Reversible lipid-mediated pH-gating of connexin-46/50 by cryo-EM

Domain: biology
Published: 2026-01-12
Editorial outcome: minor_revision

Abstract
Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to intracellular pH changes, closing under acidic conditions to limit the spread of cytotoxic signals during cellular stress, such as ischemia. Using cryo-electron microscopy (cryo-EM), we uncover insights into the structural mechanism of pH-gating in native lens connexin-46/50 (Cx46/50) gap junctions. Mild acidification drives lipid infiltration into the channel pore, displacing the N-terminal (NT) domain and stabilizing pore closure. Lipid involvement is shown to be both essential and fully reversible. Structural transitions involve an ensemble of gated states formed through non-cooperative NT domain movement as well as minor populations of a distinct destabilized open-state. These findings provide molecular insights into pH-gating dynamics, illustrating how structural changes may regulate gap junction function under cellular stress and linking Cx46/50 dysregulation to age-related cataract formation.

Introduction
Connexins form intercellular channels, known as gap junctions, that enable direct cell-to-cell communication, allowing tissues to function as a syncytium by coordinating long-range electrical and metabolic activity1,2. These channels are critical in numerous physiological processes, and their dysfunction is linked to diverse diseases, including blindness, deafness, arrhythmia, stroke, neuropathy, skin disorders, and cancer3–5.

In addition to propagating cellular signals, gap junctions play a vital role in cellular stress responses. They dilute cytotoxic signals (e.g., potassium buffering by glial cells), and may also uncouple damaged cells from healthy tissue, preventing bystander effects or the so-called “kiss of death”6,7. Uncoupling is triggered by stress-induced gating mechanisms sensitive to transjunctional voltage and cytosolic chemical signals, like H+ and Ca2+8–14. Intracellular acidification, a hallmark of ischemia and cataract, is a universal regulator of gap junctional gating15–18. However, despite recent advances19, the molecular mechanisms underpinning pH-induced gating remain poorly defined.

Structurally, gap junctions are composed of twelve connexin subunits, forming either homomeric or heteromeric/heterotypic assemblies depending on isoform composition (21 isoforms in humans)20,21. Each hemichannel, formed by six connexins, docks with its counterpart in an adjacent cell to create a large-pore intercellular channel (~1 kDa cutoff) capable of passaging ions, metabolites, and signaling molecules22. Each connexin subunit consists of four transmembrane helices (TM1–4), two extracellular loops (EC1–2) involved in junction formation, and an N-terminal domain (NT) implicated in channel selectivity and gating23–26 (see Fig. 1a). Intracellular loop (ICL) and C-terminal (CT) regions involved in trafficking and regulation are largely disordered and highly variable among isoforms20,21.

Early models of pH-gating based on studies of Cx43 and Cx50 proposed a “ball-and-chain” mechanism, linking the CT in pore occlusion27–31. However, subsequent studies have shown that pH gating in several isoforms, including Cx46 and Cx50, does not require the CT32–35. More recently, the NT domain has been implicated as a pH-gating particle, supported by cryo-electron microscopy (cryo-EM) studies of Cx2619. Though the limited resolution of this study precluded atomic-level details of the gating mechanism, the study describes pH-induced conformational changes in the NT that result in pore occlusion. The NT’s involvement in pH-gating aligns with its conserved nature and its suggested role in other gating mechanisms, such as “fast” voltage-gating, underscoring its central function in connexin regulation23,25,26,36–38.

Recent cryo-EM studies of various connexin isoforms have revealed diverse NT conformations influenced by environmental conditions and suggest a potential role for lipids in modulating NT conformational states (reviewed39). However, the origination and reversibility of pore-bound lipids in these studies remains unclear, as hydrophobic molecules can diffuse into the large pores of these channels during detergent extraction or reconstitution into lipid nanodiscs, complicating interpretation of their potential functional relevance.

Here, using single-particle cryo-EM, we investigate the pH-gating mechanism of native Cx46/50 channels from aged lens tissue. We show that at neutral pH, channels predominantly adopt a stabilized open-state devoid of pore-bound lipids, consistent with prior studies40,41. When this sample is exchanged to mildly acidic conditions known to gate these channels in electrophysiology studies32–35, structural changes induce lipid infiltration into the pore, displacing the NT and driving an ensemble of gated states. Lipid infiltration originates from the surrounding lipid environment, is essential for gating, fully reversible upon return to neutral pH, and we propose a link to protonation of conserved histidines. Structural deconvolution reveals non-cooperative NT movements contributing to multiple gated states, alongside minor populations of stabilized and destabilized open-states. Collectively, these findings provide critical insights into the dynamic interplay between lipids and pH-gating in Cx46/50, offering molecular insights into age-related cataract formation and establishing a framework for understanding connexin regulation under cellular stress.

Results
Low pH-gated state of connexin-46/50
Native Cx46/50 channels were purified from aged ovine lens tissue, containing native C-terminal truncations42,43, and reconstituted into MSP1E1 nanodiscs containing dimyristoyl-phosphatidylcholine (DMPC) lipids at pH 7.4, as previously described41. The reconstituted channels were then buffer-exchanged to pH 5.8 via gel filtration, to mimic low-pH conditions shown to gate C-terminal truncation variants of Cx46 and Cx50 channels in cellular functional studies32–35 (Supplementary Figs. 1, 2). Cryo-EM single-particle analysis resolved a low-pH gated-state structure of Cx46/50 at 2.3 Å global resolution with applied D6 symmetry (Fig. 1b; Supplementary Figs. 3, 4, and Table 1; Collection #1). Asymmetric refinement of the dodecameric channel (resolved to 2.8 Å) showed similar overall features (Fig. 1b).

Heteromeric/-typic assembly patterns of Cx46 and Cx50 could not be distinguished from the cryo-EM data, consistent with our prior studies40,41, reflecting the >80% sequence identity across structured regions of the two isoforms and limitations of current cryo-EM classification methods. As such, atomic models for both isoforms were refined into a D6-symmetrized map, encompassing TM1-4, EC1-2, and the NT gating domain, while the CT and ICL regions remained largely unresolved due to intrinsic disorder (Fig. 1c; Supplementary Fig. 5 and Table 1). Over 400 lipid densities (mostly single acyl chains of DMPC) and >900 water molecules were also resolved, with extracellular leaflet lipid chains exhibiting long-range hexagonal packing, consistent with previous observations41. Given the structural similarity between Cx46 and Cx50 (Supplementary Fig. 6), Cx46 is presented as the archetype, with isoform-specific differences noted where relevant.

The pH-gated gated-state is characterized by inward displacement of the NT domain, as compared to the stabilized open state resolved under neutral pH41, shifting ~18° toward the pore center (Cα r.m.s.d. = 4.7 Å over the NT domains) (Fig. 1a, d, e; Supplementary Fig. 6). Due to limited local resolution, the NT could be modeled only to residue W4, with side chains from residues 4–16 built to the β-carbon level (Fig. 1b, f–h). Residues G2 and D3 remained unresolved, leaving their role in channel closure unclear (Fig. 1b, f–i). Nonetheless, the inward displacement moves the NT ~4.7 Å toward the pore center compared to the stabilized open state (measured at the most constricted Cα positions of D3 in the open state and S5 in the pH-gated state), further highlighting the conformational shift of the NT in the gated state (Fig. 1i).

The low pH-gated state of the NT is stabilized by infiltrating lipids, which disrupt hydrophobic interactions between the NT and TM1/TM2 that anchor the NT in the stabilized open state (Fig. 1a, c, f–i). These lipids occupy a hydrophobic pocket within the channel lumen, formed by TM1 and TM2, and intercalate beneath the NT, supporting its inward displacement. In addition to stabilizing the gated conformation, these lipids contribute to the pore-lining architecture of the channel (Fig. 1i). Although limited local resolution prevents definitive modeling of inter-subunit interactions at the constriction site, the lipid-mediated repositioning of the NT brings proximal residues S5 and F6 from adjacent subunits into close proximity (Fig. 1g), suggesting a potential structural role in facilitating channel occlusion in the pH-gated state.

NT movement is coupled with a global clockwise rearrangement of the transmembrane helices, as viewed from the cytoplasmic side, that reinforce the gated NT conformation (Fig. 1d, e). TM2 exhibits the largest displacement, facilitated by a conserved proline kink at P88 that shifts the cytoplasmic portion of TM2 by 9.0° toward the pore, enabling hydrophobic interactions between L93 (V93 in Cx50) on TM2 and A14 (V14 in Cx50) on the NT (Fig. 1e). Additionally, subtle structural variation is observed compared to the open-state in the π-helix region of TM1 (residues ~39–41) that forms a kink separating the para-helical region leading to EC1 (Supplementary Fig. 6).

Lipid stabilization of the low pH gated-state
Beyond NT movement, the most striking difference between the open and gated-states is lipid translocation into the pore and at inter-subunit interfaces (Figs. 1c; 2). Two pore-bound lipids (PL1, PL2) form a double-layer hydrophobic “gasket” stabilizing the NT domain in the gated-state, while an interstitial lipid (IL) is identified intercalated at the subunit interface (Fig. 2a, b). Although the local resolution of these lipids is somewhat limited, likely reflecting positional and/or conformational heterogeneity, substantial regions were sufficiently resolved to permit interpretation for atomic modeling.

PL1, modeled as a nine-carbon acyl chain of DMPC, occupies a hydrophobic groove formed by TM1/2 of one subunit and TM1 of a neighboring subunit, displacing NT hydrophobic residues (W4, L7, and L11) that occupy this site in the open-state (Fig. 2c, d). The cytoplasmic portion of TM2 shifts toward the center of the pore under low-pH conditions, bringing L93 (V93 in Cx50) on TM2 3.0 Å closer to L26 on TM1, contributing to NT displacement (Fig. 2c, d).

PL2 is positioned atop PL1 and forms additional interactions with TM1 of an adjacent subunit (Fig. 2a). The phosphocholine (PC) headgroup of PL2 was modeled within the solvent-exposed lumen, while the acyl chains are oriented toward the hydrophobic pocket formed by PL1, TM1, and the NT (Fig. 2e, f). Although the limited resolution of the NT restricts detailed interpretation, the positions of hydrophobic anchor residues W4, L7, and L11 suggest they contribute to stabilizing the gated NT conformation through hydrophobic contacts with PL2.

The IL, intercalated at the subunit interfaces, aligns with the intercellular leaflet of the bilayer (Fig. 2a, b). The IL exhibits splayed acyl chains, with one chain forming extensive hydrophobic contacts with TM1 and TM2/3 of adjacent subunits, with the other chain penetrating toward the NT, potentially interacting with its hydrophobic surface (Fig. 2g, h). These extensive interactions, particularly with TM2 and the NT, likely contribute to stabilizing the gated-state conformation and may also mark a potential entry or exit point for lipid translocation. Notably, the IL density is absent in the open-state structure, suggesting a pH-dependent interaction and supporting the hypothesis that this site serves as a conduit for lipid infiltration into the channel (discussed below).

pH-gating is lipid-dependent and reversible
We next sought to demonstrate that a lipid environment is required for pH-gating of Cx46/50 and that lipid recruitment into the pore is a reversible process driven by pH conditions. First, to determine lipid dependence, Cx46/50 channels were reconstituted into amphipol (PMAL-C8) at pH 7.4, following established methods for obtaining the open-state structure40, and then buffer-exchanged to pH 5.8 (mimicking the strategy for our nanodisc system, Collection #1). Cryo-EM single-particle analysis resulted in a 2.7 Å reconstruction, with NT domains resolved adopting the stabilized open-state conformation (Cα r.m.s.d. = 0.2 Å)41 with no lipids or detergent resolved inside the pore (Fig. 3a; Supplementary Figs. 2, 6, 7; Collection #2), confirming lipid dependence of the pH-gated state.

Next, to ensure lipids enter the pore as a result of low pH-induced structural changes, rather than contaminating the pore during nanodisc reconstitution, the following control experiments were conducted. Cx46/50 channels reconstituted into nanodiscs at pH 7.4 were divided into two samples and buffer-exchanged to either neutral (pH 7.4 control) or low pH (pH 5.8) conditions (Supplementary Fig. 2; Collections #3 and #4, respectively). Cryo-EM 3D reconstruction of the neutral pH sample yielded a 2.0 Å map with NT domains in the stabilized open-state and a pore free of lipids or detergent (Fig. 3b; Supplementary Fig. 8). In contrast, the low pH sample produced a 2.6 Å map, revealing PL lipids stabilizing the NT in the gated-state (Fig. 3c and inset), confirming that lipids are translocated from the local lipid environment in response to low pH-induced structural changes.

Finally, reversibility of lipid-mediated gating was tested by returning the low pH-gated channels to neutral pH conditions. Here, Cx46/50 channels reconstituted into nanodiscs at pH 7.4 were buffer-exchanged to pH 5.8 to induce the gated-state. This sample was then split into two groups: one remained at pH 5.8 during buffer exchange (pH 5.8 control), and the other was dialyzed back to pH 7.4 (reversed pH 7.4) (Supplementary Fig. 2; Collections #5 and #6, respectively). Cryo-EM reconstruction of the pH 5.8 control yielded a 2.3 Å map, confirming the NT gated-state with PL lipids (Fig. 3d and inset; Supplementary Fig. 9). In contrast, reconstruction of the reversed pH 7.4 sample produced a 2.0 Å map, showing the stabilized open-state conformation devoid of pore-bound lipids (Fig. 3e). These findings confirm that lipid translocation is reversible and essential for pH-dependent gating observed by cryo-EM.

Putative role of conserved histidine pH-sensors
Having established that pH gating in Cx46/50 is lipid-mediated, we next examined the potential mechanism underlying H+-induced conformational changes. Conserved histidine residues have been suggested to play a key role in the pH-dependent gating of several connexins33,44–46. These cytosolic sites correspond to H17 and H95 in Cx46/50 (Supplementary Fig. 6). The role of conserved histidine residues in pH gating is consistent with experimentally derived gating response pKa values of C-terminally truncated Cx46/50 (pKa ~6.6 for Cx4634 and ~7.1 for Cx5032) and the universal nature of pH sensing across connexin isoforms.

A mechanistic role for H95 in pH sensing had not been previously apparent, as it is positioned on TM2 facing the lipid environment. In the pH-gated state of Cx46/50, the imidazole ring of H95 is adjacent (<3.5 Å) to the negatively charged phosphate headgroup of the interstitial lipid (IL) (Fig. 4a). This proximity suggests that protonated H95 could form a salt-bridge with the IL, stabilizing the gated NT/TM2 conformation. Coulombic surface potential analysis shows a dramatic shift to positive potential at this binding site under acidic conditions (Fig. 4b, c). At neutral pH, deprotonation of H95 would disrupt this interaction (Fig. 4c), consistent with the absence of lipid density at this site in the open-state structures. These findings implicate H95 in recruiting the IL to its binding site and in stabilizing the gated state in a pH-dependent manner.

Notably, mutating the equivalent H95 site does not eliminate pH sensitivity in Cx4346 or Cx4633, indicating additional pH-sensing residues contribute to gating. H17, another highly conserved histidine, has also been implicated as a pH-sensor in functional studies of Cx2644 and Cx3645. H17 is positioned at the NT-TM1 hinge near the channel entrance, at a similar z-position to H95 relative to the pore axis (Fig. 4a). In the neutral pH structure, H17 forms hydrophobic interactions with V96 and L97 (A96 and V97 in Cx50) on TM2 (Fig. 4d, e). Under acidic conditions, coordinated rearrangements of the NT and TM2 appear to preserve these hydrophobic contacts (Fig. 4d, f). Notably, H17 is adjacent to E16 on the NT, which could compensate for the positive charge of a protonated H17, thus supporting persistence of these interactions even at low pH and potentially contributing to stabilization of the gated NT conformation.

Low pH induces an ensemble of gated- and open-states
Single-channel recordings show acidic conditions induce a complex “slow” gating transition, with channels fluctuating between closed and open states33,47. In contrast, at neutral pH and minimal transjunctional voltage, Cx46 and Cx50 single-channels predominantly adopt an open-state26,31,48–50. In agreement with this behavior, our structural analysis revealed pronounced conformational heterogeneity induced by low pH conditions, particularly within the NT.

To investigate the structural basis of this heterogeneity, and its potential relationship to the dynamic gating behavior, cryo-EM datasets for Cx46/50 at low pH were analyzed using 3D variability analysis (3DVA)51 (Fig. 5). The primary principal component revealed NT domains sampling both open and gated conformations symmetrically across the channel, accompanied by conformational changes in TM2 (Supplementary Movie 1), consistent with the structural rearrangements described above.

Particles corresponding to distinct states along the principal component were pooled for 3D reconstruction and used to estimate approximate state distributions (Fig. 5a; see Methods). Across all low-pH datasets (Collections #1, #4, and #5), 43.2% ± 2.3% of particles were resolved in the lipid-mediated gated-state, while 18.2% ± 5.3% represented the open-state (mean ± s.e.m.; n=3) (Fig. 5b). Notably, the open-state resolved at low pH was nearly identical to the stabilized open-state at pH 7.4 (Cα r.m.s.d. = 0.2 Å; Supplementary Fig. 6) and lacked pore-bound lipids.

In addition to the stabilized open and gated-states, a third state, termed the “destabilized open-state,” was also identified along the same principal component, accounting for 38.5% ± 6.8% of low-pH particles (n=3) (Fig. 5a, b). An atomic model built into a 3D reconstruction resolved to 2.2 Å (from Collection #1) showed the destabilized open-state resembles the stabilized open-state (Cα r.m.s.d. = 0.7 Å; Supplementary Fig. 6), but with subtle distinguishing differences in the NT and TM2 domains, as well as in the hydrogen bond patterns defining the TM1 π-helix kink at residues ~39–41 (Fig. 5c; Supplementary Figs. 4–6).

In the destabilized open-state, the NT remains anchored to the lumen by hydrophobic residues (W4, L7, and L11) but adopts a distinct conformation, with a local Cα r.m.s.d. of 1.9 Å relative to the stabilized open-state NT. TM2 shifts ~1.6 Å toward the pore center and bends by ~6.4°, resembling but less pronounced than the movements of the gated-state (Fig. 5c). This conformation introduces a pore-constriction site at S5, reducing the pore diameter to 9.7 Å in Cx46, which is narrower than the stabilized open-state (11.6 Å) but still sufficient for permeation of hydrated ions.

Rare states detected at neutral pH
Previously, under neutral pH cryo-EM conditions, we have only detected the stabilized open-state of Cx46/5040,41. Consistent with this, heterogeneous refinement and 3DVA performed on neutral pH nanodisc datasets (Collections #3 and #6), as well as the amphipol low-pH dataset (Collection #2), revealed exclusively open-state channels with minimal NT heterogeneity, with variability limited to a slight “wobble” of TM2 (Supplementary Movie 1, Supplementary Figs. 8 & 9).

However, we hypothesized that this approach may have lacked the sensitivity to detect rare conformational states that could still be present at neutral pH. To enhance our ability to resolve potential low-abundance states, we combined particles from both neutral and low pH nanodisc datasets (Collections #3, #4, and #5, #6), thereby increasing the representation of various conformational states in the pool of particles. We then reanalyzed the combined particle set using 3DVA, leveraging the well-sampled conformational states from the low pH particles to guide the detection of rare, structurally similar states within the neutral pH subset. This strategy successfully resolved all three states (stabilized open, destabilized open, and gated), allowing for their classification and independent reconstruction from particles derived exclusively from the neutral pH dataset (Fig. 5a; see Methods).

This analysis further detail the differences in state distributions between neutral and low pH datasets. At neutral pH, 87.0% ± 9.5% of particles were in the stabilized open-state, 10.7% ± 8.1% in the destabilized open-state, and only 2.4% ± 1.4% in the lipid-mediated gated-state (n=2) (Fig. 5a, b). In the sample that was never exposed to low pH (Collection #3), the stabilized open-state accounted for 96.4% of particles, with the destabilized open-state and lipid-mediated gated-state representing 2.6% and 1.0%, respectively (Fig. 5a, b; Supplementary Fig. 8).

The distribution of open-state channels at neutral pH closely aligns with single-channel electrophysiology studies conducted under minimal transjunctional voltage26,31,48–50, underscoring the agreement between structural and functional analyses. In contrast, the observed proportion of low pH-gated-state channels deviates from expectations from electrophysiology studies, which would predict a predominance of closed channels at the low pH conditions used for cryo-EM32–35. These discrepancies may stem from the absence of transjunctional voltage in cryo-EM conditions, as well as other methodological differences and limitations to cryo-EM classification methods52. Indeed, the population states identified in our analyses are intended to reflect overall trends rather than precise measurements, given the inherent noise in the raw data. Alternatively, they may indicate additional underlying heterogeneity in pH-gating transitions that was not fully captured by this approach (see below).

Non-cooperative gating of the NT-domain
In addition to symmetric gating movements of the NT on both sides of the Cx46/50 intercellular channel, 3DVA of the low-pH datasets (Collections #4 and #5) also revealed asymmetric gating movements of the NT domain, with each hemichannel operating independently (Fig. 6a; Supplementary Movie 1). To further deconvolute the gated-state heterogeneity and to investigate potential cooperativity within hemichannels, cryo-EM density for individual subunits was isolated using symmetry expansion and signal subtraction, followed by 3D classification on the individual subunits. This approach distinguished subunits in the gated and open-states based on NT characteristics, though differences between stabilized and destabilized open-states could not be confidently resolved (Fig. 6b; Supplementary Figs. 3, 8, 9). Among signal-subtracted subunits from low pH datasets, 67.0% ± 0.5% (mean ± s.e.m.; n=3; Collections #1, #4 and #5) were identified in the gated-state. In contrast, the same workflow applied to neutral pH datasets (Collections #3 and #6) exclusively identified subunits in the open-state conformation (Supplementary Figs. 8, 9).

To quantify the frequency of open and gated-state monomers within parent gap junction particles, metadata from the symmetry expansion protocol were analyzed (see Methods). Remarkably, fully gated states were rare, comprising only 1.7% ± 0.2% of gap junction particles and 14.4% ± 0.6% at the hemichannel level (mean ± s.e.m.; n=3) (Fig. 6c, d). Subunit distributions in the gated-state followed a Boltzmann-like pattern, with nine gated subunits being the most frequent configuration (23.4% ± 0.3%; n=3) (Fig. 6c). A similar trend was observed at the hemichannel level, with five gated subunits being most prevalent (33.3% ± 0.5%; n=3) (Fig. 6d). States containing two to four gated subunits per hemichannel were observed in multiple configurations, and their populations closely matched predicted values for random distribution (Fig. 6e).

Notably, no fully open-state channels were observed under low-pH conditions, and <0.5% were identified with 1–3 gated protomers. These findings suggest that NT pH-gating occurs independently between neighboring monomers under cryo-EM conditions, supporting a non-cooperative gating mechanism. Moreover, the prevalence of subunits adopting a gated conformation under acidic conditions more closely aligns with functional results, particularly considering models that suggest just a single subunit may be sufficient to initiate gating of gap junction channels36 (see Discussion).

Comparison to other connexin structures
The pH-gated structure of Cx46/50 shares similarities as well as notable differences with previously reported gap junction structures nominally obtained under chemical gating conditions, such as Cx26 at pH 6.419, Cx26 treated with CO2 maintained at pH 7.453,54 and Cx43 at pH 8.055. The TM and EC domains of Cx46/50 align closely with these structures, with Cα r.m.s.d. values (excluding the NT and cytoplasmic portion of TM2) ranging from 0.6–0.8 Å. In contrast, significant variations in NT conformations are evident across these structures; yet, the NT consistently contributes to the formation of the primary constriction site of the channel (Fig. 7a, b).

Among the compared structures, the NT of Cx26 treated with CO₂ at pH 7.4 is most similar to the pH-gated NT of Cx46/50 (Cα r.m.s.d. = 3.6 Å). In contrast, the largest deviation is observed in Cx43 (Cα r.m.s.d. = 13.4 Å), where the NT adopts a near-parallel orientation relative to the membrane bilayer. Interestingly, the NT of Cx46/50 shows substantial deviations from the pH-gated Cx26 structure (Cα r.m.s.d. = 11.5 Å), although their proximal positions indicate a shared role in pH gating (Fig. 7a, b). However, some discrepancy between these studies may reflect the limited resolution of the cryo-EM density used for modeling the Cx26 pH-gated state19.

Correlated with NT displacement, distinct conformational changes in TM2 were also observed. Compared to the stabilized open-state of Cx46/50, all other models display a more bent conformation localized to the cytoplasmic region of TM2 beyond the conserved proline kink (P88 in Cx46/50; P87 in Cx26 and Cx43) (Fig. 7a). When compared to this region of TM2 in the pH-gated state of Cx46/50, Cx43 at pH 8.0 showed the smallest deviation (Cα r.m.s.d. = 1.7 Å), followed by Cx26 treated with CO₂ (Cα r.m.s.d. = 4.4 Å), while Cx26 at low pH exhibited the largest deviation (Cα r.m.s.d. = 7.2 Å). These observations support a likely conserved mechanistic feature, where a hinge-like motion of TM2 is coupled to the conformational state of the NT.

Several recent connexin structures obtained under a variety of conditions have resolved lipids or detergents within the pore, though the source and conditions favoring their presence have been unclear (Fig. 7c, d)39. Similar to Cx46/50, lipids have been modeled at the NT docking site along TM1/2 in Cx2654, Cx3656,57 and Cx4355, where their presence correlates with NT displacement from the stabilized open-state observed in Cx46/50 (Fig. 7d). Additionally, stabilized lipids (or detergents) have been identified near the IL binding site of Cx46/50 in other connexin structures, including Cx2653,54, Cx3656,57, and Cx4355 (Fig. 7d), consistent with the relatively high conservation at these lipid binding sites (Supplementary Fig. 6). While the precise source of lipids from previous studies remains uncertain, these consistent observations suggest a conserved role for lipids in intercalating between subunits and modulating NT conformational states across diverse connexin isoforms.

Methods
Membrane scaffold protein expression and purification
A plasmid containing the sequence for a histidine tagged MSP1E1 was obtained from Addgene70 and the protein was expressed and purified as previously described with minor modification41,71. Freshly transformed E. coli cells (BL21 Gold-DE3, Agilent, Catalog #230130) were grown in LB medium supplemented with 50 μg mL−1 kanamycin at 37°C while being shaken at 250 r.p.m. After reaching an OD of 0.5–0.6, cultures were induced with 0.5 mM IPTG and allowed to express for 3 hrs post-induction at 37°C. Cells were harvested by centrifugation at 4000 x g for 20 min at 4°C, and cell pellets were resuspended in ~4 mL per gram of cell pellet of MSP Lysis Buffer (40 mM Tris, pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF)). Cell suspensions were flash frozen in liquid nitrogen and stored at -80°C until needed.

Frozen cell suspensions were supplemented with 1 mM PMSF while thawing on ice and lysed by sonication. Crude lysate was cleared by ultracentrifugation at 150,000 x g for 30 min at 4°C. Sodium chloride was added to the supernatant for a final concentration of 300 mM. The supernatant was filtered (Millipore, 0.22 μm) and applied to a gravity column packed with HisPur Ni-NTA resin (Thermo Fisher Sci) that was prepared with Equilibration buffer (40 mM Tris, pH 7.4, and 300 mM NaCl) at a ratio of 1 mL packed resin per gram of cell pellet. The supernatant was batch bound to the resin for 1 hour at 4°C with gentle rocking.

The MSP-bound resin was washed with 5 column volumes (CV) of Equilibration buffer, followed by 5 CV of the following buffers: Triton buffer (40 mM Tris pH 8.0, 300 mM NaCl, 1% (vol vol−1) Triton X-100), Equilibration buffer, Cholate buffer (40 mM Tris pH 8.0, 300 mM NaCl, 50 mM cholate), Equilibration buffer, Imidazole Wash Buffer (40 mM Tris pH 8.0, 300 mM NaCl, 50 mM imidazole) and Equilibration buffer. MSP1E1 was eluted from the resin with 3 CV of Elution Buffer (40 mM Tris pH 8.0, 300 mM NaCl, 500 mM imidazole).

Eluate was concentrated to ~5 mg mL−1 (UV 280) using a 20 kDa m.w.c.o. spin concentrator (Vivaspin 6, Sartorius), filtered (Millipore, 0.22 μm) and applied to a gel filtration column (ENrich SEC 70; BioRad) using a fast protein liquid chromatography (FPLC) (NGC system; BioRad) equilibrated with 20 mM HEPES pH 7.4 and 150 mM NaCl. All chromatography steps were performed at 4°C. Peak fractions were monitored by UV 280, pooled, and concentrated to 300–500 μM using a 20 kDa m.w.c.o. spin concentrator. Samples were then aliquoted, flash frozen with liquid nitrogen and stored at -80°C until needed.

Cx46/50 purification
Native Cx46/50 gap junctions were isolated from sheep lens fiber cells40,41. Fresh sheep eyes were obtained from Nebraska Scientific (Omaha, NE). Lenses were removed using a surgical blade and stored at -80°C. After thawing, the core lens fiber tissue, containing Cx46 and Cx50 intercellular channels that are C-terminally truncated (MP38)42 was dissected from the lens cortical tissue with a surgical blade. The lens core tissue was then Dounce homogenized, and stripped membranes were prepared as previously described40,41. Stripped membranes were stored at -80°C in Storage Buffer (10 mM Tris pH 8.0, 2 mM EDTA, 2 mM EGTA) at a total protein concentration of 2 mg mL−1 (BCA, Pierce).

Thawed membranes were solubilized with 10 mM Tris pH 8.0, 2 mM EDTA, 2 mM EGTA, and 1% (wt vol-1) n-decyl-β-D-maltoside (DM) for 30 min at 37°C with gentle mixing. Insoluble material was cleared by ultracentrifugation at 150,000 x g for 30 min at 4°C, followed by filtration (Millipore, 0.22 μm). The supernatant was then separated by anion exchange chromatography at 4°C (UnoQ, BioRad). Buffer A (10 mM Tris pH 8.0, 2 mM EDTA, 2 mM EGTA, 0.3% (wt vol−1) DM) was run for 20 CV after sample application, and protein was eluted with Buffer B (same a Buffer A with the addition of 1 M NaCl) using a gradient elution (to 35% Buffer B) over 20 CV. Peak fractions containing detergent-solubilized Cx46/50 were concentrated to 150–300 μM as determined by UV 280, using a 50 kDa m.w.c.o. spin concentrator (Vivaspin 6, Sartorius).

Reconstitution of Cx46/50 into lipid nanodiscs
Freshly prepared Cx46/50 was reconstituted at pH 7.4 into MSP1E1 nanodiscs using dimyristoyl-phosphatidylcholine (DMPC) lipids as previously described41, with minor modifications. Briefly, chloroform-solubilized DMPC (Avanti) was dried under a stream of nitrogen gas, creating a thin film, and left under vacuum overnight to remove residual solvent. The DMPC thin film was solubilized in 20 mM HEPES, pH 7.4, 150 mM NaCl, and 5% (wt vol−1) DM to a final concentration of 35 mM. Detergent-solubilized DMPC was then sonicated two-times for 20 minutes at 37°C with vortexing in between the sonication steps.

Solubilized Cx46/50 was mixed with the solubilized DMPC at a molar ratio of 0.6:90 (Cx46/50:DMPC) for 60 minutes with gentle rotation at 37°C. Purified MSP1E1 was then added for a final molar ratio of 0.6:1:90 (Cx46/50:MSP1E1:5 mM DMPC) for 15 minutes with gentle rotation at 37°C. Detergent was removed with an overnight incubation with SM-2 Bio-Beads (BioRad) at a ratio of 30:1 beads:DM (wt:wt) with gentle inversion at 37°C. Bio-beads were removed by filtration, and fresh beads were added for an additional 60 minutes of gentle rotation at 37°C. The beads were removed by filtration, and insoluble material was removed by centrifugation at 16,150 x g for 30 mins at 4°C.

The supernatant at pH 7.4 was filtered (Millipore, 0.22 μm) and applied to an FPLC gel filtration column (Superose 6 Increase 10/300 GL, GE Healthcare) equilibrated with Low pH buffer (20 mM Succinate pH 5.8, 150 mM NaCl, 2 mM EDTA, and 2 mM EGTA). Peak fractions containing Cx46/50 embedded in nanodiscs were combined and concentrated using a 50 kDa m.w.c.o. spin concentrator (Vivaspin 6, Sartorius) to a final concentration of 1.8–2.2 mg mL−1 as determined by UV 280. All chromatography steps were performed at 4°C.

We note that although a different buffer system was used at low pH (succinate versus HEPES), the absence of gating transitions for Cx46/50 in amphipols under these same low-pH conditions (described below) support the conclusions made in this work that the observed conformational changes are pH-dependent rather than buffer-specific.

Reconstitution of Cx46/50 into amphipol
Freshly purified Cx46/50 was exchanged from DM to amphipol (PMAL-C8, Anatrace) as previously described40, with minor modification. Peak fractions containing Cx46/50 (determined by SDS-PAGE) from anion exchange chromatography (see above) were combined and applied to a gel filtration column (ENrich SEC 650, Bio-Rad) equilibrated with 20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA and 0.3% DM (wt vol-1). Peak fractions containing DM solubilized Cx46/50 were pooled and combined with amphipol at a ratio 3:1 amphipol:Cx46/50 (wt:wt). The mixture was gently rotated overnight at 4°C, then Bio-beads were added at a ratio of 30:1 (wt:wt) beads:DM for 60 minutes at 4°C with gentle rotation. The beads were removed by filtration and further cleared by centrifugation at 16,150 x g for 30 mins at 4°C. The supernatant containing Cx46/50 in amphipol at pH 7.4 was then filtered (Millipore, 0.22 μm) and then applied to a gel filtration column (ENrich SEC 650, Bio-Rad) equilibrated with Low pH buffer (20 mM Succinate pH 5.8, 150 mM NaCl, 2 mM EDTA, and 2 mM EGTA). Peak fractions containing Cx46/50 in amphipol were pooled and spin concentrated (Vivaspin, 50 kDa m.w.c.o., Satorius) to 1.3 mg mL−1. Protein concentration was determined by UV 280, and sample purity was assessed by SDS-PAGE. All chromatography was done at 4°C.

Controls for lipid-mediated pH-gating
After nanodisc reconstitution of Cx46/50 and removal of Bio-beads at pH 7.4, as described above, the sample was then filtered (Millipore, 0.22 μm) and split in half. Half the reconstitution was applied to an FPLC gel filtration column (Superose 6 Increase 10/300 GL, GE Healthcare) equilibrated with Low pH buffer (20 mM Succinate pH 5.8, 150 mM NaCl, 2 mM EDTA, and 2 mM EGTA). The other half was applied to a gel filtration column equilibrated with Neutral pH buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, and 2 mM EGTA). For each pH condition, peak fractions containing Cx46/50 embedded in nanodiscs were combined and concentrated to a final concentration of 1.8–2.2 mg mL−1 as determined by UV 280 using a 50 kDa m.w.c.o. spin concentrator (Vivaspin 6, Sartorius) and sample purity was assessed by SDS-PAGE. All chromatography steps were performed at 4°C.

Reversible lipid-mediated pH-gating
To test whether lipid mediated gating was reversible, fresh Cx46/50 was reconstituted into nanodiscs at pH 7.4 as described above with the minor modification that after reconstitution the entire sample of Cx46/50 embedded in nanodiscs was applied to a gel filtration column (Superose 6, GE Healthcare) equilibrated in Low pH buffer (pH 5.8) to put the channels under gating conditions. Peak fractions containing Cx46/50 embedded in nanodiscs at low pH were pooled, divided in half, and then dialyzed (0.5–1 kDa m.w.c.o. Float-A-Lyzer G2; Spectrum Laboratories) overnight at 4°C with gentle stirring in either Neutral pH buffer or Low pH buffer (control). The ratio of dialysis buffer to sample was 100:1 (vol:vol). Dialysis buffer was exchanged after 2 hrs, and again after dialyzing overnight at 4°C with gentle stirring. After 2 hrs of dialysis with fresh buffer the following morning, the sample was recovered and concentrated to 1.8–2.1 mg mL−1 as determined by UV 280, using a spin concentrator (Vivaspin, 50 kDa m.w.c.o., Satorius).

Negative stain electron microscopy
Cx46/50 embedded in lipid nanodiscs or solubilized with amphipol were prepared for negative stain EM, as previously described40,41. Briefly, 3 μL of sample (~0.02 mg mL−1) was applied to a glow-discharged continuous carbon-coated EM grid (Ted Pella, G400), blotted with filter paper, and washed three times with gel filtration buffer. The sample was then stained with freshly prepared uranyl formate (0.75% wt vol-1, SPI-Chem). Negatively stained specimens were imaged on a 120 kV TEM (iCorr, Thermo Fisher Scientific) at a nominal 49,000 x magnification at the specimen level using a 2k x 2k CCD camera (Eagle 2K TEM CCD, Thermo Fisher Scientific) with a calibrated pixel size of 4.37 Å and with a nominal defocus of -1.5 μm, performed at OHSU’s Multiscale Microscopy Core.

Cryo-EM specimen preparation and data collection
Samples were prepared for cryo-EM using 3 μL of freshly purified Cx46/50 embedded in nanodisc (1.8–2.2 mg mL-1) or amphipol (1.3 mg mL-1) applied to glow-discharged holey carbon grids (Quantifoil, R1.2/1.3 or R2/1). All cryo-samples were vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific) at 20°C with 100% humidity and were blotted for 4–4.5 sec prior to being plunged into liquid ethane and stored in liquid nitrogen.

All cryo-EM specimens were imaged at the Pacific Northwest Cryo-EM Center (PNCC) on a Titan G4 Krios cryo-TEM (Thermo Fisher) operated at 300 kV. Movies for the amphipol dataset were recorded on a Gatan K2 summit direct electron detector at a nominal magnification of 29,000 x (calibrated pixel size = 0.665 Å pixel−1) in counting mode, with an energy filter set to a 20 eV slit width. Nanodisc datasets were collected on a Gatan K3 direct electron detector at 29,000 x or 105,000 x nominal magnification (calibrated pixel size = 0.788 or 0.822 Å pixel−1) in super resolution mode, with energy filter slit widths of 10-20 eV. Data collections used 45 to 55 frames, and total doses ranged from 40–50 e-Å-2. Datasets of 555–12423 movies were collected using SerialEM72, with nominal defocus ranges of -0.8 to -2.2 μm or -0.5 to -1.8 μm. See Supplementary Table 1 for details of each dataset.

Cryo-EM image processing
All datasets were processed in CryoSPARC (v4.4.1)73, and pre-processing was carried out in the same fashion across all datasets. Dose weighting and beam-induced motion correction were done using Patch motion correction. Micrographs were binned to their nominal pixel size (2x super resolution pixel size) during Patch motion correction, here referred to as the unbinned pixel size. Contrast transfer function (CTF) estimation was performed by Patch CTF estimation.

All datasets were processed using the same routine and the same masks were used across datasets. Processing of Cx46/50 at low pH embedded in DMPC nanodiscs (Collection #1) will be described in detail as the representative dataset. Details for individual datasets are shown in the image processing workflow (Supplementary Figs. 3, 7–9). Using a subset of micrographs, semi-automated particle picking was used to create a preliminary 3D reconstruction for template particle picking on the entire dataset, resulting in 5,572,213 particle picks from 7072 micrographs. After multiple rounds of 2D classification and removing duplicate particles, 685,022 bona-fide particles (binned to 2.4 Å pixel-1) were selected to generate an ab-initio model.

This was followed by a round of homogeneous refinement using D6 symmetry, unbinning of the particles (to 0.802 Å pixel−1) and a round of non-uniform refinement (D6 symmetry), leading to a 2.14 Å consensus reconstruction. The particles and map from the consensus refinement were used in heterogeneous refinement with 6 classes (D6 symmetry) with a target resolution of 4 Å. Non-uniform refinements were then applied to all six classes, independently, using unbinned particle stacks and applying D6 symmetry. Particle stacks from 3D refinements that went below 3 Å (classes #0, #1, and #4, 476,719 particles total) were combined, down-sampled (1.54 Å pixel-1, box 200 pixels), and subjected to another round of non-uniform refinement (D6 symmetry), resulting in a 3.16 Å resolution 3D reconstruction. The variability of this down-sampled stack of particles was then assessed at the whole particle level by 3D Variability Analysis (3DVA)51 to guide the classification of various states for final 3D refinement.

3D variability analysis and classification of states
3DVA in CryoSPARC was used to parse through the conformational heterogeneity in the NT-gating domain at the gap junction level, and classify the gated-, destabilized open-, and stabilized open-states. The particle stack from the binned non-uniform refinement (box 200 pixels, 1.54 Å/pix) was symmetry expanded using D6 symmetry, yielding a particle stack of 5,720,628 particles prior to 3DVA. A custom 10 Å low-pass filtered mask that excluded the nanodisc was generated in CryoSPARC with an 8 Å resolution molmap created in ChimeraX using Cx46 (7JKC) that had additional residues placed into the pore to generate density to cover the channel entrances. 3DVA was performed using a target resolution of 4 Å, and principal components (PC) of interest were selected and further analyzed. Ultimately, in every dataset, the primary PC was analyzed, as well as PCs that demonstrated asymmetric gating (Collections #4 and #5).

The intermediate 3DVA display job type was then used to classify the states of interest. The 3DVA display job was used to separate the PC into nine frames/particle stacks by setting the rolling window to zero and outputting particle subsets, generating a unique stack of particles along the PC of interest. For PC1, which demonstrated symmetric opening/closing on both sides of the channel, each frame’s particle stack was independently locally refined (D6 symmetry) using the same mask used for 3DVA, duplicate particles were removed, and then particles were unbinned for a final round of non-uniform refinement with D6 symmetry applied. Particle stacks yielding reconstructions of similar states (i.e., similar NT conformation) were then combined. Three distinct states were identified (gated, destabilized open, and stabilized open). The stabilized open-state map had a final resolution of 2.5 Å with 42,646 particles, the destabilized open-state map had a final resolution of 2.2 Å with 218,088 particles, and the gated-state map had a final resolution of 2.2 Å with 198,875 particles (gold-standard FSC) (Supplementary Table 1). Maps corresponding to the stabilized-open and destabilized-open states were used for final modeling. For final refinement of the gated-state conformation, a final map was obtained by the subunit enrichment protocol, described below.

For the asymmetrically gated state, where only one hemichannel of the intercellular channel is gated, both endpoint frames (i.e., frame 0 and frame 8) were independently subjected to local refinement using C6 symmetry. These refinements were performed using the same mask applied for 3DVA. Duplicate particles were removed from both particle stacks, which were then unbinned and subjected to a final round of non-uniform refinement with C6 symmetry imposed. This approach yielded two heterotopically gated 3D reconstructions: frame 0 yielded a 2.4 Å map from 162,334 particles, and frame 8 yielded a 2.5 Å map from 121,746 particles. The reconstruction corresponding to frame 0 was selected for model building (Supplementary Table 1). Pooling particles from both frames did not substantially improve the reconstructions.

Merging cryo-EM datasets for identification of rare neutral pH states
To computationally isolate rare states at neutral pH, datasets from neutral and low pH conditions were merged and subjected to 3DVA. This was achieved by merging cryo-EM datasets that were prepared from the same specimen, collected on the same cryo-electron microscope with the same settings (i.e., Collections #3 and #4 were merged, and Collections #5 and #6 were merged). As an example, Collections #5 and #6, belonging to the reversible lipid-mediated pH-gating experiment, will be described in detail. The unbinned particle stacks (0.8016 Å pixel−1, box 384 pixels) obtained after heterogeneous refinement from the respective datasets were merged. The low pH control dataset (Collection #5) contributed 764,205 particles, and the neutral pH particle stack (Collection #6) contributed 427,873 particles, for a total of 1,192,078 particles.

The merged particle stack was down-sampled (box 160, 1.97 Å/pix) and subjected to a round of non-uniform refinement (D6 symmetry), and then symmetry expanded using D6 symmetry prior to 3DVA. The symmetry expanded particles were then analyzed by 3DVA with a target resolution of 4 Å, using the same mask used for the 3DVA on the independent datasets described above. PCs of interest were split into 9 frames/particle stacks along the trajectory using the intermediate 3DVA display output. Each particle stack, containing particles from both low and neutral-pH datasets, was locally refined independently. The particles were then reverted to their original datasets and deduplicated to undo symmetry expansion.

The separated datasets were then unbinned, and individual particle stacks were subjected to non-uniform refinement (D6 symmetry). Particle stacks demonstrating similar states were pooled, and a final round of non-uniform refinement (D6 symmetry) was performed. Using this approach, all three states (gated, destabilized open, and stabilized open) were identified in the neutral pH datasets, revealing rare populations of the gated-state at neutral pH (1% for Collection #2 and 4% for Collection #5), which were previously not observed in these datasets using other common approaches of 3D classification. To assess for influence of initial map bias on end state maps, initial maps of either open or gated states and initial low-pass filters (16 Å) were both assayed as starting models, yielding similar results.

Subunit classification
Subunit-level heterogeneity was analyzed using signal subtraction and 3D classification in CryoSPARC. This workflow was applied to particles from Collections #1, #3, #4, #5, and #6. The description here focuses on the workflow for Collection #4, as the exemplar approach.

A non-uniform refinement particle stack containing 476,719 high-resolution particles was down-sampled by 2x binning (1.6 Å pixel−1, 240-pixel box size). A subset of 100,000 randomized particles (~21% of the total) was symmetry expanded (D6 symmetry), generating 1.2 million particles that were then subjected to local refinement using D6 symmetry. A mask for signal subtraction was generated in ChimeraX by creating an 8 Å molecular map (molmap), generated from overlaid monomer models of the stabilized open and gated states. After signal subtraction, particles were re-centered and subjected to 3D classification without alignment. Classification parameters were optimized as follows: three classes, a target resolution of 3 Å, an O-EM learning rate of 0.3, an O-EM batch size of 2000 particles, no per-particle scale factors, an initial low-pass filter resolution of 8 Å, and hard classification. Open and gated states were distinguished based on the position of the NT gating domain.

To determine the distribution of open and gated-state monomers within gap junction particles, metadata from the signal-subtracted 3D classified particles were analyzed. The following metadata fields were extracted into a CSV file from each class’s exported particle.cs file using our “Metadata_Extraction” python script: uid, sym_expand/idx, and sym_expand/src_uid. Metadata from similar classes were combined to generate concatenated files, representing open and gated states, respectively.

The frequency of open and gated-states within gap junction particles and hemichannels was analyzed independently. An in-house developed bash script, “Subunit State Frequency”, was used to calculate the frequency of ‘sym_expand/src_uid’ occurrences to determine the distribution of open and gated monomers in gap junctions. For example, if a gated monomer’s ‘sym_expand/src_uid’ appeared 4 times, then 4 of the 12 subunits in the gap junction were classified as gated. To analyze hemichannels, another custom bash script, “Gap Junction Splitter”, was employed to split gap junction particles into their respective hemichannels based on the ‘sym_expand/idx’ metadata. The resulting hemichannel metadata was then processed using the same Subunit State Frequency script to evaluate the distribution of open and gated monomers within the individual hemichannels of the parent gap junction particles. Frequency data for both gap junctions and hemichannels were subsequently plotted to visualize the distribution of gated states across the dataset.

To investigate potential cooperativity of subunit gating across hemichannels within gap junction particles, a custom bash script, “Hemichannel Arrangement”, was developed to identify the relative positions of open/gated conformers and applied to the gated-state particle metadata. Initially, gap junction particle metadata was split into hemichannels using the Gap Junction Splitter script described above. Subsequently, the metadata for each hemichannel was analyzed independently to evaluate the relative positions of gated subunits in hemichannels containing two, three, or four gated subunits. The Hemichannel Arrangement script determined the number of gated subunits in each hemichannel by counting the occurrences of unique ‘sym_expand/src_uid’ IDs. For hemichannels with two to four gated subunits, the script analyzed the specific arrangements of gated subunits based on the combination of ‘sym_expand/idx’ IDs. Each unique arrangement was tallied to quantify the distribution of gated-state configurations within the hemichannel metadata. These results provided insights into the spatial organization and potential cooperativity of subunit gating across the hemichannels.

Subunit enrichment of gated-state channels
Subunit-level heterogeneity was analyzed using signal subtraction and 3D classification in CryoSPARC. This workflow was applied to particles from Collections #1, #3, #4, #5, and #6. The description here focuses on the workflow for Collection #4, as the exemplar approach.

A non-uniform refinement particle stack containing 476,719 high-resolution particles was down-sampled by 2x binning (1.6 Å pixel−1, 240-pixel box size). A subset of 100,000 randomized particles (~21% of the total) was symmetry expanded (D6 symmetry), generating 1.2 million particles that were then subjected to local refinement using D6 symmetry. A mask for signal subtraction was generated in ChimeraX by creating an 8 Å molecular map (molmap), generated from overlaid monomer models of the stabilized open and gated states. After signal subtraction, particles were re-centered and subjected to 3D classification without alignment. Classification parameters were optimized as follows: three classes, a target resolution of 3 Å, an O-EM learning rate of 0.3, an O-EM batch size of 2000 particles, no per-particle scale factors, an initial low-pass filter resolution of 8 Å, and hard classification. Open and gated states were distinguished based on the position of the NT gating domain.

The 3D classification results were exported from CryoSPARC, and the particle.cs files corresponding to gated subunits, identified through visual inspection in ChimeraX, were extracted using the “Metadata_Extraction” script (described above). These gated-state metadata were then processed using a “Metadata_Enrichment” script we developed to select for gap junctions containing 10, 11, or 12 gated subunits, as determined by empirical testing, to yield the highest-quality map features for the gated-state. This enrichment strategy resulted in a final dataset of 106,741 gap junction particles selected for final refinement.

To generate a particle stack for refinement, the enriched gated-state src_uids were used to filter an exported particle.cs metadata file from a previous unbinned 3D consensus refinement using a “Metadata_Filter” python script we developed. The filtered metadata were reimported into CryoSPARC, and final non-uniform refinements were performed using either D6 or C1 symmetry. The final D6-symmetrized 3D reconstruction of the enriched gated-state was refined to a global resolution of 2.3 Å (compared to 2.8 Å with C1 symmetry). This reconstruction, along with its locally filtered post-processed volume, was used for atomic model building of the low pH gated-state structure (Supplementary Table 1).

Atomic modeling and validation
Previously published models of Cx46 (7JKC) and Cx50 (7JJP) in the stabilized open-state were used as starting points for atomic modeling in Coot74. For the gated and destabilized open states, the NT domains were pruned to the 20th residue and rebuilt into the respective maps. To avoid bias, all histidines were modeled in the protonated state for low pH datasets. Models were then iteratively refined in Phenix75, Isolde76, and Coot until convergence of statistics determined by MolProbity77. After the Cx46 models' statistics converged, point mutations were introduced in Coot to convert to the Cx50 isoform, and the model refinement/validation process was repeated until convergence (Supplementary Table 1). All final models lack residues corresponding to the intracellular loop (ICL; residues 105-138 in Cx46 and 105-150) and the CT domain (beyond residue 223 in Cx46 and 235 in Cx50), which were unresolved in the density maps. For the gated-state models of both Cx46 and Cx50, residues 2–3 were not modeled, and the side chains of residues 4-16 in the NT domain were truncated to the Cβ position, reflecting limited local resolution in this region.

Structural analysis and visualization of Coulombic surface potentials were performed in ChimeraX78. Pore profiles were analyzed using the program HOLE79. For this analysis, the sidechains were pruned to the Cα positions to normalize for differences in side chain completeness. Sequence alignments were created using Clustal-w80 and visualized in Jalview81.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Please find the attached doc file.", "reviewer_2": "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_3": "The manuscript by Jarodsky et al is a very interesting article describing the difference in structures of the connexin46/50 gap junction at high and low pH. In a lipid environment (nanodiscs made of DMPC, rather than the native environment) the change of pH causes the infiltration of lipids into the pore and causes apparent closure of the channels caused by an upward movement of the N-terminal helix. Surprisingly, this is reversible.\n\nThe study is interesting. The possible involvement of lipids in the regulation of connexins and other large pore channels appears to be a recurring theme in the recent structural studies that have been carried out. The link between pH and lipids has not been observed before. Within the limits of the experimentation (using nanodiscs with DMPC rather than native lipids) the experiments seem well done with extensive classification of cryo-EM data sets.\n\nThere are a few points that need to be addressed:\n\nThe high pH buffer is HEPES. However, instead of using a similar buffer such as MES, (that could be used at pH 5.8), the authors use a completely different buffer by swapping to succinate. It is therefore possible that the change in buffering system affects the structure as well as, or even instead of, the pH? Given that they see the same conformation in amphipols using “Low pH buffer” as they see in HEPES, this is probably not an issue, but I note in their methods that the “Low pH buffer” is not explicitly defined when discussing the amphipols experiments. The buffer needs to be defined and some comment should be made regarding the differing buffering systems.\n\nThey discuss the possibility of His95 and His17 being important for pH sensitivity and gating. As they note in the results section, the literature surrounding this is not clear cut. However, the discussion is written in such a way that leads the reader to believe that the structures reported here explain how the residues contribute to pH sensitivity. This is slightly misleading.\n\nThere is only minimal comparison with other connexin structures. While I wouldn’t expect a detailed comparison of all the different structures, the movement of TM2 around the proline seems similar to that reported for Cx26 (Brotherton, et al Structure, 2022, eLife, 2024) and movements of TM2 are also mentioned for Cx43 (Lee et al, Nat Comm, 2023).\n\nIn Figure 1b, a “pore plug” is mentioned at low threshold levels. Is this perhaps an artefact of the symmetry?"}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "Reviewer Report\n\nSummary\nJarodsky et al. investigate the mechanism of pH-dependent gating of connexin-46/50 using cryo-electron microscopy and complementary biochemical analyses. While the topic is timely and of general interest, the manuscript lacks conceptual novelty, methodological rigor, and a coherent mechanistic narrative. Several conclusions are speculative and not sufficiently supported by the data presented.\n\nMajor Comments\n1. Inconsistencies between hypothesis\nThe authors initially identify connexin-46/50 as forming homomeric gap junctions and present maps and models generated using D6 symmetry. They later combine datasets and identify asymmetric channel conformations, concluding that the presence of fully symmetric gated gap junctions are rare. However, Figures 6c and 6d indicate that only about 1.7% of the particles adopt a symmetric gated conformation, while the majority are asymmetric.\n\nIn the first part of the manuscript, the discussion focuses solely on the D6-symmetry imposed maps, which are later revealed to represent a mixture of symmetric and asymmetric conformations. Therefore, drawing mechanistic conclusions solely from the D6-symmetry imposed “gated” state is misleading. The manuscript should clearly distinguish whether the hemichannels or the complete gap junctions exhibit symmetric or asymmetric conformations under neutral and acidic conditions.\n\nSeparate analyses of the symmetric and asymmetric maps are necessary to achieve a more accurate and detailed mechanistic understanding. Furthermore, reporting the distinct asymmetric states (such as two-, three-, or four-gated conformations) would greatly enhance the novelty and mechanistic clarity of the study.\n\n2. Figures and data presentation\nThe figures do not effectively support the stated conclusions. In several panels, the superposition of two structures is indistinguishable due to similar color schemes, key residues discussed in the text are missing from the figures, and the transparent surface rendering gives a misleading impression of lipid density by exaggerating weak features. Overall, the visual presentation suffers from inadequate color contrast, missing labels, and unconvincing densities, which significantly reduce the clarity and impact of the structural claims.\n\n3. Lack of a coherent narrative or conclusion\nThe manuscript reads as a collection of loosely connected observations rather than a cohesive mechanistic story. The discussion reiterates prior literature but does not integrate the new results into a unified model. There is no clear mechanism explaining how pH modulates gating through structural transitions.\n\nAdditionally, connexin-43 (Cx43) structures (e.g., Lee et al., Nat. commun., 2023: PMID: 36805660) have demonstrated multiple hemichannel conformations and well-defined lipid densities that are consistently positioned along the transmembrane helices. In contrast, the lipids modeled in the present work appear in opposite orientations, with no supporting hypothesis or discussion about lipid entry, exit, or rearrangement during conformational change. This omission represents a major conceptual gap, as the supposed lipid-mediated gating cannot be reconciled with the structural evidence or prior connexin studies.\n\n4. Lack of novelty and conceptual advance\nThe manuscript reiterates mechanisms already reported for connexin-46/50 and connexin-26 (e.g., Flores et al., 2020; Brotherton et al., 2022; Meigh et al., 2013). The central premise of lipid-mediated gating under acidic conditions has been previously proposed and partially resolved at comparable resolution in other connexin studies(Lee et al., Nat. commun., 2023). The current study does not significantly extend that understanding—either mechanistically or structurally—and thus offers limited conceptual advancement.\n\nComparative context:\nSimilar pH-dependent gating transitions have been described in multiple connexins.\nConnexin-26 exhibits proton-induced N-terminal helix rearrangements leading to a closed conformation (Brotherton et al., 2022, Nat. Commun.), while connexin-43 and connexin-32 show analogous low-pH gating transitions modulated by protonation of cytoplasmic residues (Meigh et al., 2013, J. Biol. Chem.; Nielsen et al., 2017, eLife). The “neutral destabilized open” and “acidic gated” states proposed here are thus not unique to connexin-46/50 and were previously observed in both homomeric and heteromeric gap junctions. Consequently, the mechanistic insight presented does not constitute a substantive advance over these earlier studies and largely restates already known features of connexin gating.\n\nReferences:\n• Flores, J. E. et al. Structural basis of pH gating in connexin-46/50 gap junction channels. Nat. Commun. 11, 505 (2020).\n• Brotherton, D. H. et al. Proton-dependent conformational rearrangements underlie connexin-26 gating. Nat. Commun. 13, 2750 (2022).\n• Meigh, L. et al. pH-dependent modulation of connexin-32 and connexin-43 by intracellular protons. J. Biol. Chem. 288, 22758–22767 (2013).\n• Nielsen, M. S. et al. Structural basis of pH gating and regulation in connexins. eLife 6, e20213 (2017).\n• Myers, J. B. et al. Structural basis of lipid-mediated gating and multiple conformational states of connexin-43 hemichannels. Science 381, 512–518 (2023).", "reviewer_2": "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_3": "The authors have addressed the reviewers comments."}, "author_response": "Point-by-point Response\nWe thank the reviewers for their thoughtful and constructive feedback, which has significantly improved the clarity and quality of our manuscript. In response, we have revised the text, figures, and methods, as well as additional new data to address all major concerns.\nNotably, we have further refined our gated-state structure and now present a more conservative model that better reflects the local resolution limitations within the NT gating domain. We have also expanded structural comparisons to other connexin isoforms and gating mechanisms, clarified key interpretations and methodological details, and strengthened our discussion of pH- and lipid-mediated gating. In addition, we now explicitly acknowledge key limitations of the current study. A detailed, point-by-point response to each reviewer comment is provided below.\nReviewers #1/2:\nThe authors have used sophisticated cryo-EM techniques to investigate the reversible, lipid-mediated pH-gating of connexin-46/50. While they have attempted several approaches to resolve various states, there are several critical and major concerns that need to be addressed. In general, to support most of their conclusions, they need to improve the quality of the maps in certain important regions, which are central to the discussion in this manuscript. Many statements are overly speculative and not supported by map densities. The authors either need to collect more data and classify structural heterogeneity more effectively or moderate their claims accordingly.\nMajor Concerns:\n1. MSP Belt Tightness & Nanodisc Stability\no The MSP belt appears too tight, which may influence conformational changes in the N-terminal (NT) domain, particularly at low pH, where it is proposed to be stabilized by lipids.\no Nanodiscs do not behave well at low pH, and MSP stability decreases under such conditions, potentially affecting lipid entry. As a result, the lipid density observed near the NT may not be physiologically relevant.\nResponse: We thank the reviewers for raising these concerns regarding the use of nanodiscs and the potential influence of MSP belt tightness and pH-dependent instability on our structural observations, which we address below. As for the overarching concern, that our in vitro nanodisc systems may not fully replicate the physiological membrane environments, we certainly agree and we explicitly acknowledge this limitation in the manuscript, pg. 12:\nHowever, it is important to note that a direct role of lipids in gap junction gating remains to be validated through cellular functional studies.\npg. 15\nMoreover, as noted above, the physiological relevance of lipid-mediated gating remains to be established in cellular systems, including investigations into potential lipid specificity and defining the translocation pathway(s) into and out of the pore.\nRegarding MSP belt tightness: While the reviewers don’t provide their rationale for making this assertion, we respectfully note that the use of MSP1E1 in our system resulted in highly homogeneous populations of gap junction channels in the open state under neutral pH conditions. Notably, our structures of Cx46/50 in MSP1E1 nanodiscs remain, to our knowledge, the only system to provide such a well-defined and consistent open-state conformation of the NT-domain for gap junction channels under non-gating conditions. Furthermore, our 3D variability analysis (3DVA) demonstrates that even under these conditions conformational flexibility persists, especially in TM2 and other TMs, indicating that intrinsic dynamics are preserved and not overly constrained by the nanodisc scaffold. In fact, we have unpublished data showing that nanodiscs using MSP1E1 can contain more than one Cx46/50 channel, and still preserve the channels in a stabilized open-state.\nRegarding MSP behavior at low pH: we understand the concern that potential destabilization of the MSP belt could, in principle, influence lipid redistribution. However, several lines of evidence strongly argue against this, or for it being a dominant contributor to the observed lipid infiltration or NT gating:\n1. Reversibility: A key finding of our study is the reversible nature of the lipid-dependent gating transition. Upon restoring samples from low pH to neutral pH, >96% of channels return to the open conformation, with concomitant removal of pore-bound lipids (a key result and aspect of rigor that has been missing from other gap junction structural studies that observe lipids inside the channel pore). This reversibility strongly supports a non-artifactual mechanism and preserved integrity of our nanodisc system.\n2. Detergent Controls: In parallel experiments using amphipol-reconstituted channels, we show that low pH alone was insufficient to induce pore closure, strongly supporting our conclusion that lipids are required for this gating transition.\n3. Stability at Low pH: Although not shown in the original manuscript, we have additional data demonstrating that Cx46/50 nanodiscs reconstituted with MSP1E1 and DMPC are stable at pH 5.8 for extended periods (≥1 week at 4°C), as assessed by negative stain EM (image now included in our revised Extended Data Fig. 1). These findings further support the stability and suitability of our system under the experimental conditions used.\n4. We would further note that this same MSP system has recently been used by our lab to capture a distinct gating mechanism in Cx46/50 in response to Ca2+, where NT movement is similarly induced but without clear evidence of lipid infiltration (Flores et al. BioRxiv 2025).\nTaken together, we believe the combined biochemical, structural, and control data, and orthogonal gating studies all support the validity of our conclusions that pH-gating of Cx46/50 is reversibly facilitated by lipids when using the selected MSP-nanodisc system. Nevertheless, we have revised the text to more clearly acknowledge the limitations regarding physiological relevance.\n2. NT Helix Density & Pore Radius Calculation\no Despite the overall high resolution, the NT region exhibits weak density, indicating flexibility and structural heterogeneity.\no Discussions regarding NT interactions lack strong map support.\no The weak density in this region also calls into question the accuracy of pore radius calculations, which may lead to erroneous interpretations.\nResponse: We appreciate the reviewers’ careful assessment of the NT domain and agree that this region remains structurally heterogeneous, limiting our initial interpretations. As part of our revised analysis, we undertook more extensive particle classification routines to further enrich for the fully gated conformation (see response at the end, for details), yet the NT-domain continued to exhibit limited resolvability for many sidechain features, consistent with their intrinsic flexibility and/or positional variability under gating conditions.\nTo better reflect the limitations in map quality within the NT, we have revised our structural models of the gated state by pruning unresolved NT sidechains to their Cβ atoms, consistent with the lack of resolvable sidechain features in this region. Residues 2-3 also remain unresolved and are not modeled, consistent with our original models. These modeling decisions are now explicitly stated in the revised Methods section, Main text and related Figure legends.\nIn response to the reviewers’ valid concern regarding interpretation of NT interactions, we have removed prior descriptions of inter-subunit NT interactions from both the main text and Fig. 1, as we agree that the available map density does not support such specific conclusions.\nRegarding the pore radius calculations: we also agree with this concern. As such, the previous pore profile analyses (Fig. 1i and Fig. 7b) have been replaced with revised panels (new Fig. 1i and Fig. 7b) that are meant to illustrate the displacement of the NT helices from the open-state conformation, measured as the change in Cα position. We believe this more conservative and structurally grounded analysis more accurately reflects the intended purpose of this analysis for comparing the observed conformational changes of the NT-domain.\n3. Lipid Density & Model Reliability\no The authors claim to have built 400 lipid molecules, but most of the observed density corresponds only to the acyl chains, making this an overstatement.\no The modelled head groups lack clear supporting density, making inferred interactions unreliable.\no Weak lipid density at the pore suggests low occupancy. Further classification or additional data collection may reveal particle classes without lipids, which would impact the proposed model.\nResponse: We thank the reviewers for raising these points, addressed below.\nClarification of number of lipids. We agree, our initial wording may have unintendedly implied the extent to which full lipid molecules were resolved. As revised, we now explicitly state that the majority of lipid densities correspond primarily to the acyl chain regions, with a limited subset displaying resolved lipid headgroups. Our updated description on pg. 4 now reads:\nOver 400 lipid densities (mostly single acyl chains of DMPC) and >900 water molecules were also resolved, with extracellular leaflet lipid chains exhibiting long-range hexagonal packing, consistent with previous observations41.\nRegarding lipid headgroups/interactions. As part of our revised analysis, we performed more focused particle classification to enrich for channels with gated subunit conformations (see concluding response, below). This led to modest improvements in map quality for the subset of lipid headgroups that were resolved. We have accordingly updated our main text and Figures to reflect these new map densities, and we address the remaining limitations with revised descriptions of lipid interactions to emphasize proximity to protein residues, rather than definitive atomic distances. For example, we now state that certain lipid headgroups are “located in close proximity to” or “adjacent to” protein residues, rather than asserting precise distances.\nPore-bound lipid densities. Even after further refinement and improvement of the pore-bound lipid densities, we agree that they remain weaker than expected for a well-defined/stabilized interaction. However, we interpret this not as an indication of low lipid occupancy, but rather as a reflection of conformational heterogeneity in lipid binding within the pore.\nThis interpretation is supported by multiple observations:\n1. No lipid-free gated state detected: Across all classifications and datasets, we do not observe a gated NT conformation in the absence of pore-bound lipid density (even at neutral pH, the rare gated-state that was classified showed pore-bound lipid densities).\n2. Amphipol control: In amphipol-reconstituted samples, where lipid entry into the pore is excluded, low pH alone does not induce the NT gating transition – highlighting the necessity of lipids for the observed conformational change.\n3. Energetic considerations: The gated NT conformation exposes hydrophobic surfaces on both the NT and TM1/2 lining the pore lumen, which would be energetically unfavorable in the absence of lipid occupation. Thus, it is unlikely that the observed conformation could be stabilized without the presence of lipids – or some other molecule/driving force to support this state.\nWe have updated our discussion of these results and interpretations – see for example, pg. 5:\nAlthough the local resolution of these lipids is somewhat limited, likely reflecting positional and/or conformational heterogeneity, substantial regions were sufficiently resolved to permit interpretation for atomic modeling.\n4. Plug-Like Density & Symmetry Artifacts\no The central plug-like density, interpreted as pore occlusion, was identified using D6 symmetry. However, symmetry axes often exhibit artificial densities that may not reflect the true protein structure. The authors should verify whether this density persists in symmetry-free reconstructions.\nResponse: We agree. Notably, in our initial analysis, the plug-like density within the pore was observed in both D6 and C1 reconstructions, leading us to consider it a potentially relevant feature. However, following the reviewers’ suggestion and as part of our revised analysis, we performed additional particle classification to further enrich for gated-state channels, and re-refined both C1 and D6 maps using these more focused particle sets. Both the C1 and D6 maps are now prominently displayed in Fig. 1. Notably, in the resulting maps, the central plug-like density is no longer observed in either symmetry condition, indicating that this feature was either a reconstruction artifact or reflected a component of heterogeneity that had been removed upon further classification. We have therefore removed all references to the plug-like density and its potential interpretation from the revised manuscript, including associated figure panels and discussion.\n5. Heterogeneous Structural States & Data Processing\no The manuscript discusses multiple states leading up to the \"Low pH Induced an Ensemble of Gated and Open States\" but later concludes that these states are mixtures of different conformations.\no Since the earlier structures likely represent averaged density maps, their significance is diminished.\no The study should focus on asymmetric subunit conformations rather than enforcing D6 symmetry after identifying asymmetric particles.\no Structural heterogeneity is still evident, as indicated by standard deviations. The authors must precisely isolate symmetric or asymmetric but homogeneous particles for refinement to ensure the final maps represent distinct states accurately.\nResponse: We thank the reviewers for raising this point. Indeed, the structural heterogeneity (particularly at low pH) is a key feature of this system and we agree that this complicates interpretation of symmetry-imposed reconstructions. In response, we have revised the manuscript to better delineate the distinct insights gained from both symmetric and asymmetric processing strategies.\nThe D6-symmetrized reconstructions were critical for defining the high-resolution features of the most primary conformational states, specifically the stabilized-open, destabilized-open and gated-state configurations. These idealized models allowed us to identify key structural rearrangements, including pore-lining lipids and the more nuanced changes in the NT and TM2 for the destabilized-open state, which we were not able to resolve at lower resolutions achieved by symmetry-expansion and focused refinement methods performed on single subunits.\nImportantly, by leveraging D6-symmetry, this approach also enabled us to detect rare gated-state and destabilized-open state populations under neutral pH conditions (transitions that were also not evident in our asymmetric processing approach).\nHowever, we fully acknowledge that these D6-refined structures do not represent the dominant conformational state of channels under low pH conditions. Our subunit-focused classification and symmetry expansion analyses revealed that most channels exist in asymmetric, heterogeneous ensembles consisting of both open and gated subunits. This ensemble-level heterogeneity was essential for demonstrating that under cryo-EM conditions gating in Cx46/50 is not a fully concerted or cooperative process.\nMoreover, we would like to emphasize that this level of conformational heterogeneity, and the use of both symmetric and asymmetric processing approaches, reflects an ongoing challenge in the field of gap junction structural biology. Similar degrees of structural heterogeneity have been reported in other connexin structures, including Cx26, Cx32, Cx36 and Cx43, where authors similarly employed symmetry-imposed reconstructions to resolve high-resolution states, alongside C1 or subunit-level analyses to reveal asymmetric features. Our approach is therefore consistent with established methodological practices used to address the inherent complexity of these high-order oligomeric membrane proteins.\nWe have updated the manuscript to:\n• Clearly state that D6 reconstructions represent idealized endpoints rather than the dominant ensemble.\n• Clarify how each analytical approach (D6 vs. C1) contributes complementary insights.\n• Reference comparable examples in the field where similar methodological strategies have been employed.\npg. 13-14\nThis ensemble-level heterogeneity also highlights an important methodological point. It is important to emphasize that the D6- (or C6-) symmetrized cryo-EM reconstructions presented in this work do not represent the dominant conformations present under low pH conditions. Rather, these idealized reconstructions serve as high-resolution endpoints that highlight key structural rearrangements, including lipid interactions and NT/TM2 conformational changes, which were otherwise obscured at the lower resolutions obtained through our focused subunit-level analyses. Consistent with this, our subunit-level classification approaches revealed that the majority of channels adopt heterogeneous assemblies composed of both open and gated subunits. Together, these complementary strategies provide a more complete view of the gating process, where D6/C6-symmetrized reconstructions define high-resolution detail, while subunit-based analyses capture ensemble-level heterogeneity. Similar methodological strategies have been employed in recent studies of other connexins (e.g., Cx26, Cx32, Cx36, Cx43)53-56,67, underscoring both the complexity of gap junction structural characterization and the value of integrating symmetric and asymmetric approaches.\nWe appreciate the reviewers’ constructive critique, which has helped us clarify the interpretation and limitations of our data and to place our work in the broader context of current methodological approaches to connexin structural studies.\n6. Neutral pH Data Processing\no What happens when the neutral pH dataset is processed separately?\no Would this yield a neutral pH open state? Why was it combined with the low pH dataset?\nResponse: We thank the reviewers for the opportunity to clarify these points regarding our data processing strategy.\nThe neutral pH datasets (collections #3 and #6) were indeed processed independently prior to employing our mixed-dataset approach for classification of rare-states, as described in our Methods section. When analyzed separately (e.g., using heterogeneous refinement, 3DVA, or symmetry expansion and signal subtraction followed by focused 3D classification) we consistently observed only the primary conformation of the stabilized open-state (see Fig. 3, Extended Data Fig. 8, Extended Data Fig. 9, and Supplemental Movie 1). These findings are consistent with our previously published structural characterization of Cx46/50 in MSP1E1 nanodiscs under similar neutral pH conditions (Flores et al., 2020).\nTo enhance our ability to identify and resolve minor states that we hypothesized may be present, but only poorly sampled under these conditions, we implemented a combined dataset processing strategy. Specifically, we pooled particles from both neutral and low pH datasets to leverage the dominant state captured under each condition (i.e., open at neutral pH and gated at low pH). This approach allowed us to leverage the major population from one condition to help isolate the minor population from the other. Notably, this strategy was successful and enabled us to extract a rare population of lipid-mediated gated-state particles present under neutral pH conditions, which we were unable to resolve using conventional approaches on the neutral dataset alone.\nWe hope this explanation resolves any confusion. We have further clarified this rationale and strategy in the revised text on pg. 9.\nRare states detected at neutral pH\nPreviously, under neutral pH cryo-EM conditions, we have only detected the stabilized open-state of Cx46/5040,41. Consistent with this, heterogenous refinement and 3DVA performed on neutral pH nanodisc datasets (Collections #3 and #6), as well as the amphipol low-pH dataset (Collection #2), revealed exclusively open-state channels with minimal NT heterogeneity, with variability limited to a slight “wobble” of TM2 (Supplemental Movie 1, Extended Data Fig. 8 & 9).\nHowever, we hypothesized that this approach may have lacked the sensitivity to detect rare conformational states that could still be present at neutral pH. To enhance our ability to resolve potential low-abundance states, we combined particles from both neutral and low pH nanodisc datasets (Collections #3, #4 and #5, #6), thereby increasing representation of various conformational states in the pool of particles. We then reanalyzed the combined particle set using 3DVA, leveraging the well-sampled conformational states from the low pH particles to guide the detection of rare, structurally similar states within the neutral pH subset. This strategy successfully resolved all three states (stabilized open, destabilized open, and gated) allowing for their classification and independent reconstruction from particles derived exclusively from the neutral pH dataset (Fig. 5a; see Methods).\n7. Resolution Claims & Model Interpretation\no Line 133: The claim that heteromeric/-typic assembly patterns of Cx46/50 were indistinguishable is questionable.\no At 2.2 Å resolution, individual residues should be distinguishable. If they are not, the reported resolution may be overestimated or your maps are still averaged density.\nResponse: We appreciate the reviewers’ comments and welcome the opportunity to clarify these points.\nAt the global resolution of 2.2 Å (now 2.3 Å in our revised gated-state map), most side chains (particularly in well-ordered regions) are clearly resolved, as shown in Extended Data Fig. 5. At positions conserved between Cx46 and Cx50, side chains are well defined. In contrast, density is weaker or ambiguous at isoform-divergent positions, consistent with averaging across two nearly identical sequences rather than a limitation of global map quality.\nOur statement that heteromeric/-typic assemblies are “indistinguishable” refers not to resolution, but to the intrinsic limitations of current cryo-EM classification methods when applied to systems with high sequence identity and compositional heterogeneity. Although our prior cross-linking mass spectrometry confirmed the presence of heteromeric Cx46/50 channels (Myers et al., Nature 2018), isoform-specific features remain too subtle and spatially dispersed to support reliable classification. Cx46 and Cx50 share over 80% sequence identity across structured regions, and the few differing residues are often solvent-exposed (i.e., conformationally dynamic), and sparsely distributed – further reducing the signal available to discriminate subunit identity.\nThis challenge is further compounded by the combinatorially large number of possible subunit arrangements in a dodecameric assembly. Similar issues have been reported for co-expressed Cx26 and Cx32, where high-resolution AFM and Fab-labeling (Naulin et al., J. Biol. Chem. 2021) demonstrated largely stochastic subunit incorporation, underscoring the broader challenge of resolving compositional heterogeneity in such systems by cryo-EM.\nNotably, these limitations persist even at higher resolution. In our prior study using datasets refined to 1.9 Å (Flores et al., Nat. Commun. 2020), we observed similar ambiguity at isoform-divergent positions, and provide detailed map-to-model comparisons to illustrate these limitations. The challenges/limitations of this system have also been described in detail in our inaugural structural characterization (see Myers et al. Nature 2018).\nWe are currently developing Fab-based fiducial labeling strategies to enable isoform-specific assignment in future cryo-EM studies, though these efforts are still in early stages and beyond the scope of the current work. Given our previous detailed analysis and description of these limitations with native Cx46/50 channels, we do not feel the need to delve into such detail in this current work. We have revised the manuscript text to clarify that the inability to distinguish heteromeric/-typic assemblies arises from both the high structural similarity of the isoforms and the methodological limitations of cryo-EM, rather than an overstatement of resolution. We also display map-to-model quality at these sites of sequence similarity and difference in Extended Data Fig. 5.\npg. 4\nHeteromeric/-typic assembly patterns of Cx46 and Cx50 could not be distinguished from the cryo-EM data, consistent with our prior studies40,41, reflecting the >80% sequence identity across structured regions of the two isoforms and methodological limitations of current cryo-EM classification methods.\nMinor Points:\n• The methods section should clearly explain how asymmetric maps were generated.\nDone\n• Lines 40–43: The statement is unclear and should be reworded for clarity.\nDone\n• Line 1162: Specify which signal was subtracted.\nDone\n• Lines 965–967: The observed density alone is insufficient to confirm occlusion.\nRemoved\n• Line 290: Indicate the corresponding supplementary movie number.\nDone\n• Line 555: Clarify whether affinity purification or ion exchange chromatography was used.\nDone\n• Figure 6: Adjust the lipid color scheme for better visibility.\nDone\nIn summary, the authors need to improve map quality, particularly in critical regions, to support their conclusions. Additional data collection and classification could resolve heterogeneity and strengthen structural interpretations.\nResponse: We appreciate the reviewers’ overarching concern and agree that addressing structural heterogeneity is essential for supporting the mechanistic conclusions of our study. As noted, the degree of conformational variability in our data is both a challenge and, in our view, an informative feature of the system, shedding light on the ensemble-based nature of pH- and lipid-mediated gating in connexin channels.\nIn response to the reviewers’ recommendation, we have implemented additional data processing strategies and expanded our classification efforts beyond what was included in the original submission. Specifically, to improve the refinement of the fully gated state, we applied a revised workflow that builds on our subunit-level classification approach. This included a substantial increase in data usage: instead of a 100k particle subset, we now applied symmetry expansion, signal subtraction, and 3D classification across the full low-pH dataset (476,719 particles, symmetry-expanded to over 5 million subunits).\nWe then filtered for channels containing ≥10 gated subunits (empirically determined to be optimal for maximizing particle count while limiting contamination by ungated subunits), yielding a set of 106,741 channels, which we refined to generate a more homogenous representation of the fully gated state. While this approach did not significantly improve the resolvability of NT sidechains, it did enhance the definition of pore-bound lipid headgroup density – an important feature of our lipid-mediated gating model. This revised refinement also led to the disappearance of the previously reported plug-like density at the symmetry axis (as described in responses to Points #4 and #5 above), further improving the interpretability of this conformation. This updated approach is detailed in the revised Methods section, and associated Extended Data Figure 3.\nWe also note that despite these advances, the subunit-level classification approach did not resolve the nuanced difference between the stabilized- and destabilized open-states. For this reason, we have retained the original models for the open and destabilized-open states, which were derived using 3DVA-based classification.\nWe hope the reviewers recognizes our continued and rigorous efforts to resolve the substantial intrinsic heterogeneity of Cx46/50 at low pH. We believe our combined use of high-resolution symmetry-imposed models and subunit-level ensemble analysis represents the most comprehensive and robust depiction of the gating transitions accessible from these datasets.\n---\nReviewer #3:\nThe manuscript by Jarodsky et al is a very interesting article describing the difference in structures of the connexin46/50 gap junction at high and low pH. In a lipid environment (nanodiscs made of DMPC, rather than the native environment) the change of pH causes the infiltration of lipids into the pore and causes apparent closure of the channels caused by an upward movement of the N-terminal helix. Surprisingly, this is reversible.\nThe study is interesting. The possible involvement of lipids in the regulation of connexins and other large pore channels appears to be a recurring theme in the recent structural studies that have been carried out. The link between pH and lipids has not been observed before. Within the limits of the experimentation (using nanodiscs with DMPC rather than native lipids) the experiments seem well done with extensive classification of cryo-EM data sets.\nThere are a few points that need to be addressed:\n1. The high pH buffer is HEPES. However, instead of using a similar buffer such as MES, (that could be used at pH 5.8), the authors use a completely different buffer by swapping to succinate. It is therefore possible that the change in buffering system affects the structure as well as, or even instead of, the pH? Given that they see the same conformation in amphipols using “Low pH buffer” as they see in HEPES, this is probably not an issue, but I note in their methods that the “Low pH buffer” is not explicitly defined when discussing the amphipols experiments. The buffer needs to be defined and some comment should be made regarding the differing buffering systems.\nResponse: We thank the reviewer for this insightful comment. Indeed, MES would have provided a more chemically similar buffering system to HEPES and could have minimized the possibility of buffer-specific effects in our experimental design. However, as the reviewer also notes, the lack of gating transition of Cx46/50 in amphipol under low pH conditions using the same succinate-based buffer system strongly suggests that the observed gating transition is driven by pH and not an artifact of the buffer composition.\nWe have revised the Methods section to explicitly define the low pH buffer used in our amphipol experiments. Additionally, we now include a brief comment in the manuscript noting that, although a different buffer system was used at low pH, the absence of gating transitions for Cx46/50 in amphipols under these same low-pH conditions supports the conclusion that the observed conformational changes are pH-dependent rather than buffer-specific.\npg. 19\nWe note, that although a different buffer system was used at low pH (succinate versus HEPES), the absence of gating transitions for Cx46/50 in amphipols under these same low-pH conditions (described below) supports the conclusions made in this work that the observed conformational changes are pH-dependent rather than buffer-specific.\n2. They discuss the possibility of His95 and His17 being important for pH sensitivity and gating. As they note in the results section, the literature surrounding this is not clear cut. However, the discussion is written in such a way that leads the reader to believe that the structures reported here explain how the residues contribute to pH sensitivity. This is slightly misleading.\nResponse: We agree, our original discussion may have inadvertently implied a more definitive role for His95 and His17 in pH sensing than is currently supported by the structural data or literature. We have revised the relevant section of the Discussion to clarify that while these residues are candidates for contributing to pH sensitivity, their roles remain unresolved and require further experimental validation. We now emphasize that our structures provide a framework to guide future studies but do not yet establish a direct mechanistic role for these histidines in gating.\npg. 15\nWhile these structural interactions support our proposed gating mechanism and underscore the potential importance of conserved histidine residues in pH-sensing, the functional roles of these histidine residues remain unresolved and further studies are needed to validate the specific protonation events involved and their mechanistic consequences. This includes exploring the possible contribution of non-histidine residues, such as N-terminal acidic residues68,69.\n3. There is only minimal comparison with other connexin structures. While I wouldn’t expect a detailed comparison of all the different structures, the movement of TM 2 around the proline seems similar to that reported for Cx26 (Brotherton, et al Structure, 2022, eLife, 2024) and movements of TM 2 are also mentioned for Cx43 (Lee et al, Nat Comm, 2023).\nResponse: We appreciate this suggestion and have expanded our comparative analysis in our revised Results subsection, “Comparison to other connexin structures.” In this section, in addition to comparing NT conformation and lipid binding sites, we include both structural and quantitative assessments of TM2 orientation, highlighting conserved movement around the hinge-proline (P88 in Cx46/50), which aligns with prior observations in Cx26 and Cx43 (Brotherton et al., 2022, eLife, 2024; Lee et al., 2023).\npg. 11\nCorrelated with NT displacement, distinct conformational changes in TM2 were also observed. Compared to the stabilized open-state of Cx46/50, all other models display a more bent conformation localized to the cytoplasmic region of TM2 beyond the conserved proline kink (P88 in Cx46/50; P87 in Cx26 and Cx43) (Fig. 7a). When compared to this region of TM2 in the pH-gated state of Cx46/50, Cx43 at pH 8.0 showed the smallest deviation (Cα r.m.s.d. = 1.7 Å), followed by Cx26 treated with CO₂ (Cα r.m.s.d. = 4.4 Å), while Cx26 at low pH exhibited the largest deviation (Cα r.m.s.d. = 7.2 Å). These observations support a likely conserved mechanistic feature, where a hinge-like motion of TM2 is coupled to the conformational state of the NT.\nWe also discuss this NT–TM2 coupling in our discussion, where we further note that the role of the conserved proline in voltage gating was originally proposed in Cx26 and 32 (Suchyna et al., 1993; Ri et al., 1999), and our recent cryo-EM study of Cx46/50 under Ca2+-gating conditions (Flores et al., 2025) reveals a similar displacement of TM2. These comparisons reinforce a broader model in which NT-TM2 coupling serves as a conserved mechanism for connexin gating across different isoforms and stimuli.\npg. 13\nOur findings, together with prior cryo-EM studies, highlight a consistent pattern of coordinated movement between TM2 and the NT, facilitated by a conserved proline kink (P88 in Cx46/50) that serves as a hinge element19,53-55,62. This hinge confers structural flexibility of TM2, allowing it to accommodate NT rearrangements during pH-dependent gating transitions (Fig. 8a,b). Notably, missense mutations at this site in Cx50 have been linked to congenital cataracts63,64, underscoring the physiological relevance. Mutagenesis studies at the homologous proline site in Cx26 and Cx32 previously implicated this site as a transduction element in voltage-gating65,66. Consistently, TM2 displacement originating from P88 is also observed in our recent cryo-EM structure of Cx46/50 in the presence of Ca2+ 62. Collectively, these observations support a conserved mechanism of NT-TM2 coupling via the P88 hinge that modulates connexin gating under diverse physiological conditions.\n4. In Figure 1b, a “pore plug” is mentioned at low threshold levels. Is this perhaps an artefact of the symmetry?\nResponse. We agree (see also our response to Reviewers 1/2 on this topic). We thank both sets of reviewers for prompting this clarification.\nNotably, in our initial analysis, the plug-like density within the pore was observed in both D6 and C1 reconstructions, leading us to consider it a potentially relevant feature. However, following the reviewers’ suggestion and as part of our revised analysis, we performed additional particle classification to enrich for fully gated channels, and re-refined both C1 and D6 maps using these focused particle sets. Both C1 and D6 maps are now prominently displayed in Fig. 1. In the resulting maps, the central plug-like density is no longer observed in either symmetry condition, indicating that this feature was either a reconstruction artifact or reflected a component of heterogeneity that had been removed upon further classification. We have therefore removed all references to the plug-like density and its potential interpretation from the revised manuscript, including associated figure panels and discussion.\nPoint-by-Point Response\nReviewer #3 confirmed all their concerns were fully resolved and now supports publication of the revised manuscript.\nReviewer #1/2 provided additional comments, which we address in our point-by-point response below. In doing so, we highlight clear examples of fabricated references used by these Reviewers to question the novelty and significance of our findings. This behavior constitutes a serious breach of scientific integrity and substantially undermines the credibility of the reviewers’ entire evaluation, which as we detail below, is problematic throughout.\n--\nReviewer Report\nSummary\nJarodsky et al. investigate the mechanism of pH-dependent gating of connexin-46/50 using cryo-electron microscopy and complementary biochemical analyses. While the topic is timely and of general interest, the manuscript lacks conceptual novelty, methodological rigor, and a coherent mechanistic narrative. Several conclusions are speculative and not sufficiently supported by the data presented.\nMajor Comments\n1. Inconsistencies between hypothesis\nThe authors initially identify connexin-46/50 as forming homomeric gap junctions and present maps and models generated using D6 symmetry. They later combine datasets and identify asymmetric channel conformations, concluding that the presence of fully symmetric gated gap junctions are rare. However, Figures 6c and 6d indicate that only about 1.7% of the particles adopt a symmetric gated conformation, while the majority are asymmetric.\nIn the first part of the manuscript, the discussion focuses solely on the D6-symmetry imposed maps, which are later revealed to represent a mixture of symmetric and asymmetric conformations. Therefore, drawing mechanistic conclusions solely from the D6-symmetry imposed “gated” state is misleading. The manuscript should clearly distinguish whether the hemichannels or the complete gap junctions exhibit symmetric or asymmetric conformations under neutral and acidic conditions.\nSeparate analyses of the symmetric and asymmetric maps are necessary to achieve a more accurate and detailed mechanistic understanding. Furthermore, reporting the distinct asymmetric states (such as two-, three-, or four-gated conformations) would greatly enhance the novelty and mechanistic clarity of the study.\nResponse: This concern was already raised by the reviewers in their first round of comments. We responded to it directly and in detail in our original point-by-point rebuttal, and we made extensive revisions to the manuscript to address it.\nWe believe these efforts have fully addressed this concern. For completeness and transparency, we refer the editorial team and reviewers to our initial response, included as an Appendix below.\n2. Figures and data presentation\nThe figures do not effectively support the stated conclusions. In several panels, the superposition of two structures is indistinguishable due to similar color schemes, key residues discussed in the text are missing from the figures, and the transparent surface rendering gives a misleading impression of lipid density by exaggerating weak features. Overall, the visual presentation suffers from inadequate color contrast, missing labels, and unconvincing densities, which significantly reduce the clarity and impact of the structural claims.\nResponse: We disagree:\n• Structural Superpositions & Contrast: The structures being compared are clearly distinguished using high-contrast representations (e.g., white versus dark gray – see Fig. 1d, Fig. 4d).\n• Residue Labeling: All residues and protein domains discussed in the main text are appropriately labeled in the corresponding figures.\n• Lipid Density Representation: Contrary to the reviewers’ statement, lipid densities are shown in full opacity in all main figures. Additionally, model-to-density fitting for these key lipids is provided in Extended Data Fig. 5, supporting our interpretations.\n3. Lack of a coherent narrative or conclusion\nThe manuscript reads as a collection of loosely connected observations rather than a cohesive mechanistic story. The discussion reiterates prior literature but does not integrate the new results into a unified model. There is no clear mechanism explaining how pH modulates gating through structural transitions.\nAdditionally, connexin-43 (Cx43) structures (e.g., Lee et al., Nat. commun., 2023: PMID: 36805660) have demonstrated multiple hemichannel conformations and well-defined lipid densities that are consistently positioned along the transmembrane helices. In contrast, the lipids modeled in the present work appear in opposite orientations, with no supporting hypothesis or discussion about lipid entry, exit, or rearrangement during conformational change. This omission represents a major conceptual gap, as the supposed lipid-mediated gating cannot be reconciled with the structural evidence or prior connexin studies.\nResponse: The claims of omission and that our manuscript lacks mechanistic detail are completely unfounded. We provide extensive structural evidence and interpretation regarding lipid orientation, our proposed mechanism of proton-mediated lipid entry/exit, and detail lipid rearrangements involved in the gating transition. Critically, we demonstrate reversibility of the lipid-mediated gating in response to pH changes, a central and novel finding that underscores the physiological relevance of the mechanism. To our knowledge, this study provides the first high-resolution structural characterization of a pH-dependent conformational change in any connexin isoform. Thus, our work offers a substantial mechanistic and conceptual advance in the field of gap junction biology.\nWe encourage editorial team and the reviewers to revisit the relevant sections of the manuscript, which directly address the concerns raised.\n4. Lack of novelty and conceptual advance\nThe manuscript reiterates mechanisms already reported for connexin-46/50 and connexin-26 (e.g., Flores et al., 2020; Brotherton et al., 2022; Meigh et al., 2013). The central premise of lipid-mediated gating under acidic conditions has been previously proposed and partially resolved at comparable resolution in other connexin studies(Lee et al., Nat. commun., 2023). The current study does not significantly extend that understanding—either mechanistically or structurally—and thus offers limited conceptual advancement.\nComparative context:\nSimilar pH-dependent gating transitions have been described in multiple connexins. Connexin-26 exhibits proton-induced N-terminal helix rearrangements leading to a closed conformation (Brotherton et al., 2022, Nat. Commun.), while connexin-43 and connexin-32 show analogous low-pH gating transitions modulated by protonation of cytoplasmic residues (Meigh et al., 2013, J. Biol. Chem.; Nielsen et al., 2017, eLife). The “neutral destabilized open” and “acidic gated” states proposed here are thus not unique to connexin-46/50 and were previously observed in both homomeric and heteromeric gap junctions. Consequently, the mechanistic insight presented does not constitute a substantive advance over these earlier studies and largely restates already known features of connexin gating.\nReferences:\n• Flores, J. E. et al. Structural basis of pH gating in connexin-46/50 gap junction channels. Nat. Commun. 11, 505 (2020).\n• Brotherton, D. H. et al. Proton-dependent conformational rearrangements underlie connexin-26 gating. Nat. Commun. 13, 2750 (2022).\n• Meigh, L. et al. pH-dependent modulation of connexin-32 and connexin-43 by intracellular protons. J. Biol. Chem. 288, 22758–22767 (2013).\n• Nielsen, M. S. et al. Structural basis of pH gating and regulation in connexins. eLife 6, e20213 (2017).\n• Myers, J. B. et al. Structural basis of lipid-mediated gating and multiple conformational states of connexin-43 hemichannels. Science 381, 512–518 (2023).\nResponse: The assertions made in this critique are demonstrably false and appear to be constructed around the titles of fabricated references (presumably hallucinated by a generative AI tool). The titles cited in support of the reviewers’ arguments (highlighted above) do not exist in the scientific literature and are deeply misleading.\nStrikingly, several of these fictitious citations list members of our own research group as first authors (e.g., Flores, JE; Myers, JB), despite the fact that no such studies have been written, submitted, or published by our lab. One of the citations, attributed to Flores et al., even appears to be a fabrication of the title of our current manuscript, and used as the basis for arguing a lack of novelty.\nThis egregious misconduct constitutes a fundamental breach of scholarly integrity and severely compromises the validity and motivation of the reviewers’ arguments.\nAPPENDIX\nHere, we provide our initial response to these Reviewers concerns regarding the presentation, analysis, and interpretation of structural heterogeneity, which we believe has already been fully and faithfully addressed in our revised manuscript.\n5. Heterogeneous Structural States & Data Processing\no The manuscript discusses multiple states leading up to the \"Low pH Induced an Ensemble of Gated and Open States\" but later concludes that these states are mixtures of different conformations.\no Since the earlier structures likely represent averaged density maps, their significance is diminished.\no The study should focus on asymmetric subunit conformations rather than enforcing D6 symmetry after identifying asymmetric particles.\no Structural heterogeneity is still evident, as indicated by standard deviations. The authors must precisely isolate symmetric or asymmetric but homogeneous particles for refinement to ensure the final maps represent distinct states accurately.\nResponse: We thank the reviewers for raising this point. Indeed, the structural heterogeneity (particularly at low pH) is a key feature of this system and we agree that this complicates interpretation of symmetry-imposed reconstructions. In response, we have revised the manuscript to better delineate the distinct insights gained from both symmetric and asymmetric processing strategies.\nThe D6-symmetrized reconstructions were critical for defining the high-resolution features of the most primary conformational states, specifically the stabilized-open, destabilized-open and gated-state configurations. These idealized models allowed us to identify key structural rearrangements, including pore-lining lipids and the more nuanced changes in the NT and TM2 for the destabilized-open state, which we were not able to resolve at lower resolutions achieved by symmetry-expansion and focused refinement methods performed on single subunits. Importantly, by leveraging D6-symmetry, this approach also enabled us to detect rare gated-state and destabilized-open state populations under neutral pH conditions (transitions that were also not evident in our asymmetric processing approach).\nHowever, we fully acknowledge that these D6-refined structures do not represent the dominant conformational state of channels under low pH conditions. Our subunit-focused classification and symmetry expansion analyses revealed that most channels exist in asymmetric, heterogeneous ensembles consisting of both open and gated subunits. This ensemble-level heterogeneity was essential for demonstrating that under cryo-EM conditions gating in Cx46/50 is not a fully concerted or cooperative process.\nMoreover, we would like to emphasize that this level of conformational heterogeneity, and the use of both symmetric and asymmetric processing approaches, reflects an ongoing challenge in the field of gap junction structural biology. Similar degrees of structural heterogeneity have been reported in other connexin structures, including Cx26, Cx32, Cx36 and Cx43, where authors similarly employed symmetry-imposed reconstructions to resolve high-resolution states, alongside C1 or subunit-level analyses to reveal asymmetric features. Our approach is therefore consistent with established methodological practices used to address the inherent complexity of these high-order oligomeric membrane proteins.\nWe have updated the manuscript to:\n• Clearly state that D6 reconstructions represent idealized endpoints rather than the dominant ensemble.\n• Clarify how each analytical approach (D6 vs. C1) contributes complementary insights.\n• Reference comparable examples in the field where similar methodological strategies have been employed.\npg. 13-14\nThis ensemble-level heterogeneity also highlights an important methodological point. It is important to emphasize that the D6- (or C6-) symmetrized cryo-EM reconstructions presented in this work do not represent the dominant conformations present under low pH conditions. Rather, these idealized reconstructions serve as high-resolution endpoints that highlight key structural rearrangements, including lipid interactions and NT/TM2 conformational changes, which were otherwise obscured at the lower resolutions obtained through our focused subunit-level analyses. Consistent with this, our subunit-level classification approaches revealed that the majority of channels adopt heterogeneous assemblies composed of both open and gated subunits. Together, these complementary strategies provide a more complete view of the gating process, where D6/C6-symmetrized reconstructions define high-resolution detail, while subunit-based analyses capture ensemble-level heterogeneity. Similar methodological strategies have been employed in recent studies of other connexins (e.g., Cx26, Cx32, Cx36, Cx43)53-56,67, underscoring both the complexity of gap junction structural characterization and the value of integrating symmetric and asymmetric approaches.\nWe appreciate the reviewers’ constructive critique, which has helped us clarify the interpretation and limitations of our data and to place our work in the broader context of current methodological approaches to connexin structural studies.\n[…]"}]

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