Connexins form hexameric channels in the plasma membrane known as hemichannels, which can either function as regulated passageways between the cell and its environment, or dock with a hemichannel from another cell to form a dodecameric intercellular channel, or gap junction channel (GJC). Connexins have been shown to be directly regulated by various stimuli such as voltage (Valiunas, 2002; Young and Peracchia, 2004), pH (Bevans and Harris, 1999; Khan et al., 2020; Yu et al., 2007) or indirectly via intracellular calcium ion concentrations (Peracchia, 2004). Recent reports based on structural data also suggest that lipids may be involved in regulation (Lee et al., 2023a; Lee et al., 2023b; Qi et al., 2023a). We have shown, however, that connexin26 (Cx26) and other similar β-connexins (Cx30, Cx32) can be regulated by the direct action of physiological concentrations of carbon dioxide independently of pH (Huckstepp et al., 2010; Meigh et al., 2013). Mutants of Cx26 are a leading cause of congenital deafness (Xu and Nicholson, 2013). While many of the mutations are non-syndromic, others lead to severe diseases such as keratitis ichthyosis deafness syndrome (KIDS) (Xu and Nicholson, 2013). Hemichannels are known to have different properties to GJCs (Stout et al., 2004) and our previous results show that an increase in the partial pressure of CO₂ (PCO₂) will open Cx26 hemichannels (Huckstepp et al., 2010) but close Cx26 GJCs (Nijjar et al., 2021), which are open under physiological conditions of PCO₂.

There are 20 connexin genes in the human genome (Abascal and Zardoya, 2013) and several structures have now been published (Bennett et al., 2016; Brotherton et al., 2022; Flores et al., 2020; Khan et al., 2020; Lee et al., 2023a; Lee et al., 2020; Lee et al., 2023b; Maeda et al., 2009; Myers et al., 2018; Qi et al., 2023a; Qi et al., 2023b). The connexin subunit, which is common to all, consists of four transmembrane helices (TMs) with a cytoplasmic N-terminal helix that in the hexameric arrangement of the hemichannel points towards the central pore (Maeda et al., 2009). In structures of the dodecameric GJC, the extracellular part involved in docking is well defined, whereas the cytoplasmic region is much more variable. A large cytoplasmic loop between TM2 and TM3, shown to be involved in regulation, has not been visible in any structure. Structures of the connexins either have the N-terminal helices tucked back against the wall of the channel (Flores et al., 2020; Lee et al., 2023a; Lee et al., 2023b; Myers et al., 2018), in a raised position (Lee et al., 2023a; Lee et al., 2020; Qi et al., 2023a), in an intermediate position (Brotherton et al., 2022; Lee et al., 2023a; Maeda et al., 2009), or not well defined in the density (Bennett et al., 2016; Brotherton et al., 2022; Khan et al., 2020; Lee et al., 2023b; Qi et al., 2023b). The position of the N-terminal helix is thought to be important in the regulation of channel permeability. We have shown previously for human Cx26 GJCs, that the position of the N-terminus is dependent on PCO₂ at constant pH (Brotherton et al., 2022). By examining structures from protein vitrified at different levels of PCO₂, we observed that under conditions of high PCO₂ the conformation of the protein was biased towards a conformation where the N-terminus protrudes radially into the pore to form a constriction at the centre (N_Const). Two distinct conformations of the protein were seen with the predominant difference between them in the cytoplasmic portion of TM2 (Brotherton et al., 2022) where an anticlockwise rotation of TM2 correlated with more definition of the density for the N-terminus. On the other hand, under low PCO₂ conditions the conformation with the more defined N-terminus was not observed and the channel appeared more open (N_Flex).

Based on a wealth of mutational data it has been hypothesised that the regulation of hemichannel opening by CO₂ is through a carbamylation reaction of a specific lysine (Meigh et al., 2015; Meigh et al., 2013; Nijjar et al., 2021). This post-translational modification is a reversible and highly labile reaction of CO₂ (Lorimer, 1983) that effectively changes the charge of a neutral lysine residue to make it negative. A so-called ‘carbamylation motif’ was identified in CO₂-sensitive connexins (Dospinescu et al., 2019; Meigh et al., 2013) that when introduced into a related CO₂-insensitive connexin rendered the protein CO₂-sensitive (Meigh et al., 2013). In Cx26 this motif has the sequence K₁₂₅VRIEG₁₃₀. In the crystal structure of the Cx26 GJC that was published in 2009 (Maeda et al., 2009), Lys125, which is conserved amongst β-connexins that are known to be modulated by CO₂ (Dospinescu et al., 2019), is positioned near to the N-terminus of TM3 within ~6 Å of Arg104 of TM2 of the neighbouring subunit, at either side of the disordered cytoplasmic loop. It was suggested that upon carbamylation, the negative charge of the modified lysine would attract Arg104 causing a conformational change (Meigh et al., 2013). In hemichannels, mutation of Lys125 to glutamate, so mimicking the charge of the carbamylated lysine (K125E), results in constitutively open hemichannels consistent with elevated PCO₂, whereas the corresponding K125R mutation results in hemichannels that cannot be opened by CO₂ (Meigh et al., 2013). In GJCs, the K125R mutation results in the protein not closing in response to CO₂, though importantly, this mutation does not prevent closure by acidification (Nijjar et al., 2021).

While our previous structures (Brotherton et al., 2022) demonstrated an effect of PCO₂ on the conformation of the protein, neither Lys125 nor Arg104 were visible in the density. Here, we probe this further. By solubilising the protein in the detergent lauryl maltose neopentyl glycol (LMNG) rather than dodecyl β-D-maltoside (DDM), we obtain much improved density for the cytoplasmic region of the protein. We refine two conformationally diverse structures of the protein, where we see much more defined differences in the cytoplasmic region of the protein than we were able to observe previously. These data suggest a mechanism for closure involving the concerted movements of TM2 and the KVRIEG motif. We show that the structure with the K125E mutation matches the more closed conformation of the protein.

Connexins open and close to various stimuli. Despite a number of recent high-resolution structural studies of connexins, the structural basis for pore closure or opening in response to a stimulus is still poorly understood. Cx26 is affected by PCO₂. We have previously shown that there is a correlation in the closure of the Cx26 GJC with the level of PCO₂ and also with the position of TM2 (Brotherton et al., 2022). Our improved data show that, not only does pore closure and the position of the N-terminal helix correlate with TM2, but also with the conformation of TM1 and importantly, the KVRIEG motif on the regulatory cytoplasmic loop.

The conformation of the KVRIEG motif and the cytoplasmic loop is interesting. In the original low-resolution crystal structure of Cx26 (Cx26-xtal) (Maeda et al., 2009), the KVRIEG motif is modelled as part of TM3. However, in most other structures of connexin GJCs, the equivalent residues are not observed in the density, and it appears there is a breakdown in helical conformation at this point. In the N_Const structures, the KVRIEG motif forms an extended conformation mimicking the conformations seen in AlphaFold2 structures. In fact, the conformation of the complete loop that we observe in the subunit focussed classification is similar to that seen in AlphaFold2 structures (Varadi et al., 2022). Essentially the KVRIEG motif can be described as a break in TM3 as the helix extends at either side of this extended motif. Although the low resolution of the density of the complete loop prevents the accurate positioning of the residues, the combination of our structure and the predicted models paves the way for further studies of the importance of particular interactions in the loop.

From a comparison of our structures with those of other connexins it is apparent that the position of TM2 seen in the N_Const structures is an outlier, with a rotation of the cytoplasmic region of the helix compared to other structures (Figure 6a). With respect to the N-terminus the various reported structures can be considered in three main categories: those with the N-terminus folded into the channel pore (N-down) (Flores et al., 2020; Lee et al., 2023a; Lee et al., 2023b; Myers et al., 2018); those where the N-terminus is lifted (N-up) (Lee et al., 2020; Qi et al., 2023a); and those where the N-terminus is flexible (N-flex) (Bennett et al., 2016; Brotherton et al., 2022; Khan et al., 2020; Lee et al., 2023b; Qi et al., 2023b). The conformation of the Cx26-N_Const structure most resembles the structures with N-down as these have similar conformations of TM1 (Figure 6). However, whereas the N-terminus in these latter structures folds flat against the pore of the channel to give an open aperture, in the Cx26-N_Const structure, it would be prevented from doing so by the position of TM2 and so is forced outwards to form a much more constricted aperture. The conformation of TM1 in the Cx26-N_Flex structures, which we consider to be open, is more similar to structures of other connexins in the N-flex or N-up conformations. For Cx36 and Cx43 these conformations have been suggested to represent the closed conformation (Lee et al., 2023a; Qi et al., 2023a), though for Cx32 this has been described as an open conformation (Qi et al., 2023b). The apparent outlier amongst the Cx26 structures is the Cx26-xtal structure where the N-terminus points into the pore, but the overall conformation is more similar to the Cx26-N_Flex conformation. However, relative to this latter structure the helix is pulled back (Figure 6b) so is more open than seen in the Cx26-N_Const structure.

Figure 6

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Previously, when examining our Cx26 maps from DDM solubilised protein we noted a lipid-like molecule in the pore of the protein and we questioned whether this would be detergent or lipid and whether it would interfere with the position of the N-terminal helix (Brotherton et al., 2022). The fact that similar density remains when the protein is solubilised with LMNG rather than DDM strongly suggests that this molecule cannot be detergent and is more likely to be a lipid. Though the presence of the molecule may still be an artefact of either the solubilisation process or heterologous expression in insect cells, it is interesting to note that a lipid-like molecule in this position appears to be a constant feature of all the high-resolution cryo-EM maps associated with the structures of connexins where the N-terminus is not in the N-down position. This includes both GJCs (Lee et al., 2023a; Lee et al., 2023b; Qi et al., 2023a; Qi et al., 2023b) and hemichannels (Lee et al., 2020; Qi et al., 2023b) and is irrespective of the solubilisation method or whether the structure has been solved in the presence of detergent or nanodiscs. It seems that the lipid is only displaced when the N-terminus adopts the pore lining position. As this position is not possible in the Cx26-N_Const structures due to the presence of TM2, the lipid remains.

It must be asked whether the N_Const structures that we observe represent the closed state. It is difficult to model the first three residues of the N-terminus unambiguously, presumably due to the breakdown in sixfold symmetry at this point, but with minor changes to the side-chains, the centre of the pore could be closed. The presence of the lipid would seal any other apertures between the neighbouring N-termini. Rather surprisingly given that we can isolate a symmetrical conformation of the protein using C6 symmetry, reconstructions of the full dodecamer following the focussed classifications of the single subunit do not show an influence of the conformation of that subunit on its neighbours. While this is consistent with structural studies on Cx43 gap junctions (Lee et al., 2023a) the stochastic nature of the subunit conformations contrasts with studies of Cx26 hemichannels in cell membranes where significant cooperativity has been observed (de Wolf et al., 2017).

There is a clear correlation between the results of the dye transfer assays and the structural results, supporting the interpretation that the N_Const conformation is more like the closed state. The effect on the structure of Cx26 in changing K125 to a glutamate both in CO₂/HCO₃^- and in HEPES buffer is consistent with the hypothesis that the CO₂-mediated closure of the gap junctions is caused by the carbamylation of K125. It would also suggest that even under high PCO₂ conditions the WT protein is not completely carbamylated during vitrification. Given that the carbamylation reaction is highly labile, this is perhaps not surprising. The hypothesis has been that the negative charge introduced onto the lysine side-chain would enable it to form a salt bridge with Arg104 from the neighbouring subunit (Meigh et al., 2013). Consistent with this, mutation of Arg104 to alanine abolishes CO₂ sensitivity in both GJCs (Nijjar et al., 2021) and hemichannels (Meigh et al., 2013), similar to the mutation of Lys125. In models from AlphaFold2, which differ in conformation from the structure upon which the hypothesis was based, it is also notable that the lysine is located next to the arginine (Figure 3a). In the N_Const structures the position of the Lys/Glu125 main chain is near to Arg104, even though TM2 moves further from TM3 in moving from the N_Flex to the N_Const conformations. Though there is no clear interaction between the two residues, with minor adjustments of the side-chains, the residues could be made to interact and the absence of a definite interaction may be due to distortions during the vitrification process or radiation damage to which acidic residues are more prone. Glutamate is also much shorter than the carbamylated lysine, so that while the charges would be equivalent, the two residues might not be able to make the same specific interactions. Our results here show that the negative charge at residue 125 is important for altering conformation. For hemichannels, a direct interaction between the two residues (Meigh et al., 2015) is strongly supported by a mutant in which both R104 and K125 are replaced by cysteines, allowing a potential cross-link through a disulphide bond. This mutant responds to a change in redox potential in a similar way to which the WT protein responds to CO₂ (Meigh et al., 2015). However, although both Lys125 and Arg104 are necessary for CO₂-dependent gap junction closure, attributing the induction of the conformational change in the gap junction to a single salt bridge between Lys125 and Arg104 may be an over-simplification. For example, there may be other interactions involved and it is possible that multiple carbamylation events contribute to gap junction sensitivity (Brotherton et al., 2020). Given that we observe some protein in the N_Const conformation even when Arg125 is mutated to arginine, our data would be consistent with a conformationally flexible protein, in which the introduction of a negative charge would stabilise one particular conformation, rather than causing a conformational change per se (Figure 7).

Figure 7

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Mutations in Cx26 lead to both syndromic and non-syndromic deafness (Xu and Nicholson, 2013). While these mutations have been mapped on the structure previously, the position of Ala88, mutation of which to Val causes KIDS, is interesting with regard to the new conformations of TM1. This mutation, which is at the flexion point of TM2 (Figure 2b), results in leaky hemichannels (Mhaske et al., 2013) and has been shown to prevent CO₂ sensitivity either to the gap junctions, in closing the channels (Nijjar et al., 2021), or to the hemichannels in opening the protein (Meigh et al., 2014). In the N_const structures the C_β of Ala88 lies within 4.2 Å of Trp24, which moves during the conformational change of TM1. Replacement of the alanine with the bulkier valine would hinder this conformation from being adopted and therefore, would disfavour the closed conformation. Interestingly, the alanine and the tryptophan are located next to Arg143. Mutation of Arg143 to tryptophan is a very common mutation that leads to non-syndromic hearing loss (Xu and Nicholson, 2013). Overall our data, summarised in Figure 7, provide important mechanistic insight into the conformational changes behind pore closure in Cx26, which is useful for understanding how mutations in the protein can lead to disease.

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-18290 (K125E90), EMD-18295 (K125R90), EMD-18296 (K125EHEPES), EMD-18297 (WTHEPES), EMD-18291 (LMNG-NConst), EMD-18292 (LMNG-NFlex), EMD-18293 (LMNG-NConst-mon), EMD-18294 (LMNG-NFlex-mon). Structure models have been deposited in the RCSB Protein Data Bank under accession numbers 8Q9Z, 8QA1, 8QA0, 8QA2, 8QA3. Figure 4—source data 1 contain the numerical data used to generate the figure.

1. 1. Brotherton DH
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   (2024) RCSB Protein Data Bank

   ID 8QA0. Cryo-EM structure of Cx26 solubilised in LMNG - hemichannel classification - NConst conformation.

   https://www.rcsb.org/structure/8QA0
2. 1. Brotherton DH
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   (2024) RCSB Protein Data Bank

   ID 8QA1. Cryo-EM structure of Cx26 solubilised in LMNG - Hemichannel classification NFlex conformation.

   https://www.rcsb.org/structure/8QA1
3. 1. Brotherton DH
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   (2024) RCSB Protein Data Bank

   ID 8QA2. Cryo-EM structure of Cx26 solubilised in LMNG: classification on subunit A; Nconst-mon conformation.

   https://www.rcsb.org/structure/8QA2
4. 1. Brotherton DH
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   (2024) RCSB Protein Data Bank

   ID 8QA3. Cryo-EM structure of Cx26 solubilised in LMNG: classification on subunit A; NFlex conformation.

   https://www.rcsb.org/structure/8QA3
5. 1. Brotherton DH
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   (2024) RCSB Protein Data Bank

   ID 8Q9Z. Cryo-EM structure of Cx26 gap junction K125E mutant in bicarbonate buffer (classification on hemichannel).

   https://www.rcsb.org/structure/8Q9Z
6. 1. Brotherton DH
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   (2024) Electron Microscopy Data Bank

   ID EMD-18290. Cryo-EM structure of Cx26 gap junction K125E mutant in bicarbonate buffer (classification on hemichannel).

   https://www.ebi.ac.uk/emdb/EMD-18290
7. 1. Brotherton DH
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   (2024) Electron Microscopy Data Bank

   ID EMD-18291. Cryo-EM structure of Cx26 solubilised in LMNG - hemichannel classification - NConst conformation.

   https://www.ebi.ac.uk/emdb/EMD-18291
8. 1. Brotherton DH
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   (2024) Electron Microscopy Data Bank

   ID EMD-18292. Cryo-EM structure of Cx26 solubilised in LMNG - Hemichannel classification NFlex conformation.

   https://www.ebi.ac.uk/emdb/EMD-18292
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   (2024) Electron Microscopy Data Bank

   ID EMD-18293. Cryo-EM structure of Cx26 solubilised in LMNG: classification on subunit A; Nconst-mon conformation.

   https://www.ebi.ac.uk/emdb/EMD-18293
10. 1. Brotherton DH
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    ID EMD-18294. Cryo-EM structure of Cx26 solubilised in LMNG: classification on subunit A; NFlex conformation.

    https://www.ebi.ac.uk/emdb/EMD-18294
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    https://www.ebi.ac.uk/emdb/EMD-18295
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    https://www.ebi.ac.uk/emdb/EMD-18296
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    (2024) Electron Microscopy Data Bank

    ID EMD-18297. Cryo-EM reconstruction of Cx26 gap junction WT in HEPES buffer.

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Author details

1. Deborah H Brotherton

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Writing – original draft, Protein purification, Grid preparation, Data collection, Processing and structure refinement

   Competing interests

   No competing interests declared

2. Sarbjit Nijjar

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   Data curation, Investigation, Writing – review and editing, Dye transfer assays

   Competing interests

   No competing interests declared

3. Christos G Savva

   Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom

   Contribution

   Investigation, Writing – review and editing, Data collection

   Competing interests

   No competing interests declared

4. Nicholas Dale

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Writing – original draft

   For correspondence

   n.e.dale@warwick.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2196-2949

5. Alexander David Cameron

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Writing – original draft

   For correspondence

   a.cameron@warwick.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8776-3518

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