Structural mechanisms for inhibition and activation of human small-conductance Ca^2+-activated potassium channel SK2

Domain: physics
Published: 2026-01-15
Editorial outcome: minor_revision

Abstract
The small-conductance calcium-activated potassium (SK1-3 or KCa2) channels regulate the intrinsic excitability and firing frequency of excitable cells. SK channels are modulated by a variety of distinct modulators; however, the underlying mechanisms remain elusive. Here, we present four cryo-electron microscopy structures of the human SK2-calmodulin complex bound with apamin, UCL1684, AP30663, and CAD-1883, elucidating their distinct binding sites and regulatory mechanisms. Apamin and UCL1684 compete for a similar binding site above the selectivity filter, which is formed by the distinct S3-S4 linker of SK2. CAD-1883 glues the N-lobe of calmodulin and the S4-S5 linker of SK2, reinforcing the open state. In contrast, AP30663 resides in the central cavity of SK2, blocking ion conductance. This study reveals multiple modulation sites in SK2 and the molecular mechanisms for the inhibition and potentiation of SK channels, which could advance rational drug design targeting SK2 channel for the treatment of cardiovascular and neurological disorders.

Introduction
The small-conductance calcium-activated potassium (SK) channels play pivotal roles in regulating cellular excitability and calcium signaling in many tissues by governing the medium after-hyperpolarization (mAHP) phase of action potentials 1–4. SK channels are involved in numerous physiological activities, including synaptic plasticity, learning and memory, and cardiac pacemaker control 5–7. The SK channel family comprises three small-conductance channels, SK1 (KCa2.1, encoded by KCNN1), SK2 (KCa2.2, encoded by KCNN2), and SK3 (KCa2.3, encoded by KCNN3) with a single-channel conductance of ~10pS 8,9, and one intermediate-conductance channel, SK4 (IK1 or KCa3.1, encoded by KCNN4) with a single-channel conductance of ~40pS 10,11. SK1-3 share a high degree of amino acid identity of over 80%, whereas they are less than 48% identical to SK4. SK1-3 are widely expressed in central and peripheral neurons, but SK4 is largely absent in brain neurons 9,12. Despite these differences, SK channels are solely activated by submicromolar Ca2+ via calmodulin (CaM), which constitutively binds to the intracellular calmodulin-binding domain (CaM-BD) of the channel 13,14. The structural study of SK4 has revealed the overall homotetrameric architecture and the Ca2+-activation mechanism 15; that is, the C-lobe of CaM permanently binds to the CaM-BD of SK4, whereas the CaM N-lobe is flexible without Ca2+; after sensing and binding to Ca2+, the N-lobe moves to the S4–S5 linker and pulls the pore-lining S6 to open the gate. Consistent with this mechanism, recent structural studies of rat SK2/CaM 16 and human chimeric SK2-4/CaM 17 revealed similar architecture and assembly to that of the SK4/CaM complex.

The peptide neurotoxin apamin from bee venom is considered the most selective and potent blocker of SK1-3, which has been widely used as an invaluable tool for investigating SK channel properties 6,18–20. Apamin preferentially blocks SK2 over SK1 and SK3 with at least 10-fold higher affinity and, remarkably, is ineffective on SK4 8,10,11,21,22. Mutagenesis studies suggest that apamin sensitivity is largely determined by several residues in the outer vestibule 23–26. Moreover, d-tubocurarine, dequalinium and its bis-quinolinium cyclophane derivatives UCL1684 and UCL1848 also block SK1-3 via occupation of the apamin site 23,27–30. However, the precise binding site of apamin in SK channels and its mechanism of action remain undefined.

Because of their physiological importance, SK1-3 have been implicated as attractive therapeutic targets for the treatment of atrial fibrillation, ataxia, epilepsy, anxiety disorders, and neurodegenerative diseases 1,3,4,31,32. Many small-molecule modulators that alter SK channel functions are emerging 32, several of which have advanced to clinical trials to explore their therapeutic potential. For instance, AP30663, a novel nonselective SK1-3 negative modulator 33, has recently completed Phase I and Phase II clinical trials to evaluate its efficacy in treating atrial fibrillation 34–36, demonstrating positive results. Moreover, CAD-1883 (also known as rimtuzalcap), a positive modulator of SK channels, has undergone two Phase II clinical trials to investigate its potential to alleviate movement disorders, including spinocerebellar ataxia (Phase II: NCT04301284) and essential tremor (Phase II: NCT03688685). These clinical investigations highlight the growing interest in SK channels as therapeutic targets. Previous structural analysis suggested that SK channel activators such as NS309 and riluzole bind to the interface of CaM and the isolated CaM-BD of SK2 37–39; however, this model contradicts the binding mode of CaM revealed by the full-length SK4/CaM structure 15. Therefore, the mechanisms of action for the inhibition and potentiation of SK channels by these clinical compounds are unknown, and the lack of the desired SK-drug complex structure hampers further optimization of these compounds to achieve better efficacy.

In this work, we have solved four cryoelectron microscopy (cryo-EM) structures of the human SK2-calmodulin complex bound with apamin, UCL1684, CAD-1883, and AP30663, respectively, revealing three distinct modulation sites in SK2 and their detailed modes of action. Augmented with mutagenesis studies and electrophysiological functional analysis, this work revealed that apamin sensitivity is endowed by the distinct S3–S4 linker of SK2 and provides the structural basis for the activation, inhibition, potentiation, and pharmacology of SK channels, which opens new avenues for precise therapeutic development to treat a variety of SK channel-related diseases.

Results
Structures of SK2–CaM bound with modulators
To explore the structural pharmacology of SK2, we purified human wild-type (WT) full-length SK2 coexpressed with human CaM in HEK293F cells and analyzed the detergent-purified samples by cryo-EM. However, the human WT SK2 exhibited structural heterogeneity in the intracellular domain, which hindered high-resolution structure determination. After optimization, we found that the addition of 2 mM Ca2+ and excess purified CaM substantially improved the sample quality of human SK2/CaM, as reflected by the association of CaM with SK2 through purification (Supplementary Fig. 1a, b). We next performed cryo-EM single-particle analysis of the purified samples in the presence of apamin, UCL1684, CAD-1883 or AP30663 and determined the SK2-CaM structures in complex with CAD-1883 (SK2-CAD-1883), apamin (SK2-apamin), UCL1684 (SK2-UCL1684), and AP30663 (SK2-AP30663) at resolutions of 3.35 Å, 3.23 Å, 2.96 Å, and 3.34 Å, respectively (Supplementary Figs. 1–4). The density maps permit reliable model building of the SK2 TM core region for all four structures and the placement of the respective modulators; however, CaM can be assigned only to the activator-bound SK2-CAD-1883, whereas the C-lobe of CaM is partly flexible, and the N-lobe is completely invisible in the other three structures (Fig. 1a, b, Supplementary Fig. 5), indicating that SK2-CAD-1883 adopts an open conformation, whereas SK2-apamin, SK2-UCL1684, and SK2-AP30663 are in a closed state. Because the CAD-1883-bound structure contains a more complete model for both SK2 and CaM than the other three structures do, it was selected for subsequent structural description and comparison.

The overall structure of SK2-CaM resembles the SK4-CaM structure (PDB ID: 6CNN) 15 with a root mean square deviation (r.m.s.d.) of 2.6 Å between 1833 aligned Cα atoms. The homotetrameric SK2 is organized in a domain nonswapped manner, and each protomer comprises a voltage sensor-like domain (S1–S4) and a pore domain (S5–S6), which are connected by the S4–S5 linker consisting of two short α helices (SA and SB). The pore lining S6 is extended into two long, chopstick-like HA and HB helices that are parallel to the membrane plane. Notably, the hairpin-like S3–S4 linker is clearly resolved and points to the central axis, four of which together constitute the outer vestibule of SK2 (Fig. 1b, c, Supplementary Figs. 2f, 3d). The C-lobe of CaM holds onto the HA helix of one protomer, whereas the N-lobe engages with the SA helix of the opposing protomer (Fig. 1b, c), which is consistent with the observations in the Ca2+-bound SK4–CaM structure 15.

The superposition of the SK2 and SK4 structures revealed that the TM core regions closely resemble each other, but the loop regions exhibited marked structural differences. In particular, the S3–S4 linker of SK2 forms a pair of antiparallelly arranged β sheets; in contrast, the S3–S4 linker of SK4 is unstructured and invisible to cryo-EM (Fig. 1d). The outer vestibule formed by these linkers in SK2 plays a vital role in apamin binding, as shown below. Additionally, S5 and S6 of SK2 are covalently linked by a disulfide bond between C332 and C370, which is absent in SK4 (Fig. 1d). Furthermore, an extra density in the space between S4 and S6 of SK2 suggests the binding of a possible cholesterol molecule (Fig. 1d), whereas a detergent-like molecule binds to SK4 15. Interestingly, the density maps at the equivalent position of the three inhibitor-bound closed SK2 structures fit a possible phospholipid, likely because of local conformational shifts between the open and closed SK2 structures (Supplementary Fig. 4h, i).

Structural basis for SK2 blockade by apamin and UCL1684
Apamin is an 18-residue peptide neurotoxin that can potently and selectively inhibit SK2 8,21. Structural analysis revealed that apamin consists of an N-terminal loop and a C-terminal helix 40–42, which are linked by two cysteine disulfide bonds, Cys1-Cys11 and Cys3-Cys15 (Fig. 2a). Our whole-cell patch-clamp recording results revealed that apamin gradually reduced SK2 currents, resulting in an IC50 of 120.6 ± 29.2 pM (Fig. 2b), which is comparable to the results of previous studies 8,21,22,26.

A large piece of strong density was observed in the outer vestibule of SK2-apamin but not in SK2-CAD-1883 or SK2-AP30663, which fits the entire C-terminal helix and partly covers the N-terminal loop of apamin, demonstrating the binding of one apamin to the tetrameric SK2 (Figs. 1a, 2c, Supplementary Fig. 5a, f). Because of this configuration, we only imposed C1 symmetry during density map refinements. However, the poor density of the N-loop is more likely due to its intrinsic flexibility, not the use of C1 symmetry (Supplementary Figs. 1e, 5a). The outer vestibule of SK2 has a negatively charged surface potential that matches the positive side of apamin, reinforcing their strong interactions (Supplementary Fig. 5c). The SK2-apamin structure clearly elucidates that apamin binding completely blocks the ion pathway from the extracellular side.

Apamin is tightly embraced by the four S3–S4 linkers, which are flexible in SK4, explaining why SK4 is insensitive to apamin (Figs. 1d, 2d). The long antiparallel S3–S4 linkers are stabilized by extensive interactions. Specifically, R240 participates in electrostatic interactions with the backbone carbonyl oxygens of Q339 and D340 from the neighboring protomer; Y245 interacts with the backbone carbonyl of A242 (Fig. 2e). In addition, H336 of the pore loop forms a hydrogen bond interaction with S248, and van der Waals contacts with W237; N367 of the pore loop engages in polar interactions with the backbone nitrogen of A242 (Fig. 2e). We speculate that these specific interactions stabilize the conformation of the S3–S4 linker, providing a proper chemical and geometric environment for the binding of apamin. Consistent with this hypothesis, although H336 and N367 have no direct contact with the bound apamin in the SK2-apamin structure, previous studies have shown that mutating H337 at the equivalent position in rat SK2 with Asn or Arg greatly impaired apamin potency 23,25. The nonconserved N367 is substituted by His in SK1 and SK3 (Supplementary Fig. 6a). We showed that 300 nM apamin inhibited the H336N and N367H mutants by only ~20% and ~60%, respectively (Fig. 2g, h, Supplementary Fig. 7h). We further generated the R240A, A242F, and Y245A mutations and investigated the inhibitory effects of apamin on these functional mutants (Supplementary Fig. 7h). Although these residues are not involved in apamin binding, A242F is insensitive to 300 nM apamin, and the apamin dose–response curves revealed IC50 values of 11.8 ± 4.5 nM in R240A and 3.7 ± 1.2 nM in Y245A, which are approximately 98- and 31-fold less potent than that in WT SK2, respectively (Fig. 2g, h). The AlphaFold2 43 models suggest that the S3–S4 linkers of SK1-3 adopt a similar fold to that of our SK2 structure. We speculate that several nonconserved residues, including S244 in the S3–S4 linker and N367 in the pore loop, could affect the conformation of the S3–S4 linker, which is crucial for apamin binding, resulting in the apamin sensitivity gap in SK1-3.

A closer look at the apamin binding site revealed that Arg13 and Arg14 of apamin stick their long side chains into the space between two adjacent F243 of the S3–S4 linkers, forming cation‒π interactions with F243 and electrostatic interactions with the backbone carbonyl oxygens of G362 and D363 from the selectivity filter; meanwhile, Leu10, Gln17 and His18 interact with the channel and contribute to apamin binding (Fig. 2d). This observation coincides well with the fact that Arg13 and Arg14 are critical for apamin binding and toxic activity 44. In addition, apamin also makes close contacts with S244 of the S3–S4 linker (Fig. 2e). To validate these structural findings, we generated the S244F and F243A mutants and assayed their response to apamin. Both the S244F and F243A mutants elicited currents comparable to those of WT SK2; however, the F243A mutant was almost completely insensitive to 300 nM apamin, and the S244F was inhibited only approximately 40% by 300 nM apamin (Fig. 2g, h, Supplementary Fig. 7h). Of note, S244 is not conserved in SK1-3 and is replaced by T216 in human SK1. Previous studies demonstrated that mutating S245 at the equivalent position in rat SK2 with a Thr impaired apamin potency by ~7.5-fold and that the T216S mutation in human SK1 increased the apamin sensitivity of human SK1 24,26, indicating that S244 is one of the determinants contributing to the differences in the potency of apamin in SK1-3.

Organic compounds such as d-tubocurarine and dequalinium have been shown to competitively displace apamin binding 23,27,32. We assayed the dose-dependent inhibition of SK2 by the high-affinity bis-quinolinium cyclophane derivative UCL1684, which yielded an IC50 of 3.1 ± 1.1 nM (Supplementary Fig. 7a, b), in line with the findings of a previous study 29. To elucidate the relationship between apamin and UCL1684 binding in SK2, we determined the SK2-CaM structure in complex with UCL1684 (Supplementary Figs. 2, 7c). Similar to SK2-apamin, strong nonproteinaceous density appears in the outer vestibule of SK2-UCL1684, which could accommodate one UCL1684 molecule (Supplementary Fig. 7c, d). UCL1684 is situated in the outer vestibule, where it forms extensive contacts, mainly with the four F243 residues (Fig. 2f). The binding of UCL1684 fully occludes the ion path, reminiscent of the action of apamin (Fig. 2d, f). The superposition of SK2-apamin and SK2-UCL1684 revealed that UCL1684 binds deeper in SK2 than does apamin, and interestingly, the two positive quinolinium rings are positioned similarly to Arg13 and Arg14 of apamin (Supplementary Fig. 7e, f). This observation implies that the two shared positive rings in d-tubocurarine, dequalinium and the bis-quinolinium cyclophane derivatives play critical roles in inhibiting SK channels. Our mutagenesis results revealed that F243A and H336N greatly impaired the efficacy of 1 μM UCL1684, similar to their response to 300 nM apamin; however, the A242F, S244F, Y245A and N367H mutants could still be largely inhibited by 1 μM UCL1684, whereas their sensitivity to 300 nM apamin substantially decreased (Fig. 2g, Supplementary Fig. 7i). These results are in accordance with the structural observation that the peptide apamin participates in broader interactions with the S3–S4 linker than does UCL1684 (Fig. 2d, f).

Two recent studies revealed that apamin and UCL1684 bind to the extracellular vestibule of rat SK2 and human chimeric SK2-4 channels 16,17. Importantly, structural comparisons demonstrated very similar binding modes for apamin and UCL1684 in these SK2 structures, although the intracellular gate and selectivity filter of these structures assume different conformations (Supplementary Fig. 8a–f), suggesting that apamin and UCL1684 are state-independent blockers.

Structural basis for SK2 potentiation by CAD-1883
CAD-1883 (also called rimtuzalcap), a positive allosteric modulator of SK channels, has undergone two Phase II clinical studies for the treatment of essential tremors and spinocerebellar ataxia (Fig. 3a). To reveal the mechanism of action of CAD-1883, we first investigated the concentration-dependent potentiation of SK2 by CAD-1883 via whole-cell patch clamp recording of SK2-expressing HEK293T cells. When the internal Ca2+ concentration was ~300 nM, the currents of SK2 were gradually increased by CAD-1883, yielding an EC50 value of 2.7 ± 1.3 μM (Fig. 3b). We next determined the cryo-EM structure of SK2-CaM bound to CAD-1883 at a resolution of 3.35 Å with C4 symmetry (Supplementary Fig. 3). In contrast to the closed SK2-apamin and SK2-UCL1684, the density maps for the four complete CaM molecules are clearly resolved in SK2-CAD-1883, indicating a different state (Fig. 1a, Supplementary Fig. 5). Most importantly, the strong nonproteinaceous density in each of the CaM N-lobes unambiguously elucidated the binding of CAD-1883 (Fig. 3c, Supplementary Fig. 3d).

In the SK2-CAD-1883 complex structure, CAD-1883 resides in a pocket formed by the CaM N-lobe, S4–S5 linker helix, and HA helix from the adjacent subunit and places its difluorocyclohexane and pyrimidine moieties on the CaM N-lobe, the methylpyrazole group pointing towards SK2, and the morpholine group facing intracellular solvents (Fig. 3c, d). More specifically, the difluorocyclohexyle and pyrimidine moieties engage in extensive hydrophobic interactions with L291 from SK2 and F19, L32, M36, M51, F68, and M71 from the N-lobe of CaM; the methylpyrazole group interacts with I288, N292, and F409 from SK2 as well as L39 and Q41 from CaM (Fig. 3c, d). Sequence alignment revealed that these residues responsible for CAD-1883 binding are highly conserved among SK1-4, suggesting that CAD-1883 may be a nonspecific potentiator for SK channels (Supplementary Fig. 6). To validate this CAD-1883 binding site, we generated the L291F, L291W, and L291A/N292A mutations and assayed their currents elicited by 1 μM internal Ca2+. Although the activation kinetics of these mutants remain similar to that of WT SK2, all the mutants display reduced currents compared with WT SK2 (Supplementary Fig. 9a). However, none of them can be strongly activated by 100 μM CAD-1883 (Fig. 3e–h, Supplementary Fig. 9b), suggesting that introducing steric hindrance by L291F or L291W and abolishing the polar interaction from N292 may impair the binding of CAD-1883 into the pocket.

Our pore radius analysis clearly revealed that SK2-apamin is closed and that SK2-CAD-1883 has an intracellular gate with a van der Waals diameter of ~8 Å (Fig. 3i, j), indicating that SK2-CAD-1883 was captured in an open state. In particular, the intracellular gate of SK2-apamin is sealed by four V390 residues from the S6 helices, which undergo a conformational shift and rotation in SK2-CAD-1883, resulting in a dilated open gate (Supplementary Fig. 9c–e). Moreover, the gate of SK2-CAD-1883 resembles the open gates of rat SK2 and SK4 activated-II 15, and is notably wider than those of the closed SK4 and the activated-I SK4 (Supplementary Figs. 8k, 9f–h). Structural comparisons revealed that the CAD-1883-bound SK2 closely resembles the fully opened rat SK2 and SK4 activated-II structures and exhibits marked conformational differences in the S4-S5 linker, S6, and the N-lobe of CaM compared with the closed SK2-apamin, closed SK4, and SK4 activated-I (Fig. 3k, Supplementary Figs. 8j–l, 9i–k). Although both are in the open state, CAD-1883 binding still induces outward conformational shifts of the CaM N-lobe relative to the open SK4, facilitating closer interactions between the SA helix of SK2 and the CaM N-lobe (Fig. 3k).

These structural observations elucidate that CAD-1883 functions as a glue-like molecule that promotes the recruitment of the CaM N-lobe to the SA helix, thereby stabilizing the SK2 channel in this specific open state.

Structural basis for SK2 inhibition by AP30663
AP30663 is a potential antiarrhythmic agent that has been investigated in Phase II clinical trials for the treatment of atrial fibrillation. To understand how AP30663 inhibits SK2 channel, we first tested the concentration-dependent inhibition of SK2 by AP30663, which resulted in an IC50 value of 0.99 ± 0.14 μM, in line with a previous study 33 (Fig. 4a, b). We next purified SK2-CaM samples in the presence of AP30663 and solved the cryo-EM structure at 3.34 Å resolution with C1 symmetry imposed (Supplementary Fig. 4). The EM density map allows the model building of SK2, but the density for the CaM is weak, similar to that in SK2-apamin and SK2-UCL1684 (Fig. 4c, Supplementary Fig. 5). We observed strong extra density in the central cavity of SK2-AP30663, which can accommodate an AP30663 molecule and is not present in the central cavity of SK2-apamin or SK2-UCL1684 (Fig. 4d). The density map is not sufficient for unambiguous placement of AP30663, presumably because of the binding of an asymmetric blocker on the fourfold symmetric axis of the channel, a similar situation that has also been reported in a recent AP14145-bound rat SK2 study 16 and other fourfold symmetric channels 45. The SK2-AP30663 structure strongly indicates that AP30663 binds to the central cavity beneath the selectivity filter and serves as a pore blocker (Fig. 4c, e).

AP30663 resides in the central cavity with an orientation nearly perpendicular to the membrane plane and is sandwiched by four S358 residues from the top and four A383 and four T386 residues from the bottom (Fig. 4e). In this potential binding pose, the cyano group of AP30663 forms a possible polar interaction with the backbone carbonyl oxygen of G379, and the compound’s core region engages in van der Waals interactions with S358, A383, and T386 (Fig. 4e). S358 and A383 are not conserved in SK4, which are substituted by T250 and V275, respectively, which potentially explains the finding that the S508T/A532V double-site mutation in rat SK3 (corresponding to S358 and A383 in SK2) reduces AP14145 (a close analog of AP30663) inhibition by ~70%, and the T250S/V275A mutation confers AP14145 sensitivity to the insensitive human SK4 channel 46 (Supplementary Fig. 6a). To further probe the functional role of A383 and to the best of our knowledge the previously uncharacterized T386, we assayed the inhibitory effects of AP30663 on the A383F, A383L, T386L, T386F and T386A mutants and found that 30 μM AP30663 strongly inhibited the A383L mutant, similar to WT SK2, whereas it inhibited the A383F, T386L, and T386F mutants by only ~25%, ~15%, and ~40%, respectively (Fig. 4f, Supplementary Fig. 10a–e). T386A, however, greatly reduced the peak current of SK2 to less than 0.5 nA, which prevented further investigation of its effect on AP30663 (Supplementary Fig. 10f). We next determined the concentration‒response inhibition curve for A383L, which yielded an IC50 value of 2.1 ± 0.6 μM, suggesting a moderate reduction in the potency of AP30663 in the inhibition of A383L compared with WT SK2 (Fig. 4g, h). Collectively, these functional results demonstrate that AP30663 exerts its inhibitory effect on SK1-3 channels by directly blocking the central cavity and that three key residues (S358, A383, and T386) play critical roles in AP30663 binding and specificity against SK4.

Ca2+-activation and modulation mechanisms of SK2
The SK2 cryo-EM structures of this study and two recent SK2 studies 16,17 revealed a conserved Ca2+-dependent gating mechanism in SK channels, which is consistent with the SK4 channel 15. In the resting state, the C-lobe of CaM constitutively binds to the HA helix of the CaM-BD, whereas the N-lobe of CaM is mobile; thus, the intracellular gate of SK assumes a closed conformation (Fig. 5a). When intracellular Ca2+ surges to submicromolar concentrations, Ca2+ ions bind to the N-lobe of CaM and induce conformational changes in CaM, resulting in engagement between the N-lobe and the SA helix of the adjacent SK2 subunit and pulling the SA helix downwards by ~3 Å (measured at the Cα atom of N292). This displacement forces the SB and S6 helices to move away from the pore axis, thereby dilating the cytoplasmic activation gate from a radius of 5.3 Å to 10.7 Å, which allows the permeation of hydrated K+ ions (Fig. 5b, Supplementary Fig. 9c–e).

Our modulator-bound SK2 complex structures reveal three distinct modulation sites in SK2 (namely, Sites 1–3). At Site 1, apamin and UCL-1684 can bind to the extracellular vestibule formed by the distinct S3–S4 loop of SK2 channel, which physically blocks the exit of K+ ions (Fig. 5c). The central site is referred to as Site 2, in which AP30663 and its analogs reside, preventing K+ from accessing to the selectivity filter (Fig. 5c). Unlike physical blockade by inhibitors bound to Sites 1 and 2, Site 3 harbors positive allosteric modulators such as CAD-1883 and NS309, which facilitate the opening of SK2 and stabilize the open state (Fig. 5c).

In the closed structure of SK2-apamin, strong EM densities indicate that four putative K+ ions are coordinated by the backbone carbonyl oxygen atoms of the conserved “XXGYG” motif in the selectivity filter (Supplementary Fig. 11a, b). In contrast, the activated SK2-CAD-1883 structure possesses a disrupted selectivity filter, of which Y361 underwent a 180° rotation, resulting in dramatic dilation at the positions of G360 and Y361 and discontinued K+ sites in the selectivity filter (Supplementary Fig. 11c–e). The dilated selectivity filter of SK2-CAD-1883 is reminiscent of the structure of the Shaker Kv channel with a W434F mutation 47, which is proposed in a C-type inactivated state (Supplementary Fig. 11f–h). Moreover, the dilated selectivity filter with a rotation of the conserved Tyr has also been observed in the UCL1684-bound rat SK2 structure with an open intracellular gate and the chimeric human SK2-4 structures with (open) or without (closed) calcium ions 16,17 (Supplementary Fig. 8d–l). We speculate that the SK2-CAD-1883 structure may represent an inactivated state because the binding of CAD-1883 consistently activates the intracellular gate that may eventually inactivate the channel by altering the conformation of the selectivity filter.

Methods
Construct design
The cDNA fragments encoding human SK2 (KCNN2; UniProt ID: Q9H2S1) and calmodulin (CALM1; UniProt ID: P0DP23) were synthesized and inserted into the pBIG1a vector using the biGBac method with a C-terminal tag containing an HRV3C protease cleavage site, an mCherry fluorescence protein and a Twin-Strep II affinity tag. This multigene construct of the SK2/CaM complex was used for protein expression. To monitor and increase the expression of CaM, the CaM gene was cloned into a modified pEGBacMam vector, followed by an internal ribosome entry site (IRES) and a superfolder green fluorescent protein at the C-terminus. For the expression and purification of human CaM alone, the cDNA fragment encoding calmodulin was inserted into a pET28a vector, featuring an N-terminal 8×His tag and an HRV3C protease cleavage site. For electrophysiological recording, the cDNA fragments encoding SK2 were cloned into a modified pEG BacMam vector, followed by an IRES and an mCherry fluorescent protein. Point mutations were generated by a standard site-directed mutagenesis kit (Vazyme Biotech Co., Ltd.). All the constructs were confirmed by DNA sequencing.

Protein expression and purification
For CaM purification, CaM-expressing E. coli cells were resuspended in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% v/v Triton X-100, 10% v/v glycerol, 10 mM imidazole, 10 mM β-mercaptoethanol, 1 mM PMSF, 0.05 mg/ml DNase I and homogenized via high-pressure homogenization three times. The lysate was centrifuged at 100,000×g for 1 h. The supernatant was filtered through a 0.22 μm membrane and subsequently loaded onto Ni-NTA 6FF Beads (Smart-Lifesciences, China) at 4°C. The resin was washed with 10 column volumes of first wash buffer (50 mM Tris pH 8.0, 500 mM NaCl, 10% v/v glycerol, 10 mM imidazole, 10 mM β-mercaptoethanol, 1 mM PMSF) and second wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% v/v glycerol, 30 mM imidazole, 10 mM β-mercaptoethanol, 1 mM PMSF). CaM proteins were eluted with the second wash buffer supplemented with 250 mM imidazole. The 8×His tag was cleaved by 3C protease at a ratio of 1:20 (w/w). The solution was reapplied to Ni-NTA 6FF beads to remove the cleaved His-tag and protease. To eliminate imidazole, the proteins were dialyzed in buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 20% v/v glycerol, 1 mM DTT and concentrated using a 10 kDa Amicon Ultra centrifugal filter (Merck Millipore, Germany). The final CaM samples were concentrated to ~5 mg/mL and stored at −80°C.

For protein expression, recombinant baculoviruses were generated in Sf9 (Invitrogen, USA) cells using the Bac-to-Bac baculovirus expression system (Invitrogen, 11496015). Subsequently, 1% (v/v) SK2 and 1% (v/v) CaMP3 recombinant baculovirus were cotransfected into HEK293F (FreeStyle 293F cells, Gibco, USA) cells at a density of 2.5×10^6 per mL, supplemented with 1% (v/v) fetal bovine serum. After culture at 37°C with 5% CO2 for 12 h, the mammalian cells were supplied with 10 mM sodium butyrate and were further incubated for another 60 h at 30°C. The cells were then harvested by centrifugation at 1640×g for 5 min, and finally stored at −80°C after being flash-frozen in liquid nitrogen.

For all the purification processes, cell pellets expressing SK2 and CaM complexes were resuspended and homogenized in buffer A (20 mM HEPES pH 7.5, 150 mM KCl, 2 mM CaCl2, 2 mM β-mercaptoethanol (β-ME), 1 mM PMSF, 0.05 mg/mL DNase, aprotinin (2 μg/mL), leupeptin (1.4 μg/mL), pepstatin A (0.5 μg/mL)). The cell membrane was collected by ultracentrifugation at 100,000×g for 1 h. Subsequently, the membrane pellets were homogenized and solubilized with buffer B (buffer A supplemented with 1% (w/v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace), 0.15% (w/v) cholesteryl hemisuccinate (CHS, Anatrace), 5 mM MgCl2 and 2 mM ATP) for 2 h at 4°C. The insoluble material was removed by ultracentrifugation at 100,000×g for 30 min, and the supernatant was filtered through 0.22 μm filters to remove cell debris. The supernatant was then loaded onto Streptactin Beads (Smart-Lifesciences, China) via gravity flow at 4°C to enrich the protein complex. The resin was subsequently washed with 10 column volumes of buffer C (buffer A supplemented with 0.01% (w/v) Lauryl Maltose Neopentyl Glycol (LMNG, Anatrace), 5 mM MgCl2 and 2 mM ATP) and another 10 column volumes of buffer D (buffer A supplemented with 0.01% (w/v) LMNG). The protein was eluted with buffer E (buffer D supplemented with 10 mM desthiobiotin (Sigma, USA)). The mCherry and twin-strep tag were cleaved by PreScission protease at a ratio of 1:20 (w/w). Purified calmodulin was added to the purified SK2 samples to enhance complex formation at a final molar ratio of 1:4 (monomer/monomer). The mixture was incubated on ice for 2 h and then concentrated to 1 mL using a 100 kDa cut-off Amicon ultra centrifugal filter (Merck Millipore, Germany) and subjected to a size-exclusion chromatography column (Superose 6 Increase 10/300 GL (Cytiva)) pre-equilibrated with the buffer E (20 mM HEPES, pH 7.5, 150 mM KCl, 2 mM β-ME, 2 mM CaCl2, 0.005% (w/v) LMNG). The peak fractions were pooled and concentrated to ~2.5 mg/mL for cryo-EM sample preparation. To purify ligand-bound complexes, different ligands, including Apamin (MedChemExpress), UCL1684 (Merck), CAD-1883 (MedChemExpress) and AP30663 (MedChemExpress), were mixed with the SK2-CaM sample for at least 30 min, respectively. The final concentrations of apamin, UCL1684, CAD-1883 and AP30663 were controlled to 100 μM, 500 μM, 500 μM, and 500 μM, respectively.

Cryo-EM sample preparation and data acquisition
Aliquots of 4 μL purified ligand-bound SK2 complex samples were applied to holey grids (Cu R1.2/1.3, 300 mesh, Quantifoil) glow-discharged by PELCO easiGlow (Ted Pella). The grids were blotted for 3.5–5.0 s at 4°C with 100% humidity using an FEI Vitrobot Mark VI (Thermo Fisher Scientific) and were plunge-frozen in liquid ethane cooled with liquid nitrogen.

Cryo-EM data were collected using a Titan Krios G4 electron microscope operated at 300 kV (Thermo Fisher Scientific) equipped with a K3 direct electron detector (Gatan) and a Quantum GIF energy filter with a slit width of 10 e−V. All movie stacks were automatically acquired using EPU at a calibrated magnification of 105,000× under superresolution mode with a physical pixel size of 0.85 Å pix−1. Each movie was fractionated into 40 frames with an exposure time of 2.89 s. The defocus range was set between −1.0 and −2.0 μm. In total, 1268, 5043, 3837, and 2944 cryo-EM movie stacks were collected for SK2-apamin, SK2-UCL1684, SK2-CAD-1883, and SK2-AP30663, respectively.

Cryo-EM data processing
All the data were processed via CryoSPARC 48. The raw movie stacks were motion-corrected, dose-weighted and binned by two-fold using Patch Motion correction, followed by a contrast transfer function (CTF) estimation using Patch CTF. Micrographs with a resolution better than 4 Å and astigmatism lower than 500 Å were selected for further data processing. Particles were picked based on 2D templates generated by the SK4/CaM map. Subsequently, particle extraction was carried out with a box size of 320 pixels and a binning factor of 2. The particles were subjected to several rounds of 2D classification to remove ice and junk particles. A set of 2D averages with clear transmembrane features was subjected to ab initio reconstruction with 3 classes. The optimal map served as the initial reference for performing heterogeneous refinement with C1 symmetry, using all picked two-fold binned particles as input. Particles from 2D classification with clearly resolved transmembrane helices were selected, re-extracted with a box size of 320 pixels, followed by a non-uniform refinement and rounds of resolution gradient heterogeneous refinement with C1 symmetry. Finally, a round of 3D classification with a promoter mask or transmembrane mask was employed to improve the ligand density, resulting in 3D reconstruction maps at resolutions of 3.23 Å with C1 symmetry, 2.96 Å with C2 symmetry, 3.35 Å with C4 symmetry, and 3.34 Å with C1 symmetry for SK2-apamin, SK2-UCL1684, SK2-CAD-1883, and SK2-AP30663, respectively. Similar procedures were used for all data processing, as summarized in Supplementary Figs. 1–4.

Model building and refinement
The AlphaFold2 model of human SK2 and the structure of human CaM (PDB code: 6CNN) served as initial models and were placed into the EM density map of SK2-apamin by rigid-fitting in UCSF Chimera 49, followed by manual correction in Coot 50, which was used as a reference for model building of all four SK2 structures. The refinements of the SK2 models against the EM density maps were further refined in real space using phenix 51. The models and geometric constraints for UCL1684, AP30663 and CAD-1883 were generated using the eLBOW module in Phenix. The apamin structure (PDB code: 7OXF) was used as an initial model. Apamin, UCL1684, AP30663 and CAD-1883 were manually fitted into their respective density maps. All modulator-bound SK2 structures were refined and validated in Phenix with reasonable geometry restraint parameters and clash scores. The FSC curves of the model versus the map were calculated by Phenix.mtrage. The cryo-EM data collection, refinement and validation statistics are presented in Supplementary Table 1. All figures were prepared with Prism (GraphPad software), ChimeraX 52 and PyMol (Schrödinger, LLC).

Patch clamp electrophysiology
Whole-cell patch-clamp recordings were performed on HEK293T (Gibco, USA) cells transiently transfected with plasmids of WT SK2 or its variants. Currents were recorded using an EPC-10 amplifier (HEKA, Lambrecht, Germany) and acquired via Pulse/Pulsefit software (HEKA, Lambrecht, Germany). All the recordings were conducted at room temperature. Patch pipettes were pulled from borosilicate glass using a horizontal electrode puller (Model P-1000, Sutter Instruments, USA), with a resistance of 3–6 MΩ when filled with intracellular solution (compositions described below). The same bath (external) solution was used for all the current recording experiments in this study, which contained (in mM): 120 KCl, 10 HEPES, 10 EGTA, 6.19 CaCl2 (estimated free [Ca2+], 60 nM), 1.44 MgCl2, pH 7.4, adjusted with KOH. For functional validation of SK2 variants and testing the inhibitory effects of apamin, UCL1684 and AP30663, the pipette (internal) solution contained (in mM): 120 KCl, 10 HEPES, 10 ethyleneglycoltetraacetic acid (EGTA), 1.5 Na2ATP, 9.65 CaCl2 (estimated free [Ca2+], 1 μM), 2.34 MgCl2, pH 7.4, adjusted with KOH. To assess the activation efficiency of CAD-1883, a low free [Ca2+] pipette (internal) solution was used, which contained (in mM): 144 KCl, 10 Hepes, 10 EGTA, 1.205 MgCl2, and 7.5 CaCl2 (estimated free [Ca2+], 300 nM), pH 7.3, adjusted with KOH. Free [Ca2+] concentrations were calculated using MaChelator software, the software by C. Patton of Stanford University (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/downloads.htm). Currents were recorded by applying repetitive 1 s voltage ramps from −100 to +100 mV from a holding potential of 0 mV.

For the apamin-related recording experiments, apamin was dissolved in double-distilled water to prepare a stock solution of 100 μM. The stock solution was subsequently diluted with the external solution to achieve the desired working concentrations. An eight-channel (each channel having a volume capacity of 20 mL) gravity perfusion system was used for modulator delivery. After successful cell membrane rupture and establishment of a stable seal, modulators at the desired working concentrations were perfused continuously at a constant flow rate. Simultaneously, a peristaltic pump was employed to steadily remove solution from the recording chamber, enabling full exchange of the external solution throughout the recording. For UCL1684, CAD‑1883, and AP30663, these small molecules were dissolved in DMSO to prepare stock solutions with concentrations of 10 mM, 100 mM, and 10 mM, respectively. The perfusion of these three small-molecule modulators was performed similarly to that described above.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript of Bao Ma et al reports on the cryo-EM structure of the calcium-activated SK2 channel in complex with four pharmacological modulators, the outer pore blockers apamin and UCL1684, the negative gating modulator AP30663, and the activator CAD-1883. Taken together, this is a highly significant and impactful study, since SK2 channels are important drug targets for neurological and cardiovascular disease. The field urgently needs structures that can enable structure-based drug design and assist with a deeper understanding of the channel’s gating mechanisms and its pharmacological modulation. The study significantly adds to the field. Especially, the apamin structure and its description are superb. The authors provide a large amount of high-quality structural, electrophysiological and mutagenesis data and should be applauded for their success in obtaining structures for human SK2, which in contrast to rat SK2 (Nam et al. Nat Communications 2025) and a chimeric human SK2/SK4 construct (Cassell et al. eLife 2025), has proven difficult to query in structural studies. However, the authors should clarify a few points and discuss differences between their structures and previously published structures in more detail since there are some very interesting differences and similarities that are likely to provide deeper insights into the mechanisms of pharmacological modulation.\nMajor comments:\n1. In contrast to the other two structural studies using rat SK2 or a human SK2/SK4 chimera the density of the channel associated CaM in three of the four structures is weak and it is not clear from the provided figures, the text or the methods, how much calcium was present during the purification process and whether the apamin, UCL1684 and AP30663 structures contain any calcium ions in the EF hands of the four CaMs. Figure 1, which shows human SK2 in complex with CaM is showing the open structure in the presence of the activator CAD-1883. Was there any calcium visible in the CaM C-lobes in the apamin, UCL1684 and AP30663 structures? This question is of interest because the Cassell and the Nam studies captured SK2 with a wide-open inner gate when they were complexed with apamin or UCL1684. So, it is very interesting that authors obtained structures with a closed inner gate. These differences in channel state should be commented on by the authors. As a reviewer, I do not think acknowledging previous structures will distract from the significance and the impact of the authors’ work. But the authors really need to clarify how much calcium was used and whether the experiments were designed to capture open or closed channels, especially since their study does not contain an Apo structure.\n2. Did the authors also attempt to capture an Apo structure in the absence and presence of calcium? Was the resolution maybe too low? This goes towards answering the previous point. If the authors expressed and isolated SK2 protein and associated CaM at normal intracellular calcium concentrations, that could explain why only the CAD-1883 bound structure contains calcium. Like the authors correctly state, CAD-1883 increases the calcium sensitivity of SK2 and “glues” the CaM-N lobe to the S4-S5 linker. The presence and absence of calcium should be discussed.\n3. The authors should clarify if the cholesterol density shown in Figure 1 was present in all four structures.\n4. The authors might want to consider removing Supplementary Fig 9 showing a comparison of the CAD-1883 binding site at the S4-S5 linker to the “infamous” C-terminal crystal of CaM in complex with the isolated HA-HB helices. Ever since the SK4 structure was published by the MacKinnon lab, it has been clear that this C-terminal crystal is simply an artifact and this comparison might confuse younger readers. It would be much more interesting to see an overlay of the closed SK2/apamin and the closed SK2/UCL1684 structures from the authors’ work with the previously published open structures. How significant are the changes at the inner gate and in the selectivity filter? Such a figure would make a nice complement to Supplementary Figure 10 which is showing the differences in selectivity filter occupation and conformation between the apamin and the ULC1684 structures captured by the authors. This would be particularly interesting in light of the authors’ hypothesis that prolonged opening of the inner gate by CAD-1883 might eventually induce channel inactivation by altering the selectivity filter conformation.\n5.The authors state that AP30366 could not be unambiguously placed into its density. As the authors state, this is a common problem when a single drug molecule binds to a four-fold symmetric target. The authors confirm the location by mutagenesis (which is great) but should be better at explaining how the pose in Fig 4 was generate. What modeling software was used?\nMinor points:\n1. Abstract, line 33. What diseases does “these disorders” refer to? None where ever mentioned. Please edit and either delete or clarify that you mean neurological and cardiovascular disorders.\n2. Please cite the previous papers reporting on SK2 structures in the introduction.\n3. Please edit the grammar in line 78/79.\n4. Line 497 in the Methods: Why is the calcium concentration of the extracellular solution for whole-cell patch-clamp so low? Why not the usual 2 mM?\n5. Consider commenting on the altered rectification of L291W and L291F in Figure 3.\n6. Figure 4: Please clarify in the legend how much free calcium was used for the concentration response curves for AP30663. Line 6 in the legend, please replace “so” with “no”. You mean “no such density”, correct?", "reviewer_2": "In this study, Ma et al. present cryo-EM structures of human SK2/calmodulin in complex with three pore blockers (apamin, UCL1684, and AP30663) and a positive gating modulator (CAD-1883). Together with electrophysiological analyses, these data reveal structural mechanisms underlying the inhibition and modulation of SK2 channels by different drugs. This is a solid structural investigation that provides valuable insights into the pharmacological properties of SK2.\nWhile I appreciate the scientific significance of the findings, my main concern lies with the writing quality. The manuscript contains numerous grammatical errors, awkward phrasing, and imprecise statements. It therefore requires substantial editorial revision before it can be considered for publication. Additionally, several interpretations appear overstated or unclear and require correction. Below are my specific comments:\n1. Abstract, line 33: Please clarify what “these disorders” refers to.\n2. Lines 53–54: The sentence “However, the detailed structural information of SK1-3 that discriminates them from SK4 has remained unclear” is vague. Are you referring to specific structural features distinguishing SK1–3 from SK4? Please be explicit.\n3. Lines 101–107: It seems the structures were determined without Ca²⁺. Since Ca²⁺ would stabilize SK2/CaM without interfering with blocker binding, why was it excluded? Please explain.\n4. Lines 108–110: It appears that the SK2–CAD-1883 complex is used for comparison and overall structural description. If correct, please state this explicitly.\n5. Lines 120–121: The terms “subtle” and “marked” are contradictory. Please clarify which accurately describes the structural differences.\n6. Lines 126–130: Bound lipids, cholesterols, or detergents are commonly observed in membrane protein structures. Without further evidence, suggesting these represent modulation sites is speculative and should be toned down.\n7. Lines 141–144: The poor density of the N-loop is more likely due to its intrinsic flexibility, not the use of C1 symmetry. Please revise accordingly.\n8. Line 164: If T245 of rat SK1 corresponds to S244 of SK2, please make this equivalence explicit.\n9. Lines 187–189: The statement that tetramer formation amplifies conformational differences is unclear. Please explain or rephrase.\n10. Lines 380–381: Since this study does not include a Ca²⁺-activated SK2 structure, it does not directly address Ca²⁺-dependent activation. Please tone down this claim.\n11. Methods (Patch-clamp electrophysiology): The description of the four recording solutions is confusing. Please clarify which solutions were used in which experiments.", "reviewer_3": "In this manuscript, Ma et. al. investigated the regulation of human small-conductance calcium-activated potassium channel (SK) by four distinct modulators. SK channels are potassium-selective channels activated by intracellular Ca binding to calmodulin, which is constitutively associated with the channel. The SK channel family comprises four isoforms (SK1, SK2, SK3, and SK4) and this study focuses on SK2. Structural information of SK channel in complex with modulators is limited and the authors addressed this gap by determining cryoEM structures of calmodulin-bound SK2 in complex with bee venom peptide apamin and other small molecule modulators i.e. UCL1684, AP30663, and CAD-1883. These structures revealed the binding sites and regulatory mechanisms of each modulator. Apamin, UCL1684, and AP30663 act as inhibitors of SK channels while CAD-1883 is an activator. Their results show that both apamin and UCL1684 bind the outer vestibule occupying an overlapping binding site and blocks the ion permeation pathway from the extracellular side while AP30663 binds to a distinct site in the central cavity of SK2 channel blocking the pore. CAD-1883 binds the calmodulin N-lobe and the S45A helix of SK2, stabilizing the open conformation of the intracellular gate. Further, they carried out mutagenesis and electrophysiology studies to validate these structural findings.\nOverall, the study is well designed and clearly presented, providing mechanistic insights into the Ca-dependent activation and modulation of SK2 channel activity by inhibitors and activators. The findings will help further guide rational development of drugs targeting SK channels. The manuscript is suitable for publication. Some of the concerns that should be addressed before acceptance are as follows:\n1. Two recent studies reported SK channel structures with overlapping findings to those presented in the current manuscript. Nam et al. (Nature Communications, 2025) resolved cryo-EM structures of the SK2 channel in complex with calmodulin and Ca, as well as with the inhibitor UCL1684. Cassell et al. (eLife, 2025) reported the cryo-EM structure of a chimeric SK channel bound to apamin, generated by fusing the transmembrane and extracellular domains of human SK2 with the intracellular domains of human SK4. The authors should cite both studies and discuss whether their findings align with or diverge from these published results.\n2. The text between lines 167–183 is somewhat difficult to follow, as it brings together multiple interactions in a way that interrupts the logical flow of the structural description. For instance, in lines 174–176, the interactions of R240 with neighboring subunit residues are linked to stabilization of the S3–S4 linker, though this was not experimentally tested. This makes the statement feel less convincing.\nA clearer way to present this section could be to first highlight that the S3–S4 linker is ordered in the apamin-bound structure. The authors might then propose that H336, N367, A242, and Y245 contribute to stabilizing the linker based on structural evidence. Building on this, they could emphasize how mutating these residues reduces apamin inhibition, thereby supporting their role in stabilizing the loop conformation and enhancing apamin binding. Finally, the narrative could be strengthened by referencing similar findings from other groups.\n3. In lines 168–169, the authors report that 300 nM apamin inhibits the H336N and N367H mutants by only ~20% and ~60%, respectively. It would be helpful if the authors could clarify the rationale for choosing these specific substitutions—why H336 was mutated to Asn and N367 to His. Providing this context would make the functional data more informative and easier to interpret.\n4. For Fig. 4f, the author should provide representative current trace for all the mutants. Why did they mutate the interacting residues to bulkier ones only and not to Ala to abolish inhibitory effect of AP30663? Also, A383F shows reduced inhibition compared to A383L while it is reversed in case of T386 where Phe mutant shows stronger inhibition compared to the Leu mutant. What is the basis of this difference?\n5. Y245 is not labelled in Fig. 2e.\n6. In line 234, L32 is mentioned but not shown in the Fig. 3c, d.\n7. Lines 241-242 states “L291W and L291A/N292A mutants display reduced currents” However, from the data it seems that all the mutants show reduced current and not only the two mutants mentioned here.\n8. In Supplementary Fig. 2b, the Y-axis reads excitation (%). It is not clear what it means.\n9. Line 263, ‘involving of HC helix’ should be ‘involvement of HC helix’.\n10. Line 367, ‘pocket that apamin does’ should be ‘pocket than apamin does’.\n11. In the method section, it should be mentioned how different modulators were applied during electrophysiology recordings.", "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 sincerely thank all the referees for their time to evaluate our manuscript, their overall positive assessment of this study, and for their valuable suggestions and insightful criticisms on how to improve the manuscript. Following the referees’ suggestions, we have performed additional experiments and revised the manuscript substantially. After incorporating these revisions, we believe that the revised manuscript has been improved. A detailed point-by-point response in black to the referees’ comments in blue is provided below.\n[... Author responses to Reviewer 1, 2, 3, and 4 follow ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for addressing all my comments so thoroughly. Beautiful work.\nHeike Wulff", "reviewer_2": "The authors have addressed my concerns in the revision, and I am happy to support acceptance of the manuscript.", "reviewer_3": "The authors have thoroughly addressed all concerns by revising the text, incorporating additional figures, and providing new electrophysiology recordings. I recommend the manuscript for publication.", "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 greatly appreciate Dr. Heike Wulff for your important suggestions and support.\nWe greatly appreciate Reviewer 2 for your important suggestions and support.\nWe greatly appreciate Reviewer 3 for your important suggestions and support.\nWe greatly appreciate Reviewer 4 for your important suggestions and support."}]

Decision letter