\nwhole-cell recordings studying the pH dependency of channel activity in TWIK-1 and the TWIK-\n1-like Y111H mutant of TWIK-2. Interestingly, we find that the differences in the structure of TWIK-\n2 are localized to the cap region when compared with the TWIK-1 structure at pH 7.5. When\ncompared with TWIK-1 at pH 5.5, the differences lie only in the SF region, specifically the SF1-\nM2 linker that houses the pH-sensing amino acid H122 in TWIK-1. TWIK-2 has the cap region\nthat is similar to inactive TWIK-1, but has the SF loops positioned in a similar way as that of the\nactive TWIK-1, despite the residue Y111 in TWIK-2 (H122 in TWIK-1) adopting a unique ‘up’\nconformation. These new observations have been discussed in the revised version of the\nmanuscript, alongside the TWIK-2 and TWIK-1 comparisons from the older version (Fig. 1, 5).\nThe comparison of TWIK-2 with other K2P channels has been reformatted into a separate figure\n(Supplementary Figs. 4, 7).\nMinor Points\n1. - In the introduction, “Kcnk6 knockout studies have shown suppression of NLRP3\ninflammasome activation in macrophages, implicating endolysosomal localization of\nTWIK-2 in inflammation 45,46.” This sentence is a bit misleading and should be clarified.\nFrom the references cited, it is movement of TWIK2 from the endolysosome to the plasma\nmembrane that appears to be a critical step in the potassium efflux that mediates NLRP3\ninflammasome activation.\nWe thank the reviewer for the correction. We have now replaced the sentence with “Translocation\nof TWIK-2 from endosome to plasma membrane is implicated in NLRP3-mediated inflammasome\nactivation in macrophages, with Kcnk6 knockouts showing a suppressed inflammasome\nactivation”.\n2. - In figure 1 (and for nearly all electrophysiological traces shown in the manuscript),\nrecording traces are shown on a signal scale that reads “Normalized I”. It is unclear what\nthe non-transfected cells traces are normalized to? For clarity throughout the manuscript,\nit would be helpful if the authors explicitly discuss whether normalization was applied using\na universal metric (which could be described in the methods section of the manuscript) or\nad hoc for each set of recordings (in which case this should be described in each figure\nlegends)\nWe thank the reviewer for pointing out this important issue related to the normalization of\nelectrophysiological traces. In the revised manuscript, all figures now display traces showing\nactual absolute current values instead of normalized signals. This update allows for direct\ncomparison of current amplitudes across wild-type and mutant channels as well as non-\ntransfected controls. Additionally, we now include current density for each experimental condition\nwhere appropriate, ensuring that differences in current amplitudes are not influenced by variations\nin cell size. We believe these revisions significantly improve the clarity and transparency of our\ndata presentation and address the reviewer’s concern about normalization.\n3- Given that the C-terminally truncated TWIK construct was not used for structure of detailed\nfunctional studies, this data could be omitted or moved to the methods section.\nWe agree with this reviewer’s observation that the data on C-terminally truncated TWIK-2 is not\ninformative, beyond suggesting that TWIK-2 devoid of C-terminus is unstable compared to WT.\nIn accordance, we have now removed the C-term truncation data of TWIK-2 from the revised\nmanuscript.\n4 - Is it meaningful that the loop region between V75-P89 is unresolved in the cryo-EM structure?\nIn all other K2P structures, this loop is well resolved. Does this mean that the V75-P89 is dynamic\nin TWIK2, suggestive of a functional role? Or is this just a limitation of the resolutions of this\nTWIK2 structure compared to other K2P structures? Does 3D variability analysis in cryosparc\ngive any indication of what is going on in this loop region?\nThe loop V75-P89 is a part of the CH2-PH1 region that harbors two predicted glycosylation sites,\nN79 and N85. We used HEK293 GnTI- cells, which lack N-acetylglucosaminyltransferase I\nactivity, for overexpression of TWIK-2. So, it is likely that glycosylation at these sites is essential\nfor stabilizing this loop, and without such glycosylation, this loop is flexible and hence poorly\nresolved in the cryo-EM density map. Interestingly, TWIK-2 structures reported by a different\ngroup recently also lacked this region 8. Despite using glycosylation-competent Expi293F cells\nand achieving an overall higher resolution structure (~2.9 Å). Perhaps, in TWIK-2 this loop is more\ndynamic than other K2Ps. Another group reported structures of TWIK-2 while this manuscript was\nunder revision 7. They too use glycosylation-capable Expi293F cells as Khanra et. al., but were\nable to resolve the V75-P89 region in their cryo-EM map. Moreover, they find that amino acids\n75-80 in this loop actually form a short helix called ‘ear helix’ that is unique to TWIK-2, proposed\nto stabilize the TWIK-2 cap domain. As this loop region can also potentially interact with the\nkeystone inhibitor site and the K2P modulator pocket, we attempted to resolve this region using\n3DVA and subsequent classification in cryoSPARC (Response Fig. 3). However, our attempts\nwere not successful.\n5- The panel in Figure 3 showing the relative locations of T106, T214, M135, and Y111 is critically\nimportant for understanding the orientation and relevance of these residues. I would suggest\nreworking this figure to make it clearer. The inset boxes are distracting, as each one has a different\norientation relative to the main panel. Perhaps a clearly labeled “top view” showing the Y111\nresidues, “bottom view\" showing the M135 residues, and a side view like shown in the left side of\nFigure 3A would be sufficient for this.\nWe have modified the figure as per the reviewer’s suggestions.\n- “Notably, TWIK-2 channels elicited outward potassium currents that increased with\ndepolarization (over +30 mV), suggesting voltage-dependent activation of TWIK-2” Isn’t this a\nsurprising result, given that prior recordings in symmetrical potassium have shown inward\nrectification rather than outward rectification? Is there any methodologic explanation for this\ndifference in observed functional behavior?\nWhile it may seem surprising at first, the voltage-dependent activation of TWIK-2 seen in our\nrecordings agrees with previous studies, especially those indicating that TWIK-2 is one of the\nleast inwardly rectifying members of the K2P family, that inactivates and has a potential role in\nelectrogenesis9–12. Compared to strongly rectifying K2P channels, such as TREK-1 or TASK-3,\nTWIK-2 exhibits more linear or even outwardly biased I–V relationships under certain recording\nconditions. Moreover, differences in rectification behavior—particularly outward currents at\ndepolarized potentials—can depend on the expression system (e.g., Xenopus oocytes versus\nmammalian cells), ionic composition, and intracellular environment (e.g., Mg²⁺, polyamines, or\npH). In our experiments, outward currents at voltages above +30 mV are probably caused by a\ncombination of weak inherent rectification and the electrochemical driving force for K⁺ efflux under\nsymmetrical or nearly symmetrical K⁺ conditions, rather than unusual voltage-dependent gating.\nTo clarify this, we added a section in the discussion explaining how our findings align with the\nknown electrophysiological properties of TWIK-2 and reflect system-dependent variability rather\nthan an unexpected gating mechanism.\nResponse Figure 3: 3D variability analysis (3DVA) and subsequent classification to resolve V75-P89\nloop. The region shaded in gray is a part of the supplementary figure 3 in the manuscript and has been\nincluded here only for reference. Particles from the best cluster displaying all the features of TWIK-2 from\n3DVA step was used to generate an ab initio initial reference (cyan box). Next, particles from all clusters\nwith robust cap domain features (red boxes) from the 3DVA step were pooled for clean up by heterogenous\nrefinement and ab initio jobs. The final map obtained after NU-refinement, using the ab-initio reference\ngenerated from the best 3DVA cluster, resulted in a map with features and resolution that resembled the\nmap already presented in the manuscript.\n- Reference 70 in the manuscript includes data that describe the effect of mutations at the S4 site\nthreonines in K2Ps. For nearly all K2Ps, these S4 site mutations cause a loss of voltage\ndependance. It is therefore interesting that in TWIK2, the T106A and T214A mutants maintain\ntheir voltage dependent gating. Is there any rationale for why this might be the case?\nThe reference 70 reports that in channels without a dedicated voltage-sensing domain, the SF\nitself acts as a gate and sensor. They demonstrate that mutating the conserved threonines at the\nSF to cysteines causes loss of voltage-dependent behavior. However, they also show that in\naddition to just these mutations, voltage-dependent behavior is also regulated by other factors\nsuch as mechanical force, ligands, and lipids13. Regulation of voltage-dependency of K2P\nchannels by these auxiliary factors, apart from the residues at SF, has been shown in several\nK2P channels, especially the TREK family14–18. These reports suggest that the same K2P channel\ncan switch between voltage-dependent and voltage-independent current responses depending\non their conditions, perhaps a regulatory mechanism of certain K2P channels. T106A and T214A\nmutants display a reduced-voltage dependency in our experiments, but not a complete loss of\nvoltage-dependency. Because we see that the central cavity is occupied by lipid-like densities in\nour cryo-EM map, and because TWIK-2 has an ability to translocate between endosomes and\nplasma membrane and has been proposed to interact with other proteins via its C-terminus, we\nbelieve it is possible that voltage-dependency in TWIK-2 could be a context dependent\nphenomenon, in addition to being regulated by the residues at the SF. However, identification of\nall the factors in TWIK-2 that regulate its channel activity needs furthermore substantial efforts.\n- “Mutations T106A and T214A disturb these networks of hydrogen bonding and hydrophobic\ninteractions that stabilize the base of the SF and make the channel less selective and more open\n(Figure 3B-3F).” The functional experiments presented in this section use only a single ionic\ncomposition and are not suited to adequately address ion selectivity. Please support with data or\nomit this claim.\nWe agree with the reviewer that with only a single ionic composition, it is hard to address the ion\nselectivity of TWIK-2. We have rephrased this statement and added the observations from T106A\nand T214A double mutants’ whole cell recordings to this section. We find that the double mutants\ndisplay greater current density compared to single mutants and wild-type TWIK-2.\n- Is the TWIK2 Y111H mutant pH sensitive like the TWIK1 channel? This seems like an obvious\nexperiment to try and would be informative to understanding differences between TWIK1 and\nTWIK2 functions.\nAs already discovered by a few groups, H122 in TWIK-1 and H98 in TASK-3 although regarded\nas major proton-sensing amino acids in the SF1-M2 loop, are not the only determinants of pH\nsensitivity in K2P channels4,19–22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation,\nwe did not anticipate that it would single-handedly change the pH sensitivity of TWIK-2. In the\nrevised version of the manuscript, we show that the pH dependency of channel activation and\nactivity of Y111H TWIK-2 is the same as that of wild-type like TWIK-2. Furthermore, we observe\nthat in our assay setup, TWIK-1 showed pH-dependent current changes, albeit mild. These\nobservations have been included in the revised version of the manuscript in a separate figure,\nand the corresponding text has been modified accordingly.\n- More methodological detail of the automated patch clamp technique would be helpful to\nunderstand the experimental system. Are drugs applied to the intracellular face of the channel\n(via syncropatch pipette) or extracellular via bath?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the\nautomated patch clamp system. To address this, we have expanded the Methods section to\ninclude more detailed information about the experimental setup. Unless otherwise noted, all\ncompounds were applied extracellularly using an external addition protocol, following the standard\nprocedures for the SyncroPatch system. The SyncroPatch features a high-speed, multi-channel\npipetting robot that manages all liquid additions and removals during the experiments. This robot\ncan simultaneously add various external compounds—such as drugs, ligands, and chemicals—\nto each of the 384 wells, enabling a large number of experiments to be conducted simultaneously.\nAdditionally, the robot performs quick and efficient solution exchanges in the wells. Although the\nsystem permits compound delivery to the intracellular face via an intracellular exchange solution,\nthis study only examined the external application of pharmacological agents to aid in easier\napplication, better ability to perform cumulative concentration-response curves, and for clear pre-\nand post-drug comparisons. This clarification has been added to the ‘Automated patch clamp\nrecordings’ section within the Materials and Methods, to improve reproducibility and transparency\nin our data interpretation.\nReviewer #2 (Remarks to the Author):\n“The manuscript of Ma et al, provides new insights into the modulation of the K2P channel TWIK-\n2 using structural and electrophysiological data. The study provides the first cryo-EM structure of\nthe full-length TWIK-2 at 3.7Å resolution, which is accompanied by mutagenesis studies and the\nuse of a whole-cell patch clamp electrophysiology workflow. The authors show that TWIK-2 is pH-\ninsensitive but slightly voltage-gated and suggest potential different binding modes or\nmechanisms of inhibition in TWIK-2 for two of the four tested K2P modulators (ML365 and NPBA).\nThe work is well executed and well written. However, overall the study fall a bit short. The new\ninsights provided do not represent a big step forward in improving our understanding of how\nTWIK-2 (and K2P channels in general) function and are regulated. The substance and depth of\nthe study could be/have been strengthened by more structure-guided mutagenesis studies, a\nmore robust comparison of their new automated electrophysiology setup, a better literature\ncomparison with (mutagenesis studies of) TWIK-1 and other K2P channels, or even more\nstructural data or structural analysis. While I understand that addressing them all might be out of\nthe scope of this study, the manuscript requires a bit more of depth. Specific suggestion would be\n(major comments):”\nWe sincerely thank the reviewer for the constructive suggestions to increase the depth of our\nwork. We have revised the manuscript accordingly and addressed the changes point-by-point in\nour response below.\n1. In total ‘only’ four residues are identified as different/interesting and tested. Namely M135A,\nT106A, T214A and Y111H. What about other positions, e.g., around the cavity or along the ion\nconductance pathway? Is everything else conserved? What about testing mutating the same sites\nwith other substitutes or using double and triple mutations to gain more insights? Compare in\nmore depth with TWIK-1 and other K2P channels.\nWe agree with the reviewer that the mutant selection was sparse. To address this, we have\nincluded 4 more single mutants in the revised version of the manuscript, focusing on the regions\naround the SF (S213A and Y111A), the K2P modulator pocket (F98A), and hydrophobic cuff\n(M135N). Additionally, we also tested the effect of the T106A/T214A double mutant on TWIK-2\nchannel activity. We have also included an extensive comparison of TWIK-2 and TWIK-1,\nespecially the channel activity, inhibition, and differences in the structure. Lastly, we have\nreformatted the existing comparison with other K2P channels to make it more apparent. Please\nsee the remaining rebuttal, the results, and the discussion sections for more details.\n2. Most striking in this endeavor is the mutant Y111H. The authors show that in contrast to TWIK-\n1, TWIK-2 is pH-insensitive, with the histidine in that position being the pH-sensing residue in\nTWIK-1. While the authors go on and attempt to describe some of the differences observed for\nTWIK-2-Y111H, they never actually tested if this mutant has become pH-sensitive. Considering\nthe overall conservation between TWIK-1 and TWIK-2 this would be expected and highly\ninteresting to test. Further, to test the importance of the tyrosine and decouple it from a pH sensor\nposition, a mutation to another residue, e.g. Y111A and Y111L would be interesting. Finally, the\nfact that TWIK-2_Y111H mutant carries the pH sensor of TWIK-1 must be mentioned in all clarity\nalso in the results and discussion section and taking into account during interpretation.\nWe thank the reviewer for highlighting the importance of Y111H. H122 in TWIK-1, although\nregarded as a major proton-sensing amino acid in the SF1-M2 loop, is not the only determinant\nof pH4,20,22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation, we did not anticipate\nthat it would single-handedly change the pH sensitivity of TWIK-2. In the revised version of the\nmanuscript, as per the reviewer’s suggestion, we also compare the pH-dependency of TWIK-1 in\nour assay setup and test the effect of the TWIK-1-like Y111H mutant on the pH-dependence of\nTWIK-2 channel activity. We find that TWIK-1 is pH-dependent while TWIK-2 is not, despite the\nintroduction of TWIK-1 like Y111H mutation. As per the reviewer’s suggestion, we also tested the\neffect of changing the bulky Y111 to a smaller aliphatic amino acid as alanine, on TWIK-2 channel\nactivity. We found that Y111A was slow deactivating while Y111H was slow activating, compared\nto wild-type TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition.\nPerhaps, to speculate, the role of Y111 in TWIK-2 is maintaining the SF1-M2 loop integrity to\nfacilitate allosteric coupling between the bottom and top of the SF.\n3. I appreciate and highly welcome the high-throughput patch clamp workflow presented by the\nauthors. However, considering i) the discrepancies mentioned by the authors on\nelectrophysiology studies for TWIK-2 in the literature, and ii) the mentioned inconsistencies\nbetween some of their electrophysiology data with that published before, the authors should do a\nmore thorough comparison. If possible, between methods conducted by themselves with the\nsame non-glycosylated constructs (conventional patch clamp and high-throughput patch clamp).\nBut perhaps more importantly, use TWIK-1 in their automated whole-cell patch clamp\nelectrophysiology setup, for which more robust recordings have been reported. This would allow\nus to assess and compare TWIK-1 and TWIK-2 and the different workflows better.\nWe thank the reviewer for these constructive suggestions. While we agree that not all experiments\nperformed using conventional patch clamp can be adopted to a high-throughput automated patch\nclamp approach, screening of inhibitors and testing the relative change in channel activity upon\nmutagenesis has been reliably demonstrated by several groups. As there are already reports that\ncompare high-throughput APC with conventional patch clamp techniques23–25, we prioritized to\ncompare between TWIK-1 and TWIK-2. Using the same expression construct background and\nSyncropatch assay set up, we found that the overall TWIK-1 currents are comparable to TWIK-2.\nHowever, we notice that TWIK-1, unlike TWIK-2, shows pH dependency, albeit only mild. We also\nfind that TWIK-1 is faster activating than TWIK-2 and is more susceptible to inhibition by ML365\nand NPBA compared to TWIK-2. In addition to the comparison of electrophysiological properties\nof TWIK-2 and TWIK-1, in the revised version of the manuscript, we also include a detailed\nstructure comparison at the end of the results section.\n4. The slightly different impact found for two of the tested inhibitors makes the discussion a bit\nambiguous as to their potential different binding mode. I wonder if an attempt to solve an inhibitor-\nbound structure would be in the scope of this study as the authors seem to be able to obtain\nenough stable protein and the processing appears rather straightforward.\nWe agree with the reviewer that the structures of TWIK-2 with ML365 and NPBA could provide\nmore information about their mechanism of binding. We collected and analyzed cryo-EM datasets\nof TWIK-2 with ML365 or NPBA. We observed that in the presence of these ligands, the sample\nappeared to be more heterogeneous, resulting in maps of lower quality compared to the apo\nTWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound TWIK-2 maps\nare fairly similar, with one common feature being the loss of strong tubular lipid-like densities that\nwe observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is possible that loss of tubular\nlipid-like densities is a factor of poor resolution of the map. To make reasonable comparisons, we\nfiltered the apo-TWIK-2 map to ~4Å (Response Fig. 1 above in page 2 of this document).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe\nsome non-protein density immediately below the SF in the case of the NPBA dataset. The non-\nprotein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor\nresolution of the map, and the size and shape of the density, preclude us from modeling NPBA at\nthis site. Hence, we have not included these efforts in the manuscript and are presenting them\nhere in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound structures were not\nfruitful, we employed site-directed mutagenesis to identify a potential NPBA binding site by\nperforming more mutations in the SF, K2P modulator pocket, and around the hydrophobic cuff.\nWe have discussed these in the main manuscript, and our findings suggest that the potential\nbinding site for NPBA, as hinted by additional density in the map, could be below the SF. The\nmutagenesis exploration still does not identify the site for ML365 unambiguously. However, loss\nof strong tubular lipid-like densities around the hydrophobic cuff compared to apo TWIK-2 makes\nus suspect that ML365, like NPBA could bind through the cavity, albeit interacting with different\nresidues compared to NPBA.\nOther major comments:\n5. Can the authors comment on the following: With regards to the processing of the cryo-EM data,\nI wonder if the authors also tested refining the other 3 classes obtained before proceeding with a\nfinal NU-refinement of one of the classes. Considering the authors interpret their structure as an\nintermediate conductive state, I wonder if there were some hints at other conformational states\ncaptured in the data.\nWe tried refining the other 3 classes shown in the penultimate step of data processing pipeline,\nbut the resultant refined maps from these classes were of poor quality and offered no additional\nnew information. Hence, we went ahead with the map denoted as “class 0” for final refinement\nand model building (Response Fig. 4)\nResponse Figure 4: NU-refinements of all the classes post 3D classification.\n6. Could the authors comment why they mutated Y111H to mimic the corresponding TWIK-1\nresidue, but then mutated M135A instead of M135L (which would be the corresponding TWIK-1\nresidue)? What was the rationale/strategy here? And here again, it would have been ideal to have\nTWIK-1 as a control experiment, and to compare Y111H with.\nMethionine, leucine, and alanine are all hydrophobic nonpolar amino acids. Between M135L and\nM135A, the alanine substitution of methionine reduces the size of the side chain drastically\naffecting not only the size of the pocket near M135 but also affecting the relative hydrophobic\nsurface area. As we saw lipid-like densities around M135 amino acid, suspecting their role in\nregulation of channel activity, we anticipated M135A mutation to have drastic effect on TWIK-2\nactivity compared to M135L. In the revised version of the manuscript, we also tested M135N to\nexplore the role of introduction of polar amino acid in this region on TWIK-2 activity. We notice\nthat both M135A and M135N mutants exhibit slightly slower channel activation and show no\ndrastic effect on the channel activity. However, M135A shows a greater loss of NPBA inhibition\ncompared to M135N suggesting that removing the bulky amino acid at this position affects the\nefficacy of NPBA. Similarly, we also tested the effect of changing the pocket around Y111 on\nTWIK-2 channel activity using a Y111A mutant. We found that Y111A was slow deactivating while\nY111H was slow activating, compared to wild-type, like TWIK-2. Both of these mutations caused\na loss of NPBA-mediated inhibition. In the revised version of the manuscript, as per the reviewer’s\nsuggestion, we also compare pH-dependency of TWIK-1 in our assay set up and test the effect of\nTWIK-1 like Y111H mutant on the pH-dependence of TWIK-2 channel activity. We find that\nTWIK-1 is pH-dependent while TWIK-2 is not, despite the introduction of TWIK-1 like Y111H\nmutation.\n7. Could the authors comment/expand on the voltage-dependence of TWIK-2 in the, and whether\nother K2P channels have also been found to be so.\nIn a 2016 study Schewe et al. provided significant insights into the voltage-dependent gating\nmechanisms of K2P channel, challenging the traditional view that voltage-sensing channel\nrequires a canonical voltage-sensing domain. Instead, they proposed the SF to have a “check\nvalve” gating behavior. They also note that this type of voltage-dependency has been observed\nin most K2Ps except TWIK113. Apart from the residues at the SF, voltage-dependent behavior in\nK2Ps, especially the TREK family, is also regulated by other factors such as mechanical force,\nligands, and lipids 14–18. These reports suggest that the same K2P channel can switch between\nvoltage-dependent and voltage-independent current responses depending on their conditions,\nperhaps a regulatory mechanism of certain K2P channels. Because we see that the central cavity\nis occupied by lipid-like densities in our cryo-EM map, and because TWIK-2 has an ability to\ntranslocate between endosomes and plasma membrane and has been proposed to interact with\nother proteins via its C-terminus, we believe it is possible that voltage-dependency in TWIK-2\ncould be a context dependent phenomenon.\n8. While I appreciate the modeling of potential lipids and co for the interpretation and figure\nrendering of non-protein densities, considering the high ambiguity, I expect that the authors have\nno intention of including it in the to be deposited model. (in the era of AlphaFold and machine\nlearning, everything that is deposited can and will be use by AI or others)\nCompared to other K2Ps, the extra non-protein densities of TWIK-2 are a unique feature and may\nbe endogenous molecules involved in channel regulation. As noted in the manuscript, we have\nnot verified the exact nature of these lipid-like molecules, and hence we have modeled\nhydrocarbon tails in these densities. While this manuscript was under revision, two other groups\nreported structures of TWIK-2, and both groups observed tubular “non-protein” densities,\nespecially in the fenestration site and in the central cavity 7,8. The former modeled acyl chains in\nthe fenestration sites, while in the latter manuscript, the authors modeled lipids in these densities\nand proposed a mechanism of lipid-mediated regulation of TWIK-2 channel activity. Encouraged\nby multiple groups, independently, discovering lipid-like tubular densities in TWIK-2, we have\ndecided to deposit the model with hydrocarbon chains modeled into these densities.\nMinor comments:\n- Page 4: \"we identified key amino acids that differentiate TWIK-2 from TWIK-1 and other\nmembers of the K2P family\". From what I understood, only Y111 was a direct comparison with\nTWIK-1, so I suggest toning down this sentence.\nWe thank the reviewer for the correction. We have rephrased it to “We identified key amino acids\nthat regulate TWIK2 channel activity”, as we have included functional analysis of more mutations\nin the revised version of the manuscript.\n- Could the authors add details of this inhibition experiment in the Methods section (e.g. how was\nthe small molecule screening set-up, quantity/volume of compound added, (pre-) incubation time,\netc.). Could the authors show a blank/control measurement with just DMSO to account for the\nbuffer?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the\nautomated patch clamp system and the experimental setup (see above comments to reviewer 1).\nThe SyncroPatch uses 384-well chips with micro-apertures, where negative pressure captures\nindividual cells and forms high-resistance seals. A robotic pipetting unit handles liquids\nautomatically, enabling parallel compound addition, quick solution changes, and precise\nconcentration control, which ensures reliable dose–response measurements. We have updated\nthe Methods section to include more details about the inhibition experiments. Importantly, we\nclarify that the reference (control) buffer always contained the same DMSO concentration as the\ncompound solutions, accounting for any potential nonspecific effects of the solvent. As a result,\nthe vehicle condition was consistently included in all experiments, ensuring that the observed\neffects are due to the test compounds.\n- Methods section: Cloning and overexpression of recombinant TWIK-2: The text should say for\nbetter clarity: \"with an N-terminal Strep-tag-II, followed by a GFP tag and a HRV-3C protease site\"\ninstead of \"followed by an HRV-3C protease site and a GFP tag\" as it is then later stated that 3C\ncleavage results in removal of GFP tag.\nWe thank the reviewer for pointing this out. We have corrected it.\n- As the authors mention in the main text the poor heterologous expression of TWIK channels,\ncould they indicate the yield they obtained from their experiments?\nAs mentioned in the introduction, poor heterologous expression of TWIK channels has been\nreported by a few groups before us 9,12,26. We were able to obtain ~0.4 mg of protein from 1L of\nHEK293 GnTI- cells, after the affinity purification step.\n- Correct typo in: \"Mutations T106A and T214A\"\nWe have rephrased that sentence in the discussion.\nFigures:\n- Please update figure numbers so that they are cited in correct order as mentioned in the text.\nE.g S2 should be called S1; swap order of Figure 4D and 4E\nWe have made sure that the figures are in the correct order.\n- Figure S2F: could the authors explain the uV units on the y-axis of the FSEC curve\n- Specify in main text and S2 figure legend which detergent was used for solubilization of ΔC-\nTWIK-2\n- For better clarity, keep one naming for each construct between figure and figure legends (e.g.\nin figure S2: TWIK-2 is sometimes called WT or wild-type; or ΔC-TWIK-2 (C-ter 1-276) is called\nc-ter 1-276, c-ter and del1-276). In general, could the authors fix all the inconsistencies in the\nfigure legend of S2: missing periods, having a mixture of (n=15) and \",n=15\" etc.\nAs per the other reviewer’s suggestion, we removed the ΔC-TWIK-2 data from Supplementary\nFig. 2 and the legend associated with it in the revised version of the manuscript. We have fixed\nall other inconsistencies.\n- Figure 2A: could the authors improve the illustration so that the ion conduction pathway is clearer\nto the reader?\nWe have remade this figure in current version of our manuscript.\n- Figure S1: indicate the non-conserved M135 and Y111 residues on the sequence alignment\nAll the sites that have been mutated in this study, including M135 and Y111, have now been\nhighlighted in the sequence alignment figure in the current version of our manuscript.\n- Figure S3: FSC curves indicates the mask during refinement is too tight and could be optimized\nWe thank the reviewer for this suggestion. However, the unique architecture of K2P channels\nallows detergent micelles to occupy grooves formed between various secondary structure\nelements in the transmembrane region. Moreover, the detergent that we used for purification is\nDMNG. Neopentyl glycol class of detergents has low CMCs and are known to form\nheterogeneous micelles with varying sizes depending on the effective concentration of the\ndetergent 27. To avoid heterogeneity in micelle size and stochastic detergent densities from\ninterfering with refinement, we had to use a relatively tight mask to reconstruct a map with\ninterpretable features at the protein-micelle interface. We have tried to dilate the mask more, and\nthe resulting structure could not reach as high a resolution as the current map. A relatively tight\nmask is needed to effectively remove the micelle density that is tightly bound to our protein.\n- Could the authors mention/indicate the possible glycosylation sites that TWIK-2 possesses?\nThis could be added to S1.\nWe have now highlighted the glycosylation sites N79 and N85 on the sequence alignment (current\nFigure S2).\nReferences\n1. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination\nand hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Nature 414, 43–\n48 (2001).\n2. Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type\ninactivation in K(+) channels. Nature 466, 203–208 (2010).\n3. Pau, V., Zhou, Y., Ramu, Y., Xu, Y. & Lu, Z. Crystal structure of an inactivated mutant\nmammalian voltage-gated K+ channel. Nat. Struct. Mol. Biol. 24, 857–865 (2017).\n4. Turney, T. S., Li, V. & Brohawn, S. G. Structural Basis for pH-gating of the K+ channel TWIK1\nat the selectivity filter. Nat. Commun. 13, 3232 (2022).\n5. Thomson, A. S. et al. Initial steps of inactivation at the K+ channel selectivity filter. Proc. Natl.\nAcad. Sci. U. S. A. 111, E1713-22 (2014).\n6. Rohaim, A. et al. A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant\nE71V. Nat. Commun. 13, 1574 (2022).\n7. Mondal, A., Niranjan, S. & Minor, D. L., Jr. A lipid plug affects K2P6.1(TWIK-2) function.\nbioRxivorg (2025) doi:10.1101/2025.06.11.659167.\n8. Khanra, N. K., Wang, C., Delgado, B. D. & Long, S. B. Structure of the human TWIK-2\npotassium channel and its inhibition by pimozide. Proc. Natl. Acad. Sci. U. S. A. 122,\ne2425709122 (2025).\n9. Lloyd, E. E., Marrelli, S. P., Namiranian, K. & Bryan, R. M., Jr. Characterization of TWIK-2, a\ntwo-pore domain K+ channel, cloned from the rat middle cerebral artery. Exp. Biol. Med.\n(Maywood) 234, 1493–1502 (2009).\n10. Patel, A. J. et al. TWIK-2, an inactivating 2P domain K+ channel. J. Biol. Chem. 275, 28722–\n28730 (2000).\n11. Chavez, R. A. et al. TWIK-2, a new weak inward rectifying member of the tandem pore\ndomain potassium channel family. J. Biol. Chem. 274, 7887–7892 (1999).\n12. Bobak, N. et al. Recombinant tandem of pore-domains in a Weakly Inward rectifying K+\nchannel 2 (TWIK2) forms active lysosomal channels. Sci. Rep. 7, 649 (2017).\n13. Schewe, M. et al. A non-canonical voltage-sensing mechanism controls gating in K2P K(+)\nchannels. Cell 164, 937–949 (2016).\n14. Brennecke, J. T. & de Groot, B. L. Mechanism of mechanosensitive gating of the TREK-2\npotassium channel. Biophys. J. 114, 1336–1343 (2018).\n15. Harrigan, M. P., McKiernan, K. A., Shanmugasundaram, V., Denny, R. A. & Pande, V. S.\nMarkov modeling reveals novel intracellular modulation of the human TREK-2 selectivity\nfilter. Sci. Rep. 7, 632 (2017).\n16. Yang, F. A gating mechanism of K2P channels by their selectivity filter. J. Mol. Cell Biol. 14,\n(2022).\n17. Rinné, S. et al. The molecular basis for an allosteric inhibition of K+-flux gating in K2P\nchannels. Elife 8, e39476 (2019).\n18. Conrad, L. J., Proks, P. & Tucker, S. J. Effects of ionic strength on gating and permeation of\nTREK-2 K2P channels. PLoS One 16, e0258275 (2021).\n19. Morton, M. J., O’Connell, A. D., Sivaprasadarao, A. & Hunter, M. Determinants of pH sensing\nin the two-pore domain K(+) channels TASK-1 and -2. Pflugers Arch. 445, 577–583 (2003).\n20. Niemeyer, M. I., Cid, L. P., Peña-Münzenmayer, G. & Sepúlveda, F. V. Separate gating\nmechanisms mediate the regulation of K2P potassium channel TASK-2 by intra- and\nextracellular pH. J. Biol. Chem. 285, 16467–16475 (2010).\n21. Hall, P. R. et al. Structures of TASK-1 and TASK-3 K2P channels provide insight into their\ngating and dysfunction in disease. Structure 33, 115-122.e4 (2025).\n22. Lesage, F. et al. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a\nnovel structure. EMBO J. 15, 1004–1011 (1996).\n23. Li, T. et al. High-throughput electrophysiological assays for voltage gated ion channels using\nSyncroPatch 768PE. PLoS One 12, e0180154 (2017).\n24. Seibertz, F. et al. A modern automated patch-clamp approach for high throughput\nelectrophysiology recordings in native cardiomyocytes. Commun. Biol. 5, 969 (2022).\n25. Obergrussberger, A. et al. The suitability of high throughput automated patch clamp for\nphysiological applications. J. Physiol. 600, 277–297 (2022).\n26. Pountney, D. J. et al. Identification and cloning of TWIK-originated similarity sequence\n(TOSS): a novel human 2-pore K+ channel principal subunit. FEBS Lett. 450, 191–196\n(1999).\n27. Lee, H. J., Lee, H. S., Youn, T., Byrne, B. & Chae, P. S. Impact of novel detergents on\nmembrane protein studies. Chem 8, 980–1013 (2022).\nResponse to reviewer comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have now addressed all of my remaining concern and I recommend for publication\n\n======================================================================\nATTACHED REVIEWER REPORT — IMPORTANT\nThe text below is a complete reviewer report submitted as a PDF\nattachment. It appears after the Creative Commons license block.\nAssign it to whichever reviewer wrote 'see pdf' or had no inline text.\n======================================================================\n\nResponse to reviewer comments\nWe thank all the reviewers for their critical assessment of our manuscript. Our point-by-point\nresponse is below. For purposes of clarity, we have reviewer comments in red italics.\nReviewer #1 (Remarks to the Author):\n“This study determines a TWIK2 cryoEM structure and uses a commercial automated patch clamp\nsetup to assess the function of TWIK2 channel mutants and pharmacology. The structure of\nTWIK2 had not been previously determined and this study provides a first look at the TWIK2\nchannel architecture. To accompany the structure, the authors perform electrophysiological\nrecordings of TWIK2, a channel that has been notoriously difficult (though not impossible) to\nrecord from. Overall, the methodology in the structural and functional work appears to be sound\nand the writing is clear. “\nWe sincerely thank the reviewer for the overall positive feedback.\n“There are novel observations and interesting results in the manuscript but an informative\nconnection between knowledge learned from the TWIK2 structure and the functional studies\nperformed is largely lacking. The Y111H designed to mimic the TWIK1 pH sensing histidine and\nthe S4 site threonine residues are all positions that did not require a cryoEM structure to be\ninteresting candidates for mutation. While the M135A mutation is a candidate site based on the\nTWIK2 structure, it is unclear why the authors chose this position as it does not occlude the pore\nor play any clearly observable role in the TWIK2 structure. The experiments examining the effects\nof ML365 and NPBA are interesting but somewhat superficial, as the mutants show some effects\non channel pharmacology but do not yield any substantive insight into where the drugs might bind\nor how they might act.”\nWe thank the reviewer for pointing out weaknesses in this manuscript that could be improved. We\nagree with the reviewer that the mutant selection was sparse. To address this, the analysis in the\nrevised version of the manuscript includes the following single and double mutants: F98A, Y111H,\nY111A, T106A, T214A, S213A, M135N, M135A, and T106A-T214A, focusing on the regions near\nthe SF, the K2P modulator pocket (F98A), and the hydrophobic cuff (M135A and M135N). The\nalanine substitution of M135 (M135A) drastically reduces the size of the side chain, affecting not\nonly the size of the pocket near M135 but also the relative hydrophobic surface area. As we\nobserved lipid-like densities around the M135 amino acid, suggesting their role in regulating\nchannel activity, we anticipated that the M135A mutation would have a drastic effect on TWIK-2\nactivity. In the revised manuscript, we also tested M135N to explore the role of introducing a polar\namino acid in this region on TWIK-2 activity. We observe that both M135A and M135N mutants\nexhibit slightly slower channel activation, with no drastic effect on channel activity. However,\nM135A shows a greater loss of NPBA inhibition compared to M135N, suggesting that removing\nthe bulky amino acid at this position affects the efficacy of NPBA. Similarly, we also tested the\neffect of changing the pocket around Y111 on TWIK-2 channel activity using a Y111A mutant. We\nfound that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type\nTWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition, highlighting the role\nof Y111 in maintaining SF1-M2 loop integrity.\nTo study the binding modes of ML365 and NPBA, we collected and analyzed cryo-EM data using\nTWIK-2 complexed with ML365 and NPBA. We observed that in the presence of these ligands,\nthe sample appeared to be more heterogeneous, resulting in maps of lower quality compared to\nthe apo TWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound\nTWIK-2 maps are fairly similar (~ 4 Å), with one common feature being the loss of strong tubular\nlipid-like densities that we observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is\npossible that loss of tubular lipid-like densities is a factor of poor resolution of the map. To make\nreasonable comparisons, we filtered the apo-TWIK-2 map to 4Å (Response Fig. 1).\nResponse Figure 1: cryo-EM map comparison of TWIK-2 Apo, TWIK-2 ML365 and TWIK-2 NPBA.\nTwo bottom views on the right indicate the occupancy of the cavity close to the selectivity filter (top)\nand cytoplasmic site (bottom).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe\nsome non-protein density immediately below the SF in the case of the NPBA dataset. The non-\nprotein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor\nresolution of the map, and the size and shape of the density, preclude us from unambiguously\nmodeling NPBA at this site. Hence, we have not included these efforts in the manuscript and are\npresenting them here in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound\nstructures were not successful, we employed site-directed mutagenesis to identify a potential\nNPBA binding site by performing more mutations in the SF, K2P modulator pocket, and around\nthe hydrophobic cuff. We have discussed these in the main manuscript and our findings suggest\nthat the potential binding site for NPBA, as hinted by additional density in the map, could be below\nthe SF. The mutagenesis exploration still does not identify the site for ML365 unambiguously.\nHowever, loss of strong tubular lipid-like densities around the hydrophobic cuff compared to apo\nTWIK-2 makes us suspect that ML365, like NPBA could bind through the cavity, albeit interacting\nwith different residues compared to NPBA.\n“1) In the section entitled “Ion conduction pore of TWIK2”, the authors suggest that the carbonyl\noxygens of selectivity filter residues V107 and Y109 face away from the central permeation axis\nin TWIK-2 and argue for the relevance of this SF orientation to TWIK2 function. Given the ~3.7\nangstrom resolution of the overall structure, I cannot ascertain how the authors come to this\nconclusion. Using the map and model provided by the authors, I can easily model the selectivity\nfilter within the map in a conventional orientation and find that I improve the number of residues\nin favored orientations when the SF is modeled more conventionally.\nIf there is a biophysical argument to be made that the positioning of the valine and tyrosine\nsidechains in the TWIK2 map somehow promote an unconventional SF backbone configuration,\nthis should be provided. Simply based on the map provided by the authors, I find this section to\nbe unsupported by experimental results. If the authors do feel their selectivity figure configuration\nis accurate, a supplemental figure showing how the experimental map is better fit by the outward\nrather than inward-facing carbonyls at V107 and Y109 would be extremely helpful to make the\nauthors' case on this point.”\nWe thank the reviewer for a thorough review of our structure. In our TWIK-2 structure, the SF has\nbeen modeled with carbonyl oxygens of three amino acids – V107, Y109, and L217 – facing away\nfrom the ion-conductance pore. While we agree that at ~ 3.7 Å resolution it could be challenging\nto resolve the orientation of carbonyl oxygens in the dynamic loops of the SF region\nunambiguously, we would like to highlight that the local resolution for this region is much better\nthan 3.5 Å (Supplementary Fig. S3F in the manuscript). As per the reviewer’s critique, we\nattempted to model these three carbonyls in various combinations of them facing away or towards\nthe SF. However, just based on visually comparing the fits of these various models to density, it\nis not straight forward to identity one particular combination of the carbonyl oxygens that fits better\nto the map (see figures & table below). Hence, based on a comparison of the model statistics\nsuch as bond RMSDs, Ramachandran outliers, Rama-Z score, & CaBLAM outliers for the SF\nregion, and the overall model quality statistics for the whole protein, we believe that the model as\nit has been presented in the manuscript is the best possible fit (Response Table 1 in this rebuttal).\nIn K2P channels, the two conserved threonines at the bottom of the selectivity filter (SF) are the\nonly amino acids within the SF that coordinate K+ ions through their side chains. All other amino\nacids at the SF coordinate K+ ions through carbonyl oxygens. It has been shown by multiple\ngroups previously that reorientation of the carbonyl oxygens away from the SF is a means to\nregulate K+ ion flow, including channel inhibition, in potassium channels. Using X-ray\ncrystallography and cryo-EM, several groups have shown that reorientation of carbonyl oxygens\naway from the SF pore affects the K+ ion coordination at the SF as part of C-type or pH-mediated\ninactivation of the channel in KcsA, Kv, and TWIK-11–4. The flipped carbonyls often co-existed\nwith reduced occupancy at the canonical K+ ion sites (S0-S4) in the SF. As noted in the\nmanuscript, our cryo-EM map allowed us to confidently model K+ ions in only three out of these\nfive sites. Similarly, MD simulations on the prototypical K+ channels like MthK and KcsA have\nshown that reorientation of the carbonyl oxygen of the conserved valine at the SF is one of the\ninitial steps that promotes dissociation of K+ from the SF and allows the SF to adopt nonconducting\nor inactivated states5,6. As pointed out by the reviewer, we had provided only a brief biophysical\nexplanation for non-canonical carbonyl positioning in the discussion in the original manuscript. To\naddress that, we have expanded on the explanation associated with flipped carbonyl observation,\nby including examples of other eukaryotic K+ channels (TWIK-1, Kv, and Kir) and their prototypic\nprokaryotic homologs (MthK and KcsA) where similar observations have been made, as a\nseparate section in the discussion, & added more references to this section.\nResponse Figure 2: Various TWIK2 selectivity filter models fit to the cryo-EM density. The model\nquality statistics for the variations 1-7, and a comparison of the statistics are provided in table 1. Cryo-\nEM density is highlighted as blue mesh and the three K+ ions as blue spheres. The two protomers,\nTWIK-2 chain A and B, are colored light magenta and light cyan. All the amino acids at the SF are\nlabeled in variation 7 where none of the carbonyls are flipped away from the SF, and in the remaining\npanels only the carbonyls that are flipped away from the SF have been labeled.\nResponse Table1: TWIK2 selectivity filter model quality statistics. Selectivity filter model quality statistics were\nobtained by running PDB validation on the amino acids 104-111 and 213-220, and K+ ions at the SF using Phenix. The\nvalues in the parentheses represent the whole PDB. As the resolution of the structure is ~3.7 Å, CaBLAM outliers, Rama-\nZ score, Bond RMSDs for the SF region, and the overall statistics of the whole PDB were compared to decide on the\ncarbonyl orientations of the amino acids V107, Y109, and L217, which surround S0/S1 and S2/S3 sites at the SF. Each\nvariation (Var. 4-6) or combination of variations are show (Var. 1-3)\nDeposited\nParameter Var. 1 Var. 2 Var. 3 Var. 4 Var. 5 Var. 6 Var. 7\n9E94\nAmino acids with\nV107,\ncarbonyl Y109 & V107 & V107 &\nY109, & L217 Y109 V107 None\noxygens flipped L217 L217 Y109\nL217\naway from SF\nBond length 0.007 0.014 0.014 0.007 0.017 0.018 0.016 0.017\nRMSD (Å) (0.009) (0.010) (0.009) (0.009) (0.010) (0.010) (0.010) (0.010)\nBond angle 1.617 2.103 2.161 1.724 2.292 2.188 2.265 2.441\nRMSD (o) (1.564) (1.651) (1.624) (1.595) (1.672) (1.662) (1.643) (1.693)\n1.97 1.49 2.01 2.53 2.05 1.97 2.48 2.45\nMolProbity score\n(1.78) (1.75) (1.78) (1.89) (1.78) (1.82) (1.86) (1.88)\nRamachandran 0.00 0.00 8.33 8.33 0.00 0.00 0.00\n0.00 (0.00)\noutliers (%) (0.00) (0.00) (0.47) (0.47) (0.00) (0.00) (0.00)\nRamachandran 8.33 8.33 25.00 41.67 8.33 50.00 58.33 50.00\nallowed (%) (6.07) (6.54) (7.01) (7.94) (6.07) (8.88) (8.88) (8.88)\nRamachandran 91.67 91.67 75.00 50.00 83.33 50.00 41.67 50.00\nfavored (%) (93.93) (93.46) (92.99) (91.59) (93.46) (91.12) (91.12) (91.12)\n-5.32 -6.25 -7.54 -6.18 -7.92 -6.96 -8.91 -9.44\nRama-Z (whole)\n(-3.75) (-3.79) (-3.86) (-3.78) (-3.90) (-3.81) (-3.93) (-4.00)\nRama-Z (loop) -4.05 -4.76 -5.74 -4.70 -6.03 -5.30 -6.79 -7.19\n(-4.87) (-5.04) (-5.29) (-5.06) (-5.33) (-5.09) (-5.53) (-5.64)\nCaBLAM outliers 25.00 50.00 50.00 37.50 62.50 50.00 62.50 75.00\n(%) (2.88) (3.85) (4.33) (3.37) (4.33) (3.85) (4.81) (4.81)\nClash score 8.81 2.20 4.39 10.96 6.62 2.21 8.81 8.83\n(6.76) (5.91) (6.05) (6.34) (6.34) (5.62) (6.20) (6.63)\n“2) In Figure 2B, the pore radius profile shows a constriction of the pore at around 5A from the\ncytoplasmic face of the channel. This cytoplasmic constriction does not seem to be discussed in\nthe manuscript. Is this constriction brought about by the lipids modeled in the structure? If so, is\nthis at all relevant to the function of TWIK2? If the authors believe that these lipid densities are\nimportant to shaping the TWIK2 pore, then this pore radius plot is appropriate. If the relevance of\nthese lipids is unclear and could simply be an artifact of the protein purification process, it would\nbe better to omit the lipid densities when running HOLE (or include a second HOLE profile in the\nabsence of the lipids) to show the profile of just the TWIK2 protein.\nIt would also be appropriate to discuss the lipid densities present in the map in the results section\nof the manuscript, rather than the discussion. This would be a good spot to do so.”\nThe pore radius plot in Fig. 2B was constructed using the TWIK-2 model with hydrocarbon tails\nmodeled into the tubular lipid-like densities. We have now edited Fig. 2B to show the pore radius\nprofile with and without the modeled lipid-like densities. We did not include any exogenous lipids\nduring the purification. So, we believe these tubular densities arise from the molecules that co-\npurified with TWIK-2 from HEK293 GnTI- cells. Because we could not verify the exact nature of\nthese molecules, we have only modeled hydrocarbon tails in these densities and called them lipid-\nlike in the manuscript. The discussion on the lipid-like densities has now been moved to the results\nsection in the revised version of the manuscript, as per the reviewer’s suggestion. While this\nmanuscript was under revision, another group reported a preprint suggesting a potential lipid\nmediated regulation of TWIK-2 channel activity. They argued that lipid molecules “plug” the\nchannel on the cytoplasmic side by occupying the central cavity and by physically obstructing the\npassage of ions, rendering TWIK-2 inactive. They demonstrate that R257, an amino acid towards\nthe C-terminus that lies below the hydrophobic cuff, is crucial for this lipid binding in the central\ncavity and that the R257A mutation increases the channel activity, suggesting that channel\nactivation is a result of lipid removal or repositioning in the central cavity. In our TWIK-2 structure,\nwe observe lipid-like densities in similar positions (central cavity and along the groove formed by\nthe PH2, M2, and M4 helices) as reported by Mondal et al7. However, these lipid-like densities do\nnot entirely obstruct the access to the central pore (Fig. 2). The observation of lipid-like density,\ncorroborated by a different group independently, suggests that these molecules are relevant to\nthe function. We have added a few lines discussing this preprint and its relevance in the context\nof our structure to the discussion section of the revised manuscript.\n“3) For the experiments where G/GMAX–V curves are calculated, it does not appear that the\nrecordings reach a steady state at 200 ms. The current is still rising for TWIK1 wt and this is even\nmore pronounced in the mutants that slow activation (Figure 3B). Is it possible that the differences\nin V50 are artificial, since the maximal steady state current is reached at 200ms in some of the\nmutants but not in all of them.”\nWe appreciate the reviewer’s concerns regarding the possible underestimation of steady-state\ncurrents due to incomplete activation at the end of the 200-ms depolarizing pulse, especially in\nslow-activating TWIK-2 mutants. To reinforce the validity of our conclusions regarding mutant-\ninduced shifts in voltage dependence, we performed a new analysis based on normalized tail\ncurrents, which better reflect the relative conductance by capturing the instantaneous current\nupon repolarization—thus minimizing the effects of kinetic differences in channel activation.\nSpecifically, we constructed revised G/GMAX–V curves using tail currents, enabling a more\naccurate assessment of the voltage dependence of activation across all variants. From these\ncurves, we calculated the ΔV50 values, which provide a more reliable measure of the voltage shift\ncaused by each mutation. Importantly, this alternative approach produced similar V50 shifts as\npreviously reported, confirming that the observed differences are genuine and not artifacts,\nreflecting fundamental changes in channel gating properties. These new results are now included\nin the revised Supplementary Fig. 5 and discussed in the Results section. To enable a better\ncomparison of currents across TWIK-2 variants, we have also modified Fig. 3 to now display\nwhole-cell current traces and activation time constants for all the mutants that we tested.\n“4) I think the manuscript would be greatly improved by a more direct comparison of the TWIK2\nstructure with the previously published TWIK1 structures. Does this TWIK2 structure look more\nlike the low pH or neutral pH TWIK2 structure? Are there clear differences in the positioning of\nthe cap or TM helices? The entire section of the results devoted to an overview of the TWIK2\nchannel could be redone as a more direct comparison between TWIK1 and TWIK2 and would\nsignificantly increase interest.”\nWe agree with the reviewer that a comprehensive comparison of TWIK-2 with TWIK-1 could be a\nbeneficial addition to the manuscript. In our first version of the manuscript, we had presented a\ncomparison of TWIK-2 and TWIK-1 in Figs. 5B, C and Supplementary Fig. S4, focusing only on\nthe conformational changes in pore domains, SF, and M4 helix. As TWIK-2 is physiologically\nunlike TWIK-1, for the sake of completeness, we have also included comparisons with other pH-\nsensitive and lipid-regulated K2P channels. To make these comparisons more apparent, we have\nreformatted and expanded the figures and text to include a separate comparison section of the\nTWIK-2 and TWIK-1 structures in their entirety. In addition, we have also included the results from\nwhole-cell recordings studying the pH dependency of channel activity in TWIK-1 and the TWIK-\n1-like Y111H mutant of TWIK-2. Interestingly, we find that the differences in the structure of TWIK-\n2 are localized to the cap region when compared with the TWIK-1 structure at pH 7.5. When\ncompared with TWIK-1 at pH 5.5, the differences lie only in the SF region, specifically the SF1-\nM2 linker that houses the pH-sensing amino acid H122 in TWIK-1. TWIK-2 has the cap region\nthat is similar to inactive TWIK-1, but has the SF loops positioned in a similar way as that of the\nactive TWIK-1, despite the residue Y111 in TWIK-2 (H122 in TWIK-1) adopting a unique ‘up’\nconformation. These new observations have been discussed in the revised version of the\nmanuscript, alongside the TWIK-2 and TWIK-1 comparisons from the older version (Fig. 1, 5).\nThe comparison of TWIK-2 with other K2P channels has been reformatted into a separate figure\n(Supplementary Figs. 4, 7).\nMinor Points\n1. - In the introduction, “Kcnk6 knockout studies have shown suppression of NLRP3\ninflammasome activation in macrophages, implicating endolysosomal localization of\nTWIK-2 in inflammation 45,46.” This sentence is a bit misleading and should be clarified.\nFrom the references cited, it is movement of TWIK2 from the endolysosome to the plasma\nmembrane that appears to be a critical step in the potassium efflux that mediates NLRP3\ninflammasome activation.\nWe thank the reviewer for the correction. We have now replaced the sentence with “Translocation\nof TWIK-2 from endosome to plasma membrane is implicated in NLRP3-mediated inflammasome\nactivation in macrophages, with Kcnk6 knockouts showing a suppressed inflammasome\nactivation”.\n2. - In figure 1 (and for nearly all electrophysiological traces shown in the manuscript),\nrecording traces are shown on a signal scale that reads “Normalized I”. It is unclear what\nthe non-transfected cells traces are normalized to? For clarity throughout the manuscript,\nit would be helpful if the authors explicitly discuss whether normalization was applied using\na universal metric (which could be described in the methods section of the manuscript) or\nad hoc for each set of recordings (in which case this should be described in each figure\nlegends)\nWe thank the reviewer for pointing out this important issue related to the normalization of\nelectrophysiological traces. In the revised manuscript, all figures now display traces showing\nactual absolute current values instead of normalized signals. This update allows for direct\ncomparison of current amplitudes across wild-type and mutant channels as well as non-\ntransfected controls. Additionally, we now include current density for each experimental condition\nwhere appropriate, ensuring that differences in current amplitudes are not influenced by variations\nin cell size. We believe these revisions significantly improve the clarity and transparency of our\ndata presentation and address the reviewer’s concern about normalization.\n3- Given that the C-terminally truncated TWIK construct was not used for structure of detailed\nfunctional studies, this data could be omitted or moved to the methods section.\nWe agree with this reviewer’s observation that the data on C-terminally truncated TWIK-2 is not\ninformative, beyond suggesting that TWIK-2 devoid of C-terminus is unstable compared to WT.\nIn accordance, we have now removed the C-term truncation data of TWIK-2 from the revised\nmanuscript.\n4 - Is it meaningful that the loop region between V75-P89 is unresolved in the cryo-EM structure?\nIn all other K2P structures, this loop is well resolved. Does this mean that the V75-P89 is dynamic\nin TWIK2, suggestive of a functional role? Or is this just a limitation of the resolutions of this\nTWIK2 structure compared to other K2P structures? Does 3D variability analysis in cryosparc\ngive any indication of what is going on in this loop region?\nThe loop V75-P89 is a part of the CH2-PH1 region that harbors two predicted glycosylation sites,\nN79 and N85. We used HEK293 GnTI- cells, which lack N-acetylglucosaminyltransferase I\nactivity, for overexpression of TWIK-2. So, it is likely that glycosylation at these sites is essential\nfor stabilizing this loop, and without such glycosylation, this loop is flexible and hence poorly\nresolved in the cryo-EM density map. Interestingly, TWIK-2 structures reported by a different\ngroup recently also lacked this region 8. Despite using glycosylation-competent Expi293F cells\nand achieving an overall higher resolution structure (~2.9 Å). Perhaps, in TWIK-2 this loop is more\ndynamic than other K2Ps. Another group reported structures of TWIK-2 while this manuscript was\nunder revision 7. They too use glycosylation-capable Expi293F cells as Khanra et. al., but were\nable to resolve the V75-P89 region in their cryo-EM map. Moreover, they find that amino acids\n75-80 in this loop actually form a short helix called ‘ear helix’ that is unique to TWIK-2, proposed\nto stabilize the TWIK-2 cap domain. As this loop region can also potentially interact with the\nkeystone inhibitor site and the K2P modulator pocket, we attempted to resolve this region using\n3DVA and subsequent classification in cryoSPARC (Response Fig. 3). However, our attempts\nwere not successful.\n5- The panel in Figure 3 showing the relative locations of T106, T214, M135, and Y111 is critically\nimportant for understanding the orientation and relevance of these residues. I would suggest\nreworking this figure to make it clearer. The inset boxes are distracting, as each one has a different\norientation relative to the main panel. Perhaps a clearly labeled “top view” showing the Y111\nresidues, “bottom view\" showing the M135 residues, and a side view like shown in the left side of\nFigure 3A would be sufficient for this.\nWe have modified the figure as per the reviewer’s suggestions.\n- “Notably, TWIK-2 channels elicited outward potassium currents that increased with\ndepolarization (over +30 mV), suggesting voltage-dependent activation of TWIK-2” Isn’t this a\nsurprising result, given that prior recordings in symmetrical potassium have shown inward\nrectification rather than outward rectification? Is there any methodologic explanation for this\ndifference in observed functional behavior?\nWhile it may seem surprising at first, the voltage-dependent activation of TWIK-2 seen in our\nrecordings agrees with previous studies, especially those indicating that TWIK-2 is one of the\nleast inwardly rectifying members of the K2P family, that inactivates and has a potential role in\nelectrogenesis9–12. Compared to strongly rectifying K2P channels, such as TREK-1 or TASK-3,\nTWIK-2 exhibits more linear or even outwardly biased I–V relationships under certain recording\nconditions. Moreover, differences in rectification behavior—particularly outward currents at\ndepolarized potentials—can depend on the expression system (e.g., Xenopus oocytes versus\nmammalian cells), ionic composition, and intracellular environment (e.g., Mg²⁺, polyamines, or\npH). In our experiments, outward currents at voltages above +30 mV are probably caused by a\ncombination of weak inherent rectification and the electrochemical driving force for K⁺ efflux under\nsymmetrical or nearly symmetrical K⁺ conditions, rather than unusual voltage-dependent gating.\nTo clarify this, we added a section in the discussion explaining how our findings align with the\nknown electrophysiological properties of TWIK-2 and reflect system-dependent variability rather\nthan an unexpected gating mechanism.\nResponse Figure 3: 3D variability analysis (3DVA) and subsequent classification to resolve V75-P89\nloop. The region shaded in gray is a part of the supplementary figure 3 in the manuscript and has been\nincluded here only for reference. Particles from the best cluster displaying all the features of TWIK-2 from\n3DVA step was used to generate an ab initio initial reference (cyan box). Next, particles from all clusters\nwith robust cap domain features (red boxes) from the 3DVA step were pooled for clean up by heterogenous\nrefinement and ab initio jobs. The final map obtained after NU-refinement, using the ab-initio reference\ngenerated from the best 3DVA cluster, resulted in a map with features and resolution that resembled the\nmap already presented in the manuscript.\n- Reference 70 in the manuscript includes data that describe the effect of mutations at the S4 site\nthreonines in K2Ps. For nearly all K2Ps, these S4 site mutations cause a loss of voltage\ndependance. It is therefore interesting that in TWIK2, the T106A and T214A mutants maintain\ntheir voltage dependent gating. Is there any rationale for why this might be the case?\nThe reference 70 reports that in channels without a dedicated voltage-sensing domain, the SF\nitself acts as a gate and sensor. They demonstrate that mutating the conserved threonines at the\nSF to cysteines causes loss of voltage-dependent behavior. However, they also show that in\naddition to just these mutations, voltage-dependent behavior is also regulated by other factors\nsuch as mechanical force, ligands, and lipids13. Regulation of voltage-dependency of K2P\nchannels by these auxiliary factors, apart from the residues at SF, has been shown in several\nK2P channels, especially the TREK family14–18. These reports suggest that the same K2P channel\ncan switch between voltage-dependent and voltage-independent current responses depending\non their conditions, perhaps a regulatory mechanism of certain K2P channels. T106A and T214A\nmutants display a reduced-voltage dependency in our experiments, but not a complete loss of\nvoltage-dependency. Because we see that the central cavity is occupied by lipid-like densities in\nour cryo-EM map, and because TWIK-2 has an ability to translocate between endosomes and\nplasma membrane and has been proposed to interact with other proteins via its C-terminus, we\nbelieve it is possible that voltage-dependency in TWIK-2 could be a context dependent\nphenomenon, in addition to being regulated by the residues at the SF. However, identification of\nall the factors in TWIK-2 that regulate its channel activity needs furthermore substantial efforts.\n- “Mutations T106A and T214A disturb these networks of hydrogen bonding and hydrophobic\ninteractions that stabilize the base of the SF and make the channel less selective and more open\n(Figure 3B-3F).” The functional experiments presented in this section use only a single ionic\ncomposition and are not suited to adequately address ion selectivity. Please support with data or\nomit this claim.\nWe agree with the reviewer that with only a single ionic composition, it is hard to address the ion\nselectivity of TWIK-2. We have rephrased this statement and added the observations from T106A\nand T214A double mutants’ whole cell recordings to this section. We find that the double mutants\ndisplay greater current density compared to single mutants and wild-type TWIK-2.\n- Is the TWIK2 Y111H mutant pH sensitive like the TWIK1 channel? This seems like an obvious\nexperiment to try and would be informative to understanding differences between TWIK1 and\nTWIK2 functions.\nAs already discovered by a few groups, H122 in TWIK-1 and H98 in TASK-3 although regarded\nas major proton-sensing amino acids in the SF1-M2 loop, are not the only determinants of pH\nsensitivity in K2P channels4,19–22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation,\nwe did not anticipate that it would single-handedly change the pH sensitivity of TWIK-2. In the\nrevised version of the manuscript, we show that the pH dependency of channel activation and\nactivity of Y111H TWIK-2 is the same as that of wild-type like TWIK-2. Furthermore, we observe\nthat in our assay setup, TWIK-1 showed pH-dependent current changes, albeit mild. These\nobservations have been included in the revised version of the manuscript in a separate figure,\nand the corresponding text has been modified accordingly.\n- More methodological detail of the automated patch clamp technique would be helpful to\nunderstand the experimental system. Are drugs applied to the intracellular face of the channel\n(via syncropatch pipette) or extracellular via bath?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the\nautomated patch clamp system. To address this, we have expanded the Methods section to\ninclude more detailed information about the experimental setup. Unless otherwise noted, all\ncompounds were applied extracellularly using an external addition protocol, following the standard\nprocedures for the SyncroPatch system. The SyncroPatch features a high-speed, multi-channel\npipetting robot that manages all liquid additions and removals during the experiments. This robot\ncan simultaneously add various external compounds—such as drugs, ligands, and chemicals—\nto each of the 384 wells, enabling a large number of experiments to be conducted simultaneously.\nAdditionally, the robot performs quick and efficient solution exchanges in the wells. Although the\nsystem permits compound delivery to the intracellular face via an intracellular exchange solution,\nthis study only examined the external application of pharmacological agents to aid in easier\napplication, better ability to perform cumulative concentration-response curves, and for clear pre-\nand post-drug comparisons. This clarification has been added to the ‘Automated patch clamp\nrecordings’ section within the Materials and Methods, to improve reproducibility and transparency\nin our data interpretation.\nReviewer #2 (Remarks to the Author):\n“The manuscript of Ma et al, provides new insights into the modulation of the K2P channel TWIK-\n2 using structural and electrophysiological data. The study provides the first cryo-EM structure of\nthe full-length TWIK-2 at 3.7Å resolution, which is accompanied by mutagenesis studies and the\nuse of a whole-cell patch clamp electrophysiology workflow. The authors show that TWIK-2 is pH-\ninsensitive but slightly voltage-gated and suggest potential different binding modes or\nmechanisms of inhibition in TWIK-2 for two of the four tested K2P modulators (ML365 and NPBA).\nThe work is well executed and well written. However, overall the study fall a bit short. The new\ninsights provided do not represent a big step forward in improving our understanding of how\nTWIK-2 (and K2P channels in general) function and are regulated. The substance and depth of\nthe study could be/have been strengthened by more structure-guided mutagenesis studies, a\nmore robust comparison of their new automated electrophysiology setup, a better literature\ncomparison with (mutagenesis studies of) TWIK-1 and other K2P channels, or even more\nstructural data or structural analysis. While I understand that addressing them all might be out of\nthe scope of this study, the manuscript requires a bit more of depth. Specific suggestion would be\n(major comments):”\nWe sincerely thank the reviewer for the constructive suggestions to increase the depth of our\nwork. We have revised the manuscript accordingly and addressed the changes point-by-point in\nour response below.\n1. In total ‘only’ four residues are identified as different/interesting and tested. Namely M135A,\nT106A, T214A and Y111H. What about other positions, e.g., around the cavity or along the ion\nconductance pathway? Is everything else conserved? What about testing mutating the same sites\nwith other substitutes or using double and triple mutations to gain more insights? Compare in\nmore depth with TWIK-1 and other K2P channels.\nWe agree with the reviewer that the mutant selection was sparse. To address this, we have\nincluded 4 more single mutants in the revised version of the manuscript, focusing on the regions\naround the SF (S213A and Y111A), the K2P modulator pocket (F98A), and hydrophobic cuff\n(M135N). Additionally, we also tested the effect of the T106A/T214A double mutant on TWIK-2\nchannel activity. We have also included an extensive comparison of TWIK-2 and TWIK-1,\nespecially the channel activity, inhibition, and differences in the structure. Lastly, we have\nreformatted the existing comparison with other K2P channels to make it more apparent. Please\nsee the remaining rebuttal, the results, and the discussion sections for more details.\n2. Most striking in this endeavor is the mutant Y111H. The authors show that in contrast to TWIK-\n1, TWIK-2 is pH-insensitive, with the histidine in that position being the pH-sensing residue in\nTWIK-1. While the authors go on and attempt to describe some of the differences observed for\nTWIK-2-Y111H, they never actually tested if this mutant has become pH-sensitive. Considering\nthe overall conservation between TWIK-1 and TWIK-2 this would be expected and highly\ninteresting to test. Further, to test the importance of the tyrosine and decouple it from a pH sensor\nposition, a mutation to another residue, e.g. Y111A and Y111L would be interesting. Finally, the\nfact that TWIK-2_Y111H mutant carries the pH sensor of TWIK-1 must be mentioned in all clarity\nalso in the results and discussion section and taking into account during interpretation.\nWe thank the reviewer for highlighting the importance of Y111H. H122 in TWIK-1, although\nregarded as a major proton-sensing amino acid in the SF1-M2 loop, is not the only determinant\nof pH4,20,22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation, we did not anticipate\nthat it would single-handedly change the pH sensitivity of TWIK-2. In the revised version of the\nmanuscript, as per the reviewer’s suggestion, we also compare the pH-dependency of TWIK-1 in\nour assay setup and test the effect of the TWIK-1-like Y111H mutant on the pH-dependence of\nTWIK-2 channel activity. We find that TWIK-1 is pH-dependent while TWIK-2 is not, despite the\nintroduction of TWIK-1 like Y111H mutation. As per the reviewer’s suggestion, we also tested the\neffect of changing the bulky Y111 to a smaller aliphatic amino acid as alanine, on TWIK-2 channel\nactivity. We found that Y111A was slow deactivating while Y111H was slow activating, compared\nto wild-type TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition.\nPerhaps, to speculate, the role of Y111 in TWIK-2 is maintaining the SF1-M2 loop integrity to\nfacilitate allosteric coupling between the bottom and top of the SF.\n3. I appreciate and highly welcome the high-throughput patch clamp workflow presented by the\nauthors. However, considering i) the discrepancies mentioned by the authors on\nelectrophysiology studies for TWIK-2 in the literature, and ii) the mentioned inconsistencies\nbetween some of their electrophysiology data with that published before, the authors should do a\nmore thorough comparison. If possible, between methods conducted by themselves with the\nsame non-glycosylated constructs (conventional patch clamp and high-throughput patch clamp).\nBut perhaps more importantly, use TWIK-1 in their automated whole-cell patch clamp\nelectrophysiology setup, for which more robust recordings have been reported. This would allow\nus to assess and compare TWIK-1 and TWIK-2 and the different workflows better.\nWe thank the reviewer for these constructive suggestions. While we agree that not all experiments\nperformed using conventional patch clamp can be adopted to a high-throughput automated patch\nclamp approach, screening of inhibitors and testing the relative change in channel activity upon\nmutagenesis has been reliably demonstrated by several groups. As there are already reports that\ncompare high-throughput APC with conventional patch clamp techniques23–25, we prioritized to\ncompare between TWIK-1 and TWIK-2. Using the same expression construct background and\nSyncropatch assay set up, we found that the overall TWIK-1 currents are comparable to TWIK-2.\nHowever, we notice that TWIK-1, unlike TWIK-2, shows pH dependency, albeit only mild. We also\nfind that TWIK-1 is faster activating than TWIK-2 and is more susceptible to inhibition by ML365\nand NPBA compared to TWIK-2. In addition to the comparison of electrophysiological properties\nof TWIK-2 and TWIK-1, in the revised version of the manuscript, we also include a detailed\nstructure comparison at the end of the results section.\n4. The slightly different impact found for two of the tested inhibitors makes the discussion a bit\nambiguous as to their potential different binding mode. I wonder if an attempt to solve an inhibitor-\nbound structure would be in the scope of this study as the authors seem to be able to obtain\nenough stable protein and the processing appears rather straightforward.\nWe agree with the reviewer that the structures of TWIK-2 with ML365 and NPBA could provide\nmore information about their mechanism of binding. We collected and analyzed cryo-EM datasets\nof TWIK-2 with ML365 or NPBA. We observed that in the presence of these ligands, the sample\nappeared to be more heterogeneous, resulting in maps of lower quality compared to the apo\nTWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound TWIK-2 maps\nare fairly similar, with one common feature being the loss of strong tubular lipid-like densities that\nwe observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is possible that loss of tubular\nlipid-like densities is a factor of poor resolution of the map. To make reasonable comparisons, we\nfiltered the apo-TWIK-2 map to ~4Å (Response Fig. 1 above in page 2 of this document).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe\nsome non-protein density immediately below the SF in the case of the NPBA dataset. The non-\nprotein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor\nresolution of the map, and the size and shape of the density, preclude us from modeling NPBA at\nthis site. Hence, we have not included these efforts in the manuscript and are presenting them\nhere in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound structures were not\nfruitful, we employed site-directed mutagenesis to identify a potential NPBA binding site by\nperforming more mutations in the SF, K2P modulator pocket, and around the hydrophobic cuff.\nWe have discussed these in the main manuscript, and our findings suggest that the potential\nbinding site for NPBA, as hinted by additional density in the map, could be below the SF. The\nmutagenesis exploration still does not identify the site for ML365 unambiguously. However, loss\nof strong tubular lipid-like densities around the hydrophobic cuff compared to apo TWIK-2 makes\nus suspect that ML365, like NPBA could bind through the cavity, albeit interacting with different\nresidues compared to NPBA.\nOther major comments:\n5. Can the authors comment on the following: With regards to the processing of the cryo-EM data,\nI wonder if the authors also tested refining the other 3 classes obtained before proceeding with a\nfinal NU-refinement of one of the classes. Considering the authors interpret their structure as an\nintermediate conductive state, I wonder if there were some hints at other conformational states\ncaptured in the data.\nWe tried refining the other 3 classes shown in the penultimate step of data processing pipeline,\nbut the resultant refined maps from these classes were of poor quality and offered no additional\nnew information. Hence, we went ahead with the map denoted as “class 0” for final refinement\nand model building (Response Fig. 4)\nResponse Figure 4: NU-refinements of all the classes post 3D classification.\n6. Could the authors comment why they mutated Y111H to mimic the corresponding TWIK-1\nresidue, but then mutated M135A instead of M135L (which would be the corresponding TWIK-1\nresidue)? What was the rationale/strategy here? And here again, it would have been ideal to have\nTWIK-1 as a control experiment, and to compare Y111H with.\nMethionine, leucine, and alanine are all hydrophobic nonpolar amino acids. Between M135L and\nM135A, the alanine substitution of methionine reduces the size of the side chain drastically\naffecting not only the size of the pocket near M135 but also affecting the relative hydrophobic\nsurface area. As we saw lipid-like densities around M135 amino acid, suspecting their role in\nregulation of channel activity, we anticipated M135A mutation to have drastic effect on TWIK-2\nactivity compared to M135L. In the revised version of the manuscript, we also tested M135N to\nexplore the role of introduction of polar amino acid in this region on TWIK-2 activity. We notice\nthat both M135A and M135N mutants exhibit slightly slower channel activation and show no\ndrastic effect on the channel activity. However, M135A shows a greater loss of NPBA inhibition\ncompared to M135N suggesting that removing the bulky amino acid at this position affects the\nefficacy of NPBA. Similarly, we also tested the effect of changing the pocket around Y111 on\nTWIK-2 channel activity using a Y111A mutant. We found that Y111A was slow deactivating while\nY111H was slow activating, compared to wild-type, like TWIK-2. Both of these mutations caused\na loss of NPBA-mediated inhibition. In the revised version of the manuscript, as per the reviewer’s\nsuggestion, we also compare pH-dependency of TWIK-1 in our assay set up and test the effect of\nTWIK-1 like Y111H mutant on the pH-dependence of TWIK-2 channel activity. We find that\nTWIK-1 is pH-dependent while TWIK-2 is not, despite the introduction of TWIK-1 like Y111H\nmutation.\n7. Could the authors comment/expand on the voltage-dependence of TWIK-2 in the, and whether\nother K2P channels have also been found to be so.\nIn a 2016 study Schewe et al. provided significant insights into the voltage-dependent gating\nmechanisms of K2P channel, challenging the traditional view that voltage-sensing channel\nrequires a canonical voltage-sensing domain. Instead, they proposed the SF to have a “check\nvalve” gating behavior. They also note that this type of voltage-dependency has been observed\nin most K2Ps except TWIK113. Apart from the residues at the SF, voltage-dependent behavior in\nK2Ps, especially the TREK family, is also regulated by other factors such as mechanical force,\nligands, and lipids 14–18. These reports suggest that the same K2P channel can switch between\nvoltage-dependent and voltage-independent current responses depending on their conditions,\nperhaps a regulatory mechanism of certain K2P channels. Because we see that the central cavity\nis occupied by lipid-like densities in our cryo-EM map, and because TWIK-2 has an ability to\ntranslocate between endosomes and plasma membrane and has been proposed to interact with\nother proteins via its C-terminus, we believe it is possible that voltage-dependency in TWIK-2\ncould be a context dependent phenomenon.\n8. While I appreciate the modeling of potential lipids and co for the interpretation and figure\nrendering of non-protein densities, considering the high ambiguity, I expect that the authors have\nno intention of including it in the to be deposited model. (in the era of AlphaFold and machine\nlearning, everything that is deposited can and will be use by AI or others)\nCompared to other K2Ps, the extra non-protein densities of TWIK-2 are a unique feature and may\nbe endogenous molecules involved in channel regulation. As noted in the manuscript, we have\nnot verified the exact nature of these lipid-like molecules, and hence we have modeled\nhydrocarbon tails in these densities. While this manuscript was under revision, two other groups\nreported structures of TWIK-2, and both groups observed tubular “non-protein” densities,\nespecially in the fenestration site and in the central cavity 7,8. The former modeled acyl chains in\nthe fenestration sites, while in the latter manuscript, the authors modeled lipids in these densities\nand proposed a mechanism of lipid-mediated regulation of TWIK-2 channel activity. Encouraged\nby multiple groups, independently, discovering lipid-like tubular densities in TWIK-2, we have\ndecided to deposit the model with hydrocarbon chains modeled into these densities.\nMinor comments:\n- Page 4: \"we identified key amino acids that differentiate TWIK-2 from TWIK-1 and other\nmembers of the K2P family\". From what I understood, only Y111 was a direct comparison with\nTWIK-1, so I suggest toning down this sentence.\nWe thank the reviewer for the correction. We have rephrased it to “We identified key amino acids\nthat regulate TWIK2 channel activity”, as we have included functional analysis of more mutations\nin the revised version of the manuscript.\n- Could the authors add details of this inhibition experiment in the Methods section (e.g. how was\nthe small molecule screening set-up, quantity/volume of compound added, (pre-) incubation time,\netc.). Could the authors show a blank/control measurement with just DMSO to account for the\nbuffer?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the\nautomated patch clamp system and the experimental setup (see above comments to reviewer 1).\nThe SyncroPatch uses 384-well chips with micro-apertures, where negative pressure captures\nindividual cells and forms high-resistance seals. A robotic pipetting unit handles liquids\nautomatically, enabling parallel compound addition, quick solution changes, and precise\nconcentration control, which ensures reliable dose–response measurements. We have updated\nthe Methods section to include more details about the inhibition experiments. Importantly, we\nclarify that the reference (control) buffer always contained the same DMSO concentration as the\ncompound solutions, accounting for any potential nonspecific effects of the solvent. As a result,\nthe vehicle condition was consistently included in all experiments, ensuring that the observed\neffects are due to the test compounds.\n- Methods section: Cloning and overexpression of recombinant TWIK-2: The text should say for\nbetter clarity: \"with an N-terminal Strep-tag-II, followed by a GFP tag and a HRV-3C protease site\"\ninstead of \"followed by an HRV-3C protease site and a GFP tag\" as it is then later stated that 3C\ncleavage results in removal of GFP tag.\nWe thank the reviewer for pointing this out. We have corrected it.\n- As the authors mention in the main text the poor heterologous expression of TWIK channels,\ncould they indicate the yield they obtained from their experiments?\nAs mentioned in the introduction, poor heterologous expression of TWIK channels has been\nreported by a few groups before us 9,12,26. We were able to obtain ~0.4 mg of protein from 1L of\nHEK293 GnTI- cells, after the affinity purification step.\n- Correct typo in: \"Mutations T106A and T214A\"\nWe have rephrased that sentence in the discussion.\nFigures:\n- Please update figure numbers so that they are cited in correct order as mentioned in the text.\nE.g S2 should be called S1; swap order of Figure 4D and 4E\nWe have made sure that the figures are in the correct order.\n- Figure S2F: could the authors explain the uV units on the y-axis of the FSEC curve\n- Specify in main text and S2 figure legend which detergent was used for solubilization of ΔC-\nTWIK-2\n- For better clarity, keep one naming for each construct between figure and figure legends (e.g.\nin figure S2: TWIK-2 is sometimes called WT or wild-type; or ΔC-TWIK-2 (C-ter 1-276) is called\nc-ter 1-276, c-ter and del1-276). In general, could the authors fix all the inconsistencies in the\nfigure legend of S2: missing periods, having a mixture of (n=15) and \",n=15\" etc.\nAs per the other reviewer’s suggestion, we removed the ΔC-TWIK-2 data from Supplementary\nFig. 2 and the legend associated with it in the revised version of the manuscript. We have fixed\nall other inconsistencies.\n- Figure 2A: could the authors improve the illustration so that the ion conduction pathway is clearer\nto the reader?\nWe have remade this figure in current version of our manuscript.\n- Figure S1: indicate the non-conserved M135 and Y111 residues on the sequence alignment\nAll the sites that have been mutated in this study, including M135 and Y111, have now been\nhighlighted in the sequence alignment figure in the current version of our manuscript.\n- Figure S3: FSC curves indicates the mask during refinement is too tight and could be optimized\nWe thank the reviewer for this suggestion. However, the unique architecture of K2P channels\nallows detergent micelles to occupy grooves formed between various secondary structure\nelements in the transmembrane region. Moreover, the detergent that we used for purification is\nDMNG. Neopentyl glycol class of detergents has low CMCs and are known to form\nheterogeneous micelles with varying sizes depending on the effective concentration of the\ndetergent 27. To avoid heterogeneity in micelle size and stochastic detergent densities from\ninterfering with refinement, we had to use a relatively tight mask to reconstruct a map with\ninterpretable features at the protein-micelle interface. We have tried to dilate the mask more, and\nthe resulting structure could not reach as high a resolution as the current map. A relatively tight\nmask is needed to effectively remove the micelle density that is tightly bound to our protein.\n- Could the authors mention/indicate the possible glycosylation sites that TWIK-2 possesses?\nThis could be added to S1.\nWe have now highlighted the glycosylation sites N79 and N85 on the sequence alignment (current\nFigure S2).\nReferences\n1. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination\nand hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Nature 414, 43–\n48 (2001).\n2. Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type\ninactivation in K(+) channels. Nature 466, 203–208 (2010).\n3. Pau, V., Zhou, Y., Ramu, Y., Xu, Y. & Lu, Z. Crystal structure of an inactivated mutant\nmammalian voltage-gated K+ channel. Nat. Struct. Mol. Biol. 24, 857–865 (2017).\n4. Turney, T. S., Li, V. & Brohawn, S. G. Structural Basis for pH-gating of the K+ channel TWIK1\nat the selectivity filter. Nat. Commun. 13, 3232 (2022).\n5. Thomson, A. S. et al. Initial steps of inactivation at the K+ channel selectivity filter. Proc. Natl.\nAcad. Sci. U. S. A. 111, E1713-22 (2014).\n6. Rohaim, A. et al. A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant\nE71V. Nat. Commun. 13, 1574 (2022).\n7. Mondal, A., Niranjan, S. & Minor, D. L., Jr. A lipid plug affects K2P6.1(TWIK-2) function.\nbioRxivorg (2025) doi:10.1101/2025.06.11.659167.\n8. Khanra, N. K., Wang, C., Delgado, B. D. & Long, S. B. Structure of the human TWIK-2\npotassium channel and its inhibition by pimozide. Proc. Natl. Acad. Sci. U. S. A. 122,\ne2425709122 (2025).\n9. Lloyd, E. E., Marrelli, S. P., Namiranian, K. & Bryan, R. M., Jr. Characterization of TWIK-2, a\ntwo-pore domain K+ channel, cloned from the rat middle cerebral artery. Exp. Biol. Med.\n(Maywood) 234, 1493–1502 (2009).\n10. Patel, A. J. et al. TWIK-2, an inactivating 2P domain K+ channel. J. Biol. Chem. 275, 28722–\n28730 (2000).\n11. Chavez, R. A. et al. TWIK-2, a new weak inward rectifying member of the tandem pore\ndomain potassium channel family. J. Biol. Chem. 274, 7887–7892 (1999).\n12. Bobak, N. et al. Recombinant tandem of pore-domains in a Weakly Inward rectifying K+\nchannel 2 (TWIK2) forms active lysosomal channels. Sci. Rep. 7, 649 (2017).\n13. Schewe, M. et al. A non-canonical voltage-sensing mechanism controls gating in K2P K(+)\nchannels. Cell 164, 937–949 (2016).\n14. Brennecke, J. T. & de Groot, B. L. Mechanism of mechanosensitive gating of the TREK-2\npotassium channel. Biophys. J. 114, 1336–1343 (2018).\n15. Harrigan, M. P., McKiernan, K. A., Shanmugasundaram, V., Denny, R. A. & Pande, V. S.\nMarkov modeling reveals novel intracellular modulation of the human TREK-2 selectivity\nfilter. Sci. Rep. 7, 632 (2017).\n16. Yang, F. A gating mechanism of K2P channels by their selectivity filter. J. Mol. Cell Biol. 14,\n(2022).\n17. Rinné, S. et al. The molecular basis for an allosteric inhibition of K+-flux gating in K2P\nchannels. Elife 8, e39476 (2019).\n18. Conrad, L. J., Proks, P. & Tucker, S. J. Effects of ionic strength on gating and permeation of\nTREK-2 K2P channels. PLoS One 16, e0258275 (2021).\n19. Morton, M. J., O’Connell, A. D., Sivaprasadarao, A. & Hunter, M. Determinants of pH sensing\nin the two-pore domain K(+) channels TASK-1 and -2. Pflugers Arch. 445, 577–583 (2003).\n20. Niemeyer, M. I., Cid, L. P., Peña-Münzenmayer, G. & Sepúlveda, F. V. Separate gating\nmechanisms mediate the regulation of K2P potassium channel TASK-2 by intra- and\nextracellular pH. J. Biol. Chem. 285, 16467–16475 (2010).\n21. Hall, P. R. et al. Structures of TASK-1 and TASK-3 K2P channels provide insight into their\ngating and dysfunction in disease. Structure 33, 115-122.e4 (2025).\n22. Lesage, F. et al. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a\nnovel structure. EMBO J. 15, 1004–1011 (1996).\n23. Li, T. et al. High-throughput electrophysiological assays for voltage gated ion channels using\nSyncroPatch 768PE. PLoS One 12, e0180154 (2017).\n24. Seibertz, F. et al. A modern automated patch-clamp approach for high throughput\nelectrophysiology recordings in native cardiomyocytes. Commun. Biol. 5, 969 (2022).\n25. Obergrussberger, A. et al. The suitability of high throughput automated patch clamp for\nphysiological applications. J. Physiol. 600, 277–297 (2022).\n26. Pountney, D. J. et al. Identification and cloning of TWIK-originated similarity sequence\n(TOSS): a novel human 2-pore K+ channel principal subunit. FEBS Lett. 450, 191–196\n(1999).\n27. Lee, H. J., Lee, H. S., Youn, T., Byrne, B. & Chae, P. S. Impact of novel detergents on\nmembrane protein studies. Chem 8, 980–1013 (2022)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have now addressed all of my remaining concern and I recommend for publication", "reviewer_2": "We thank and congratulate the authors for the thorough revision and the inclusion of a significant bulk of new data. The work as gained in substance, providing a solid contribution to the field and merits publication.\nMinor comments to help the authors in the final revision of the manuscript (If taken care of, no need to be checked again by me).\n1. The authors have now included the mutant Y111A alongside the originally used Y111H, which shows some differences in the activation and inhibition profile. However, the differences are not always clearly stated. One example can be found on page 12: \"Moreover, we notice a reduction in maximum inhibition for NPBA in M135A, S213A, and the T106A-T214A double mutants of TWIK-2 (Fig. 4 and Supplementary Fig. 5C).\" However, according to the figures, Y111A/H should also be added in the sentence. In particular Fig S5 shows that Y111A goes from a significant 80% NPBA-inhibition to 40%. When Y111A is added to this statement the subsequent sentences will require revision too: \"These mutants outline a pocket underneath the SF in the central vestibule (Fig. 3A). Based on these observations, we hypothesize that NPBA diffuses through the fenestration site and binds underneath the SF in the central vestibule.”\n2. Adjust statement on top of page 5: \"As we used HEK293 GnTI- cells, which lack N-acetylglucosaminyltransferase I activity, for the overexpression of TWIK-2, glycosylation at these sites is prevented. Glycosylation at these sites may be necessary for stabilization of this loop region\". --> This is not entirely true. GntI- cells limit N-linked glycosylation of expressed glycoproteins to a pattern dominated by the Man GlcNAc N-glycan, and therefore result in a more \"homogenous\" glycosylation of the protein. Hence, better to rephrase that a more complex glycosylation than the shorter and more homogenous produced by GnTI- cells might be the cause.\n3. End of page 7, top of page 8: \"We compared the IC50 values of wild-type TWIK-2 against four mutants: one each at the entry (Y111) and exit (M135) of the ion permeation pore, as well as two at the bottom of selectivity filters SF1 (T106A) and SF2 (T214A).\" --> Please indicate to which residues Y111 and M135 have been mutated into. It is indicated later in the text, so I suspect this is just an oversight.\n4. Correct sentence on Page 8: \"For ML365, the maximum inhibition remained similar to that of wild-type TWIK-2 in all variants, except for the T106A-T214A double mutant, which showed. In the case of ML365, we noticed that the maximum inhibition for all the variants remained similar to wild-type TWIK-2, except the T106A-T214A double mutant, where we saw a significant reduction\"\n5. Page 13: The original conclusion paragraph is missing. Ignore if this was intentional.\n6. Methods section, data processing: Please indicate whether the last refinement was still done with C1symmetry.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank all the reviewers for their critical assessment of our manuscript. Our point-by-point response is below. For purposes of clarity, we have reviewer comments in red italics.\n\nReviewer #1 (Remarks to the Author):\n“This study determines a TWIK2 cryoEM structure and uses a commercial automated patch clamp setup to assess the function of TWIK2 channel mutants and pharmacology. The structure of TWIK2 had not been previously determined and this study provides a first look at the TWIK2 channel architecture. To accompany the structure, the authors perform electrophysiological recordings of TWIK2, a channel that has been notoriously difficult (though not impossible) to record from. Overall, the methodology in the structural and functional work appears to be sound and the writing is clear. “\nWe sincerely thank the reviewer for the overall positive feedback.\n“There are novel observations and interesting results in the manuscript but an informative connection between knowledge learned from the TWIK2 structure and the functional studies performed is largely lacking. The Y111H designed to mimic the TWIK1 pH sensing histidine and the S4 site threonine residues are all positions that did not require a cryoEM structure to be interesting candidates for mutation. While the M135A mutation is a candidate site based on the TWIK2 structure, it is unclear why the authors chose this position as it does not occlude the pore or play any clearly observable role in the TWIK2 structure. The experiments examining the effects of ML365 and NPBA are interesting but somewhat superficial, as the mutants show some effects on channel pharmacology but do not yield any substantive insight into where the drugs might bind or how they might act.”\nWe thank the reviewer for pointing out weaknesses in this manuscript that could be improved. We agree with the reviewer that the mutant selection was sparse. To address this, the analysis in the revised version of the manuscript includes the following single and double mutants: F98A, Y111H, Y111A, T106A, T214A, S213A, M135N, M135A, and T106A-T214A, focusing on the regions near the SF, the K2P modulator pocket (F98A), and the hydrophobic cuff (M135A and M135N). The alanine substitution of M135 (M135A) drastically reduces the size of the side chain, affecting not only the size of the pocket near M135 but also the relative hydrophobic surface area. As we observed lipid-like densities around the M135 amino acid, suggesting their role in regulating channel activity, we anticipated that the M135A mutation would have a drastic effect on TWIK-2 activity. In the revised manuscript, we also tested M135N to explore the role of introducing a polar amino acid in this region on TWIK-2 activity. We observe that both M135A and M135N mutants exhibit slightly slower channel activation, with no drastic effect on channel activity. However, M135A shows a greater loss of NPBA inhibition compared to M135N, suggesting that removing the bulky amino acid at this position affects the efficacy of NPBA. Similarly, we also tested the effect of changing the pocket around Y111 on TWIK-2 channel activity using a Y111A mutant. We found that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition, highlighting the role of Y111 in maintaining SF1-M2 loop integrity.\nTo study the binding modes of ML365 and NPBA, we collected and analyzed cryo-EM data using TWIK-2 complexed with ML365 and NPBA. We observed that in the presence of these ligands, the sample appeared to be more heterogeneous, resulting in maps of lower quality compared to the apo TWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound TWIK-2 maps are fairly similar (~ 4 Å), with one common feature being the loss of strong tubular lipid-like densities that we observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is possible that loss of tubular lipid-like densities is a factor of poor resolution of the map. To make reasonable comparisons, we filtered the apo-TWIK-2 map to 4Å (Response Fig. 1).\nResponse Figure 1: cryo-EM map comparison of TWIK-2 Apo, TWIK-2 ML365 and TWIK-2 NPBA. Two bottom views on the right indicate the occupancy of the cavity close to the selectivity filter (top) and cytoplasmic site (bottom).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe some non-protein density immediately below the SF in the case of the NPBA dataset. The non-protein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor resolution of the map, and the size and shape of the density, preclude us from unambiguously modeling NPBA at this site. Hence, we have not included these efforts in the manuscript and are presenting them here in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound structures were not successful, we employed site-directed mutagenesis to identify a potential NPBA binding site by performing more mutations in the SF, K2P modulator pocket, and around the hydrophobic cuff. We have discussed these in the main manuscript and our findings suggest that the potential binding site for NPBA, as hinted by additional density in the map, could be below the SF. The mutagenesis exploration still does not identify the site for ML365 unambiguously. However, loss of strong tubular lipid-like densities around the hydrophobic cuff compared to apo TWIK-2 makes us suspect that ML365, like NPBA could bind through the cavity, albeit interacting with different residues compared to NPBA.\n“1) In the section entitled “Ion conduction pore of TWIK2”, the authors suggest that the carbonyl oxygens of selectivity filter residues V107 and Y109 face away from the central permeation axis in TWIK-2 and argue for the relevance of this SF orientation to TWIK2 function. Given the ~3.7 angstrom resolution of the overall structure, I cannot ascertain how the authors come to this conclusion. Using the map and model provided by the authors, I can easily model the selectivity filter within the map in a conventional orientation and find that I improve the number of residues in favored orientations when the SF is modeled more conventionally.\nIf there is a biophysical argument to be made that the positioning of the valine and tyrosine sidechains in the TWIK2 map somehow promote an unconventional SF backbone configuration, this should be provided. Simply based on the map provided by the authors, I find this section to be unsupported by experimental results. If the authors do feel their selectivity figure configuration is accurate, a supplemental figure showing how the experimental map is better fit by the outward rather than inward-facing carbonyls at V107 and Y109 would be extremely helpful to make the authors' case on this point.”\nWe thank the reviewer for a thorough review of our structure. In our TWIK-2 structure, the SF has been modeled with carbonyl oxygens of three amino acids – V107, Y109, and L217 – facing away from the ion-conductance pore. While we agree that at ~ 3.7 Å resolution it could be challenging to resolve the orientation of carbonyl oxygens in the dynamic loops of the SF region unambiguously, we would like to highlight that the local resolution for this region is much better than 3.5 Å (Supplementary Fig. S3F in the manuscript). As per the reviewer’s critique, we attempted to model these three carbonyls in various combinations of them facing away or towards the SF. However, just based on visually comparing the fits of these various models to density, it is not straight forward to identity one particular combination of the carbonyl oxygens that fits better to the map (see figures & table below). Hence, based on a comparison of the model statistics such as bond RMSDs, Ramachandran outliers, Rama-Z score, & CaBLAM outliers for the SF region, and the overall model quality statistics for the whole protein, we believe that the model as it has been presented in the manuscript is the best possible fit (Response Table 1 in this rebuttal).\nIn K2P channels, the two conserved threonines at the bottom of the selectivity filter (SF) are the only amino acids within the SF that coordinate K+ ions through their side chains. All other amino acids at the SF coordinate K+ ions through carbonyl oxygens. It has been shown by multiple groups previously that reorientation of the carbonyl oxygens away from the SF is a means to regulate K+ ion flow, including channel inhibition, in potassium channels. Using X-ray crystallography and cryo-EM, several groups have shown that reorientation of carbonyl oxygens away from the SF pore affects the K+ ion coordination at the SF as part of C-type or pH-mediated inactivation of the channel in KcsA, Kv, and TWIK-11–4. The flipped carbonyls often co-existed with reduced occupancy at the canonical K+ ion sites (S0-S4) in the SF. As noted in the manuscript, our cryo-EM map allowed us to confidently model K+ ions in only three out of these five sites. Similarly, MD simulations on the prototypical K+ channels like MthK and KcsA have shown that reorientation of the carbonyl oxygen of the conserved valine at the SF is one of the initial steps that promotes dissociation of K+ from the SF and allows the SF to adopt nonconducting or inactivated states5,6. As pointed out by the reviewer, we had provided only a brief biophysical explanation for non-canonical carbonyl positioning in the discussion in the original manuscript. To address that, we have expanded on the explanation associated with flipped carbonyl observation, by including examples of other eukaryotic K+ channels (TWIK-1, Kv, and Kir) and their prototypic prokaryotic homologs (MthK and KcsA) where similar observations have been made, as a separate section in the discussion, & added more references to this section.\nResponse Figure 2: Various TWIK2 selectivity filter models fit to the cryo-EM density. The model quality statistics for the variations 1-7, and a comparison of the statistics are provided in table 1. Cryo-EM density is highlighted as blue mesh and the three K+ ions as blue spheres. The two protomers, TWIK-2 chain A and B, are colored light magenta and light cyan. All the amino acids at the SF are labeled in variation 7 where none of the carbonyls are flipped away from the SF, and in the remaining panels only the carbonyls that are flipped away from the SF have been labeled.\nResponse Table1: TWIK2 selectivity filter model quality statistics. Selectivity filter model quality statistics were obtained by running PDB validation on the amino acids 104-111 and 213-220, and K+ ions at the SF using Phenix. The values in the parentheses represent the whole PDB. As the resolution of the structure is ~3.7 Å, CaBLAM outliers, Rama-Z score, Bond RMSDs for the SF region, and the overall statistics of the whole PDB were compared to decide on the carbonyl orientations of the amino acids V107, Y109, and L217, which surround S0/S1 and S2/S3 sites at the SF. Each variation (Var. 4-6) or combination of variations are show (Var. 1-3)\nDeposited Parameter Var. 1 Var. 2 Var. 3 Var. 4 Var. 5 Var. 6 Var. 7 9E94\nAmino acids with V107, carbonyl Y109 & V107 & V107 & oxygens flipped L217 Y109 V107 None away from SF L217 L217 Y109 L217\nBond length 0.007 0.014 0.014 0.007 0.017 0.018 0.016 0.017\nRMSD (Å) (0.009) (0.010) (0.009) (0.009) (0.010) (0.010) (0.010) (0.010)\nBond angle 1.617 2.103 2.161 1.724 2.292 2.188 2.265 2.441\nRMSD (o) (1.564) (1.651) (1.624) (1.595) (1.672) (1.662) (1.643) (1.693)\n1.97 1.49 2.01 2.53 2.05 1.97 2.48 2.45\nMolProbity score (1.78) (1.75) (1.78) (1.89) (1.78) (1.82) (1.86) (1.88)\nRamachandran 0.00 0.00 8.33 8.33 0.00 0.00 0.00\noutliers (%) (0.00) (0.00) (0.47) (0.47) (0.00) (0.00) (0.00) 0.00 (0.00)\nRamachandran 8.33 8.33 25.00 41.67 8.33 50.00 58.33 50.00\nallowed (%) (6.07) (6.54) (7.01) (7.94) (6.07) (8.88) (8.88) (8.88)\nRamachandran 91.67 91.67 75.00 50.00 83.33 50.00 41.67 50.00\nfavored (%) (93.93) (93.46) (92.99) (91.59) (93.46) (91.12) (91.12) (91.12)\n-5.32 -6.25 -7.54 -6.18 -7.92 -6.96 -8.91 -9.44\nRama-Z (whole) (-3.75) (-3.79) (-3.86) (-3.78) (-3.90) (-3.81) (-3.93) (-4.00)\nRama-Z (loop) -4.05 -4.76 -5.74 -4.70 -6.03 -5.30 -6.79 -7.19\n(-4.87) (-5.04) (-5.29) (-5.06) (-5.33) (-5.09) (-5.53) (-5.64)\nCaBLAM outliers 25.00 50.00 50.00 37.50 62.50 50.00 62.50 75.00\n(%) (2.88) (3.85) (4.33) (3.37) (4.33) (3.85) (4.81) (4.81)\nClash score 8.81 2.20 4.39 10.96 6.62 2.21 8.81 8.83\n(6.76) (5.91) (6.05) (6.34) (6.34) (5.62) (6.20) (6.63)\n“2) In Figure 2B, the pore radius profile shows a constriction of the pore at around 5A from the cytoplasmic face of the channel. This cytoplasmic constriction does not seem to be discussed in the manuscript. Is this constriction brought about by the lipids modeled in the structure? If so, is this at all relevant to the function of TWIK2? If the authors believe that these lipid densities are important to shaping the TWIK2 pore, then this pore radius plot is appropriate. If the relevance of these lipids is unclear and could simply be an artifact of the protein purification process, it would be better to omit the lipid densities when running HOLE (or include a second HOLE profile in the absence of the lipids) to show the profile of just the TWIK2 protein.\nIt would also be appropriate to discuss the lipid densities present in the map in the results section of the manuscript, rather than the discussion. This would be a good spot to do so.”\nThe pore radius plot in Fig. 2B was constructed using the TWIK-2 model with hydrocarbon tails modeled into the tubular lipid-like densities. We have now edited Fig. 2B to show the pore radius profile with and without the modeled lipid-like densities. We did not include any exogenous lipids during the purification. So, we believe these tubular densities arise from the molecules that co-purified with TWIK-2 from HEK293 GnTI- cells. Because we could not verify the exact nature of these molecules, we have only modeled hydrocarbon tails in these densities and called them lipid-like in the manuscript. The discussion on the lipid-like densities has now been moved to the results section in the revised version of the manuscript, as per the reviewer’s suggestion. While this manuscript was under revision, another group reported a preprint suggesting a potential lipid mediated regulation of TWIK-2 channel activity. They argued that lipid molecules “plug” the channel on the cytoplasmic side by occupying the central cavity and by physically obstructing the passage of ions, rendering TWIK-2 inactive. They demonstrate that R257, an amino acid towards the C-terminus that lies below the hydrophobic cuff, is crucial for this lipid binding in the central cavity and that the R257A mutation increases the channel activity, suggesting that channel activation is a result of lipid removal or repositioning in the central cavity. In our TWIK-2 structure, we observe lipid-like densities in similar positions (central cavity and along the groove formed by the PH2, M2, and M4 helices) as reported by Mondal et al7. However, these lipid-like densities do not entirely obstruct the access to the central pore (Fig. 2). The observation of lipid-like density, corroborated by a different group independently, suggests that these molecules are relevant to the function. We have added a few lines discussing this preprint and its relevance in the context of our structure to the discussion section of the revised manuscript.\n“3) For the experiments where G/GMAX–V curves are calculated, it does not appear that the recordings reach a steady state at 200 ms. The current is still rising for TWIK1 wt and this is even more pronounced in the mutants that slow activation (Figure 3B). Is it possible that the differences in V50 are artificial, since the maximal steady state current is reached at 200ms in some of the mutants but not in all of them.”\nWe appreciate the reviewer’s concerns regarding the possible underestimation of steady-state currents due to incomplete activation at the end of the 200-ms depolarizing pulse, especially in slow-activating TWIK-2 mutants. To reinforce the validity of our conclusions regarding mutant-induced shifts in voltage dependence, we performed a new analysis based on normalized tail currents, which better reflect the relative conductance by capturing the instantaneous current upon repolarization—thus minimizing the effects of kinetic differences in channel activation. Specifically, we constructed revised G/GMAX–V curves using tail currents, enabling a more accurate assessment of the voltage dependence of activation across all variants. From these curves, we calculated the ΔV50 values, which provide a more reliable measure of the voltage shift caused by each mutation. Importantly, this alternative approach produced similar V50 shifts as previously reported, confirming that the observed differences are genuine and not artifacts, reflecting fundamental changes in channel gating properties. These new results are now included in the revised Supplementary Fig. 5 and discussed in the Results section. To enable a better comparison of currents across TWIK-2 variants, we have also modified Fig. 3 to now display whole-cell current traces and activation time constants for all the mutants that we tested.\n“4) I think the manuscript would be greatly improved by a more direct comparison of the TWIK2 structure with the previously published TWIK1 structures. Does this TWIK2 structure look more like the low pH or neutral pH TWIK2 structure? Are there clear differences in the positioning of the cap or TM helices? The entire section of the results devoted to an overview of the TWIK2 channel could be redone as a more direct comparison between TWIK1 and TWIK2 and would significantly increase interest.”\nWe agree with the reviewer that a comprehensive comparison of TWIK-2 with TWIK-1 could be a beneficial addition to the manuscript. In our first version of the manuscript, we had presented a comparison of TWIK-2 and TWIK-1 in Figs. 5B, C and Supplementary Fig. S4, focusing only on the conformational changes in pore domains, SF, and M4 helix. As TWIK-2 is physiologically unlike TWIK-1, for the sake of completeness, we have also included comparisons with other pH-sensitive and lipid-regulated K2P channels. To make these comparisons more apparent, we have reformatted and expanded the figures and text to include a separate comparison section of the TWIK-2 and TWIK-1 structures in their entirety. In addition, we have also included the results from whole-cell recordings studying the pH dependency of channel activity in TWIK-1 and the TWIK-1-like Y111H mutant of TWIK-2. Interestingly, we find that the differences in the structure of TWIK-2 are localized to the cap region when compared with the TWIK-1 structure at pH 7.5. When compared with TWIK-1 at pH 5.5, the differences lie only in the SF region, specifically the SF1-M2 linker that houses the pH-sensing amino acid H122 in TWIK-1. TWIK-2 has the cap region that is similar to inactive TWIK-1, but has the SF loops positioned in a similar way as that of the active TWIK-1, despite the residue Y111 in TWIK-2 (H122 in TWIK-1) adopting a unique ‘up’ conformation. These new observations have been discussed in the revised version of the manuscript, alongside the TWIK-2 and TWIK-1 comparisons from the older version (Fig. 1, 5). The comparison of TWIK-2 with other K2P channels has been reformatted into a separate figure (Supplementary Figs. 4, 7).\nMinor Points\n1. - In the introduction, “Kcnk6 knockout studies have shown suppression of NLRP3 inflammasome activation in macrophages, implicating endolysosomal localization of TWIK-2 in inflammation 45,46.” This sentence is a bit misleading and should be clarified. From the references cited, it is movement of TWIK2 from the endolysosome to the plasma membrane that appears to be a critical step in the potassium efflux that mediates NLRP3 inflammasome activation.\nWe thank the reviewer for the correction. We have now replaced the sentence with “Translocation of TWIK-2 from endosome to plasma membrane is implicated in NLRP3-mediated inflammasome activation in macrophages, with Kcnk6 knockouts showing a suppressed inflammasome activation”.\n2. - In figure 1 (and for nearly all electrophysiological traces shown in the manuscript), recording traces are shown on a signal scale that reads “Normalized I”. It is unclear what the non-transfected cells traces are normalized to? For clarity throughout the manuscript, it would be helpful if the authors explicitly discuss whether normalization was applied using a universal metric (which could be described in the methods section of the manuscript) or ad hoc for each set of recordings (in which case this should be described in each figure legends)\nWe thank the reviewer for pointing out this important issue related to the normalization of electrophysiological traces. In the revised manuscript, all figures now display traces showing actual absolute current values instead of normalized signals. This update allows for direct comparison of current amplitudes across wild-type and mutant channels as well as non-transfected controls. Additionally, we now include current density for each experimental condition where appropriate, ensuring that differences in current amplitudes are not influenced by variations in cell size. We believe these revisions significantly improve the clarity and transparency of our data presentation and address the reviewer’s concern about normalization.\n3- Given that the C-terminally truncated TWIK construct was not used for structure of detailed functional studies, this data could be omitted or moved to the methods section.\nWe agree with this reviewer’s observation that the data on C-terminally truncated TWIK-2 is not informative, beyond suggesting that TWIK-2 devoid of C-terminus is unstable compared to WT. In accordance, we have now removed the C-term truncation data of TWIK-2 from the revised manuscript.\n4 - Is it meaningful that the loop region between V75-P89 is unresolved in the cryo-EM structure? In all other K2P structures, this loop is well resolved. Does this mean that the V75-P89 is dynamic in TWIK2, suggestive of a functional role? Or is this just a limitation of the resolutions of this TWIK2 structure compared to other K2P structures? Does 3D variability analysis in cryosparc give any indication of what is going on in this loop region?\nThe loop V75-P89 is a part of the CH2-PH1 region that harbors two predicted glycosylation sites, N79 and N85. We used HEK293 GnTI- cells, which lack N-acetylglucosaminyltransferase I activity, for overexpression of TWIK-2. So, it is likely that glycosylation at these sites is essential for stabilizing this loop, and without such glycosylation, this loop is flexible and hence poorly resolved in the cryo-EM density map. Interestingly, TWIK-2 structures reported by a different group recently also lacked this region 8. Despite using glycosylation-competent Expi293F cells and achieving an overall higher resolution structure (~2.9 Å). Perhaps, in TWIK-2 this loop is more dynamic than other K2Ps. Another group reported structures of TWIK-2 while this manuscript was under revision 7. They too use glycosylation-capable Expi293F cells as Khanra et. al., but were able to resolve the V75-P89 region in their cryo-EM map. Moreover, they find that amino acids 75-80 in this loop actually form a short helix called ‘ear helix’ that is unique to TWIK-2, proposed to stabilize the TWIK-2 cap domain. As this loop region can also potentially interact with the keystone inhibitor site and the K2P modulator pocket, we attempted to resolve this region using 3DVA and subsequent classification in cryoSPARC (Response Fig. 3). However, our attempts were not successful.\n5- The panel in Figure 3 showing the relative locations of T106, T214, M135, and Y111 is critically important for understanding the orientation and relevance of these residues. I would suggest reworking this figure to make it clearer. The inset boxes are distracting, as each one has a different orientation relative to the main panel. Perhaps a clearly labeled “top view” showing the Y111 residues, “bottom view\" showing the M135 residues, and a side view like shown in the left side of Figure 3A would be sufficient for this.\nWe have modified the figure as per the reviewer’s suggestions.\n- “Notably, TWIK-2 channels elicited outward potassium currents that increased with depolarization (over +30 mV), suggesting voltage-dependent activation of TWIK-2” Isn’t this a surprising result, given that prior recordings in symmetrical potassium have shown inward rectification rather than outward rectification? Is there any methodologic explanation for this difference in observed functional behavior?\nWhile it may seem surprising at first, the voltage-dependent activation of TWIK-2 seen in our recordings agrees with previous studies, especially those indicating that TWIK-2 is one of the least inwardly rectifying members of the K2P family, that inactivates and has a potential role in electrogenesis9–12. Compared to strongly rectifying K2P channels, such as TREK-1 or TASK-3, TWIK-2 exhibits more linear or even outwardly biased I–V relationships under certain recording conditions. Moreover, differences in rectification behavior—particularly outward currents at depolarized potentials—can depend on the expression system (e.g., Xenopus oocytes versus mammalian cells), ionic composition, and intracellular environment (e.g., Mg²⁺, polyamines, or pH). In our experiments, outward currents at voltages above +30 mV are probably caused by a combination of weak inherent rectification and the electrochemical driving force for K⁺ efflux under symmetrical or nearly symmetrical K⁺ conditions, rather than unusual voltage-dependent gating. To clarify this, we added a section in the discussion explaining how our findings align with the known electrophysiological properties of TWIK-2 and reflect system-dependent variability rather than an unexpected gating mechanism.\nResponse Figure 3: 3D variability analysis (3DVA) and subsequent classification to resolve V75-P89 loop. The region shaded in gray is a part of the supplementary figure 3 in the manuscript and has been included here only for reference. Particles from the best cluster displaying all the features of TWIK-2 from 3DVA step was used to generate an ab initio initial reference (cyan box). Next, particles from all clusters with robust cap domain features (red boxes) from the 3DVA step were pooled for clean up by heterogenous refinement and ab initio jobs. The final map obtained after NU-refinement, using the ab-initio reference generated from the best 3DVA cluster, resulted in a map with features and resolution that resembled the map already presented in the manuscript.\n- Reference 70 in the manuscript includes data that describe the effect of mutations at the S4 site threonines in K2Ps. For nearly all K2Ps, these S4 site mutations cause a loss of voltage dependance. It is therefore interesting that in TWIK2, the T106A and T214A mutants maintain their voltage dependent gating. Is there any rationale for why this might be the case?\nThe reference 70 reports that in channels without a dedicated voltage-sensing domain, the SF itself acts as a gate and sensor. They demonstrate that mutating the conserved threonines at the SF to cysteines causes loss of voltage-dependent behavior. However, they also show that in addition to just these mutations, voltage-dependent behavior is also regulated by other factors such as mechanical force, ligands, and lipids13. Regulation of voltage-dependency of K2P channels by these auxiliary factors, apart from the residues at SF, has been shown in several K2P channels, especially the TREK family14–18. These reports suggest that the same K2P channel can switch between voltage-dependent and voltage-independent current responses depending on their conditions, perhaps a regulatory mechanism of certain K2P channels. T106A and T214A mutants display a reduced-voltage dependency in our experiments, but not a complete loss of voltage-dependency. Because we see that the central cavity is occupied by lipid-like densities in our cryo-EM map, and because TWIK-2 has an ability to translocate between endosomes and plasma membrane and has been proposed to interact with other proteins via its C-terminus, we believe it is possible that voltage-dependency in TWIK-2 could be a context dependent phenomenon, in addition to being regulated by the residues at the SF. However, identification of all the factors in TWIK-2 that regulate its channel activity needs furthermore substantial efforts.\n- “Mutations T106A and T214A disturb these networks of hydrogen bonding and hydrophobic interactions that stabilize the base of the SF and make the channel less selective and more open (Figure 3B-3F).” The functional experiments presented in this section use only a single ionic composition and are not suited to adequately address ion selectivity. Please support with data or omit this claim.\nWe agree with the reviewer that with only a single ionic composition, it is hard to address the ion selectivity of TWIK-2. We have rephrased this statement and added the observations from T106A and T214A double mutants’ whole cell recordings to this section. We find that the double mutants display greater current density compared to single mutants and wild-type TWIK-2.\n- Is the TWIK2 Y111H mutant pH sensitive like the TWIK1 channel? This seems like an obvious experiment to try and would be informative to understanding differences between TWIK1 and TWIK2 functions.\nAs already discovered by a few groups, H122 in TWIK-1 and H98 in TASK-3 although regarded as major proton-sensing amino acids in the SF1-M2 loop, are not the only determinants of pH sensitivity in K2P channels4,19–22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation, we did not anticipate that it would single-handedly change the pH sensitivity of TWIK-2. In the revised version of the manuscript, we show that the pH dependency of channel activation and activity of Y111H TWIK-2 is the same as that of wild-type like TWIK-2. Furthermore, we observe that in our assay setup, TWIK-1 showed pH-dependent current changes, albeit mild. These observations have been included in the revised version of the manuscript in a separate figure, and the corresponding text has been modified accordingly.\n- More methodological detail of the automated patch clamp technique would be helpful to understand the experimental system. Are drugs applied to the intracellular face of the channel (via syncropatch pipette) or extracellular via bath?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the automated patch clamp system. To address this, we have expanded the Methods section to include more detailed information about the experimental setup. Unless otherwise noted, all compounds were applied extracellularly using an external addition protocol, following the standard procedures for the SyncroPatch system. The SyncroPatch features a high-speed, multi-channel pipetting robot that manages all liquid additions and removals during the experiments. This robot can simultaneously add various external compounds—such as drugs, ligands, and chemicals—to each of the 384 wells, enabling a large number of experiments to be conducted simultaneously. Additionally, the robot performs quick and efficient solution exchanges in the wells. Although the system permits compound delivery to the intracellular face via an intracellular exchange solution, this study only examined the external application of pharmacological agents to aid in easier application, better ability to perform cumulative concentration-response curves, and for clear pre- and post-drug comparisons. This clarification has been added to the ‘Automated patch clamp recordings’ section within the Materials and Methods, to improve reproducibility and transparency in our data interpretation.\nReviewer #2 (Remarks to the Author):\n“The manuscript of Ma et al, provides new insights into the modulation of the K2P channel TWIK-2 using structural and electrophysiological data. The study provides the first cryo-EM structure of the full-length TWIK-2 at 3.7Å resolution, which is accompanied by mutagenesis studies and the use of a whole-cell patch clamp electrophysiology workflow. The authors show that TWIK-2 is pH-insensitive but slightly voltage-gated and suggest potential different binding modes or mechanisms of inhibition in TWIK-2 for two of the four tested K2P modulators (ML365 and NPBA).\nThe work is well executed and well written. However, overall the study fall a bit short. The new insights provided do not represent a big step forward in improving our understanding of how TWIK-2 (and K2P channels in general) function and are regulated. The substance and depth of the study could be/have been strengthened by more structure-guided mutagenesis studies, a more robust comparison of their new automated electrophysiology setup, a better literature comparison with (mutagenesis studies of) TWIK-1 and other K2P channels, or even more structural data or structural analysis. While I understand that addressing them all might be out of the scope of this study, the manuscript requires a bit more of depth. Specific suggestion would be (major comments):”\nWe sincerely thank the reviewer for the constructive suggestions to increase the depth of our work. We have revised the manuscript accordingly and addressed the changes point-by-point in our response below.\n1. In total ‘only’ four residues are identified as different/interesting and tested. Namely M135A, T106A, T214A and Y111H. What about other positions, e.g., around the cavity or along the ion conductance pathway? Is everything else conserved? What about testing mutating the same sites with other substitutes or using double and triple mutations to gain more insights? Compare in more depth with TWIK-1 and other K2P channels.\nWe agree with the reviewer that the mutant selection was sparse. To address this, we have included 4 more single mutants in the revised version of the manuscript, focusing on the regions around the SF (S213A and Y111A), the K2P modulator pocket (F98A), and hydrophobic cuff (M135N). Additionally, we also tested the effect of the T106A/T214A double mutant on TWIK-2 channel activity. We have also included an extensive comparison of TWIK-2 and TWIK-1, especially the channel activity, inhibition, and differences in the structure. Lastly, we have reformatted the existing comparison with other K2P channels to make it more apparent. Please see the remaining rebuttal, the results, and the discussion sections for more details.\n2. Most striking in this endeavor is the mutant Y111H. The authors show that in contrast to TWIK-1, TWIK-2 is pH-insensitive, with the histidine in that position being the pH-sensing residue in TWIK-1. While the authors go on and attempt to describe some of the differences observed for TWIK-2-Y111H, they never actually tested if this mutant has become pH-sensitive. Considering the overall conservation between TWIK-1 and TWIK-2 this would be expected and highly interesting to test. Further, to test the importance of the tyrosine and decouple it from a pH sensor position, a mutation to another residue, e.g. Y111A and Y111L would be interesting. Finally, the fact that TWIK-2_Y111H mutant carries the pH sensor of TWIK-1 must be mentioned in all clarity also in the results and discussion section and taking into account during interpretation.\nWe thank the reviewer for highlighting the importance of Y111H. H122 in TWIK-1, although regarded as a major proton-sensing amino acid in the SF1-M2 loop, is not the only determinant of pH4,20,22. While the Y111H mutant of TWIK-2 is a TWIK-1-like mutation, we did not anticipate that it would single-handedly change the pH sensitivity of TWIK-2. In the revised version of the manuscript, as per the reviewer’s suggestion, we also compare the pH-dependency of TWIK-1 in our assay setup and test the effect of the TWIK-1-like Y111H mutant on the pH-dependence of TWIK-2 channel activity. We find that TWIK-1 is pH-dependent while TWIK-2 is not, despite the introduction of TWIK-1 like Y111H mutation. As per the reviewer’s suggestion, we also tested the effect of changing the bulky Y111 to a smaller aliphatic amino acid as alanine, on TWIK-2 channel activity. We found that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition. Perhaps, to speculate, the role of Y111 in TWIK-2 is maintaining the SF1-M2 loop integrity to facilitate allosteric coupling between the bottom and top of the SF.\n3. I appreciate and highly welcome the high-throughput patch clamp workflow presented by the authors. However, considering i) the discrepancies mentioned by the authors on electrophysiology studies for TWIK-2 in the literature, and ii) the mentioned inconsistencies between some of their electrophysiology data with that published before, the authors should do a more thorough comparison. If possible, between methods conducted by themselves with the same non-glycosylated constructs (conventional patch clamp and high-throughput patch clamp). But perhaps more importantly, use TWIK-1 in their automated whole-cell patch clamp electrophysiology setup, for which more robust recordings have been reported. This would allow us to assess and compare TWIK-1 and TWIK-2 and the different workflows better.\nWe thank the reviewer for these constructive suggestions. While we agree that not all experiments performed using conventional patch clamp can be adopted to a high-throughput automated patch clamp approach, screening of inhibitors and testing the relative change in channel activity upon mutagenesis has been reliably demonstrated by several groups. As there are already reports that compare high-throughput APC with conventional patch clamp techniques23–25, we prioritized to compare between TWIK-1 and TWIK-2. Using the same expression construct background and Syncropatch assay set up, we found that the overall TWIK-1 currents are comparable to TWIK-2. However, we notice that TWIK-1, unlike TWIK-2, shows pH dependency, albeit only mild. We also find that TWIK-1 is faster activating than TWIK-2 and is more susceptible to inhibition by ML365 and NPBA compared to TWIK-2. In addition to the comparison of electrophysiological properties of TWIK-2 and TWIK-1, in the revised version of the manuscript, we also include a detailed structure comparison at the end of the results section.\n4. The slightly different impact found for two of the tested inhibitors makes the discussion a bit ambiguous as to their potential different binding mode. I wonder if an attempt to solve an inhibitor-bound structure would be in the scope of this study as the authors seem to be able to obtain enough stable protein and the processing appears rather straightforward.\nWe agree with the reviewer that the structures of TWIK-2 with ML365 and NPBA could provide more information about their mechanism of binding. We collected and analyzed cryo-EM datasets of TWIK-2 with ML365 or NPBA. We observed that in the presence of these ligands, the sample appeared to be more heterogeneous, resulting in maps of lower quality compared to the apo TWIK-2 map reported in this manuscript. The resolutions of both the inhibitor-bound TWIK-2 maps are fairly similar, with one common feature being the loss of strong tubular lipid-like densities that we observed in apo-TWIK-2 near the hydrophobic cuff. Of course, it is possible that loss of tubular lipid-like densities is a factor of poor resolution of the map. To make reasonable comparisons, we filtered the apo-TWIK-2 map to ~4Å (Response Fig. 1 above in page 2 of this document).\nWhile we do not see any additional non-protein density in the ML365 dataset, we do observe some non-protein density immediately below the SF in the case of the NPBA dataset. The non-protein density that we see in the NPBA dataset is not seen in the apo TWIK-2; however, the poor resolution of the map, and the size and shape of the density, preclude us from modeling NPBA at this site. Hence, we have not included these efforts in the manuscript and are presenting them here in the rebuttal. As the efforts to obtain high-resolution inhibitor-bound structures were not fruitful, we employed site-directed mutagenesis to identify a potential NPBA binding site by performing more mutations in the SF, K2P modulator pocket, and around the hydrophobic cuff. We have discussed these in the main manuscript, and our findings suggest that the potential binding site for NPBA, as hinted by additional density in the map, could be below the SF. The mutagenesis exploration still does not identify the site for ML365 unambiguously. However, loss of strong tubular lipid-like densities around the hydrophobic cuff compared to apo TWIK-2 makes us suspect that ML365, like NPBA could bind through the cavity, albeit interacting with different residues compared to NPBA.\nOther major comments:\n5. Can the authors comment on the following: With regards to the processing of the cryo-EM data, I wonder if the authors also tested refining the other 3 classes obtained before proceeding with a final NU-refinement of one of the classes. Considering the authors interpret their structure as an intermediate conductive state, I wonder if there were some hints at other conformational states captured in the data.\nWe tried refining the other 3 classes shown in the penultimate step of data processing pipeline, but the resultant refined maps from these classes were of poor quality and offered no additional new information. Hence, we went ahead with the map denoted as “class 0” for final refinement and model building (Response Fig. 4)\nResponse Figure 4: NU-refinements of all the classes post 3D classification.\n6. Could the authors comment why they mutated Y111H to mimic the corresponding TWIK-1 residue, but then mutated M135A instead of M135L (which would be the corresponding TWIK-1 residue)? What was the rationale/strategy here? And here again, it would have been ideal to have TWIK-1 as a control experiment, and to compare Y111H with.\nMethionine, leucine, and alanine are all hydrophobic nonpolar amino acids. Between M135L and M135A, the alanine substitution of methionine reduces the size of the side chain drastically affecting not only the size of the pocket near M135 but also affecting the relative hydrophobic surface area. As we saw lipid-like densities around M135 amino acid, suspecting their role in regulation of channel activity, we anticipated M135A mutation to have drastic effect on TWIK-2 activity compared to M135L. In the revised version of the manuscript, we also tested M135N to explore the role of introduction of polar amino acid in this region on TWIK-2 activity. We notice that both M135A and M135N mutants exhibit slightly slower channel activation and show no drastic effect on the channel activity. However, M135A shows a greater loss of NPBA inhibition compared to M135N suggesting that removing the bulky amino acid at this position affects the efficacy of NPBA. Similarly, we also tested the effect of changing the pocket around Y111 on TWIK-2 channel activity using a Y111A mutant. We found that Y111A was slow deactivating while Y111H was slow activating, compared to wild-type, like TWIK-2. Both of these mutations caused a loss of NPBA-mediated inhibition. In the revised version of the manuscript, as per the reviewer’s suggestion, we also compare pH-dependency of TWIK-1 in our assay set up and test the effect of TWIK-1 like Y111H mutant on the pH-dependence of TWIK-2 channel activity. We find that TWIK-1 is pH-dependent while TWIK-2 is not, despite the introduction of TWIK-1 like Y111H mutation.\n7. Could the authors comment/expand on the voltage-dependence of TWIK-2 in the, and whether other K2P channels have also been found to be so.\nIn a 2016 study Schewe et al. provided significant insights into the voltage-dependent gating mechanisms of K2P channel, challenging the traditional view that voltage-sensing channel requires a canonical voltage-sensing domain. Instead, they proposed the SF to have a “check valve” gating behavior. They also note that this type of voltage-dependency has been observed in most K2Ps except TWIK113. Apart from the residues at the SF, voltage-dependent behavior in K2Ps, especially the TREK family, is also regulated by other factors such as mechanical force, ligands, and lipids 14–18. These reports suggest that the same K2P channel can switch between voltage-dependent and voltage-independent current responses depending on their conditions, perhaps a regulatory mechanism of certain K2P channels. Because we see that the central cavity is occupied by lipid-like densities in our cryo-EM map, and because TWIK-2 has an ability to translocate between endosomes and plasma membrane and has been proposed to interact with other proteins via its C-terminus, we believe it is possible that voltage-dependency in TWIK-2 could be a context dependent phenomenon.\n8. While I appreciate the modeling of potential lipids and co for the interpretation and figure rendering of non-protein densities, considering the high ambiguity, I expect that the authors have no intention of including it in the to be deposited model. (in the era of AlphaFold and machine learning, everything that is deposited can and will be use by AI or others)\nCompared to other K2Ps, the extra non-protein densities of TWIK-2 are a unique feature and may be endogenous molecules involved in channel regulation. As noted in the manuscript, we have not verified the exact nature of these lipid-like molecules, and hence we have modeled hydrocarbon tails in these densities. While this manuscript was under revision, two other groups reported structures of TWIK-2, and both groups observed tubular “non-protein” densities, especially in the fenestration site and in the central cavity 7,8. The former modeled acyl chains in the fenestration sites, while in the latter manuscript, the authors modeled lipids in these densities and proposed a mechanism of lipid-mediated regulation of TWIK-2 channel activity. Encouraged by multiple groups, independently, discovering lipid-like tubular densities in TWIK-2, we have decided to deposit the model with hydrocarbon chains modeled into these densities.\nMinor comments:\n- Page 4: \"we identified key amino acids that differentiate TWIK-2 from TWIK-1 and other members of the K2P family\". From what I understood, only Y111 was a direct comparison with TWIK-1, so I suggest toning down this sentence.\nWe thank the reviewer for the correction. We have rephrased it to “We identified key amino acids that regulate TWIK2 channel activity”, as we have included functional analysis of more mutations in the revised version of the manuscript.\n- Could the authors add details of this inhibition experiment in the Methods section (e.g. how was the small molecule screening set-up, quantity/volume of compound added, (pre-) incubation time, etc.). Could the authors show a blank/control measurement with just DMSO to account for the buffer?\nWe appreciate the reviewer's request for clarification regarding the delivery of compounds in the automated patch clamp system and the experimental setup (see above comments to reviewer 1). The SyncroPatch uses 384-well chips with micro-apertures, where negative pressure captures individual cells and forms high-resistance seals. A robotic pipetting unit handles liquids automatically, enabling parallel compound addition, quick solution changes, and precise concentration control, which ensures reliable dose–response measurements. We have updated the Methods section to include more details about the inhibition experiments. Importantly, we clarify that the reference (control) buffer always contained the same DMSO concentration as the compound solutions, accounting for any potential nonspecific effects of the solvent. As a result, the vehicle condition was consistently included in all experiments, ensuring that the observed effects are due to the test compounds.\n- Methods section: Cloning and overexpression of recombinant TWIK-2: The text should say for better clarity: \"with an N-terminal Strep-tag-II, followed by a GFP tag and a HRV-3C protease site\" instead of \"followed by an HRV-3C protease site and a GFP tag\" as it is then later stated that 3C cleavage results in removal of GFP tag.\nWe thank the reviewer for pointing this out. We have corrected it.\n- As the authors mention in the main text the poor heterologous expression of TWIK channels, could they indicate the yield they obtained from their experiments?\nAs mentioned in the introduction, poor heterologous expression of TWIK channels has been reported by a few groups before us 9,12,26. We were able to obtain ~0.4 mg of protein from 1L of HEK293 GnTI- cells, after the affinity purification step.\n- Correct typo in: \"Mutations T106A and T214A\"\nWe have rephrased that sentence in the discussion.\nFigures:\n- Please update figure numbers so that they are cited in correct order as mentioned in the text. E.g S2 should be called S1; swap order of Figure 4D and 4E\nWe have made sure that the figures are in the correct order.\n- Figure S2F: could the authors explain the uV units on the y-axis of the FSEC curve\n- Specify in main text and S2 figure legend which detergent was used for solubilization of ΔC-TWIK-2\n- For better clarity, keep one naming for each construct between figure and figure legends (e.g. in figure S2: TWIK-2 is sometimes called WT or wild-type; or ΔC-TWIK-2 (C-ter 1-276) is called c-ter 1-276, c-ter and del1-276). In general, could the authors fix all the inconsistencies in the figure legend of S2: missing periods, having a mixture of (n=15) and \",n=15\" etc.\nAs per the other reviewer’s suggestion, we removed the ΔC-TWIK-2 data from Supplementary Fig. 2 and the legend associated with it in the revised version of the manuscript. We have fixed all other inconsistencies.\n- Figure 2A: could the authors improve the illustration so that the ion conduction pathway is clearer to the reader?\nWe have remade this figure in current version of our manuscript.\n- Figure S1: indicate the non-conserved M135 and Y111 residues on the sequence alignment\nAll the sites that have been mutated in this study, including M135 and Y111, have now been highlighted in the sequence alignment figure in the current version of our manuscript.\n- Figure S3: FSC curves indicates the mask during refinement is too tight and could be optimized\nWe thank the reviewer for this suggestion. However, the unique architecture of K2P channels allows detergent micelles to occupy grooves formed between various secondary structure elements in the transmembrane region. Moreover, the detergent that we used for purification is DMNG. Neopentyl glycol class of detergents has low CMCs and are known to form heterogeneous micelles with varying sizes depending on the effective concentration of the detergent 27. To avoid heterogeneity in micelle size and stochastic detergent densities from interfering with refinement, we had to use a relatively tight mask to reconstruct a map with interpretable features at the protein-micelle interface. We have tried to dilate the mask more, and the resulting structure could not reach as high a resolution as the current map. A relatively tight mask is needed to effectively remove the micelle density that is tightly bound to our protein.\n- Could the authors mention/indicate the possible glycosylation sites that TWIK-2 possesses? This could be added to S1.\nWe have now highlighted the glycosylation sites N79 and N85 on the sequence alignment (current Figure S2).\nReferences\n1. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel–Fab complex at 2.0 Å resolution. Nature 414, 43–48 (2001).\n2. Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type inactivation in K(+) channels. Nature 466, 203–208 (2010).\n3. Pau, V., Zhou, Y., Ramu, Y., Xu, Y. & Lu, Z. Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel. Nat. Struct. Mol. Biol. 24, 857–865 (2017).\n4. Turney, T. S., Li, V. & Brohawn, S. G. Structural Basis for pH-gating of the K+ channel TWIK1 at the selectivity filter. Nat. Commun. 13, 3232 (2022).\n5. Thomson, A. S. et al. Initial steps of inactivation at the K+ channel selectivity filter. Proc. Natl. Acad. Sci. U. S. A. 111, E1713-22 (2014).\n6. Rohaim, A. et al. 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