{"paper_id":"e29be73a-00d5-4e6b-915a-1e4f4d132a06","body_text":"On the mechanisms of permselectivity of connexin hemichannels to small molecules \n \nAlexandra Lovatt, Jack Butler and Nicholas Dale * \n \nSchool of Life Sciences, The University of Warwick, Coventry, CV4 7AL, United Kingdom \n \n \n• Correspondence: n.e.dale@warwick.ac.uk \n \nKeywords: Connexins, hemichannels, ATP, glutamate, lactate, N-terminus, permeability \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nAbstract \nConnexins can either that act as hemichannels, to facilitate ion and small molecule movement \nfrom the cytosol to the extracellular space or as gap junction channels to provide a pathway for \nsolute exchange between cells. Connexins are ubiquitously expressed throughout the body and \nare implicated in a wide range of physiological and pathological processes. The permselectivity \nof connexin hemichannels for small neurochemicals  remains poorly understood . By \ncoexpressing genetically encoded fluorescent sensors  for ATP, glutamate and lactate with a \nrange of connexins, we examined the ability of different hemichannels to permit release of these \ncompounds under physiological conditions  and in response to physiological stimuli ( small \nchanges in PCO 2 and transmembrane depolarisation).  We found that some connexins were \nrelatively non-selective (Cx26, Cx32, Cx43, Cx31.1) allowing passage of ATP, glutamate and \nlactate. By contrast other connexins (Cx36, Cx46 and Cx50) were highly selective. Cx36 and \nCx46 allowed release of ATP, but not glutamate or lactate. This shows that size of t he \npermeating molecule is not the sole determinant of  permselectivity. By contrast, Cx50 \npermitted the release of lactate and glutamate but not ATP. We also found that the nature of the \nopening stimulus could alter the permselectivity of the hemichannel  -for some of the relatively \nnon-selective connexins, hemichannel opening via depolarisation was ineffective at allowing \nrelease of lactate. By comparing the differential selectivity of the closely related Cx46 and Cx50, \nwe showed that the charge on the N -terminus and N -terminus-TM2 interactions are key \ncontributors to permselectivity. \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nIntroduction \nThere are 21 connexin genes in the human genome  (Lucaciu et al., 2023) . Connexins form \nhexamers that, if unopposed , can act as a plasma membrane hemichannel that opens to the \nextracellular space. However, hemichannels of closely apposed cells can also dock together to \nform gap junction channels to provide an aqueous passageway between cells. The structure of \nthe hemichannel is highly conserved in all 21 isoforms; each connexin has an N -terminal helix, \n4 transmembrane helices, a cytoplasmic loop, 2 extracellular loops and a cytoplasmic C-\nterminus (Maeda et al., 2009; Myers et al., 2018; Flores et al., 2020; Brotherton et al., 2022; Lee \net al., 2023; Qi et al., 2023; Brotherton et al., 2024) . 6 subunits then co -assemble to form a \nhemichannel with a central pore, spanning ~1.2 nm that is permeable to small molecules up to \na molecular weight of about 1000.  Major differences in structure between isoforms lie within \nthe cytoplasmic loop and C -terminus, which vary in sequence and length  (Mese et al., 2007) . \nThe 6 N -terminal helices line the hemichannel pore, to form the narrowest part of the \npermeation pathway, suggesting that the N-terminus may be an important for determining the \npermeability of the channel (Oshima et al., 2007, 2008; Maeda et al., 2009; Nielsen et al., 2019; \nYue et al., 2021).  \nConnexin hemichannels have been documented to release small molecules such as ATP under \nphysiological conditions (Weissman et al., 2004; Pearson et al., 2005; Huckstepp et al., 2010b; \nChever et al., 2014; van de Wiel et al., 2020) . Yet the mechanisms that control hemichannel \npermeability to different molecules , and whether there is specificity to which molecules may \npermeate is still unclear. Traditionally, this has been investigated using various fluorescent dyes \nsuch as ethidium bromide, and the size and charge of dyes provided some evidence for \nselectivity to release of larger molecules (Li et al., 1996; Saez et al., 2010; Johnson et al., 2016). \nInvestigation of hemichannel permeability via dye fluxes, while valuable , may differ from how \nphysiological metabolites such as ATP, glutamate or lactate permeate these channels.  \nTraditionally, connexin permeability studies have used the removal of extracellular divalent \ncations to unblock the hemichannels (Hansen et al., 2014; Nielsen et al., 2019). The mechanism \nwas defined in Cx26 and Cx32 and involves a ring of 12 aspartate residues within the \nextracellular loop that provide a carboxylate cluster able to bind Ca2+ ions with millimolar affinity \n(Gomez-Hernandez et al., 2003; Bennett et al., 2016; Lopez et al., 2016) . While hemichannels \nare essentially blocked at Ca2+ concentrations over 1 mM, there are very few if any physiological \nconditions in which extracellular Ca2+ is lower than 1 mM. Thus, unblocking of hemichannels via \nCa2+ removal may open a permeation pathway that is not representative of physiological gating \nof connexin hemichannels.  Nevertheless, this method has been used to suggest differential \npermeability of Cx30 and Cx43 to a variety of small molecules  (Hansen et al., 2014; Nielsen et \nal., 2019). \nConnexin hemichannels can be opened under physiological conditions by other gating stimuli. \nAs the N-termini of connexins have charged residues and are within the membrane electric field, \nalmost all c onnexins can be opened by sufficient depolarisation, without the need to lower \nextracellular Ca 2+ (Pinto et al., 2016) . A subset of connexins is directly sensitive to the \nconcentration of gaseous CO2 and can be opened by relatively small changes in the partial \npressure of CO2 (PCO2) around the physiological norm. CO2-dependent gating was elucidated in \nCx26 (Meigh et al., 2013): K125 is carbamylated by CO2, which facilitates the formation of a salt \nbridge with R104 of the adjacent subunit to bias the channel into an open conformation. This \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nmechanism involves movements of the N -terminus, along with related movements of the \ntransmembrane helices (particularly TM2) (Brotherton et al., 2022; Brotherton et al., 2024). CO2 \ngating has subsequently been discovered in a subset of connexins: Cx30, Cx32 (Huckstepp et \nal., 2010a), Cx43 (Nijjar et al., 2025) and Cx50, since they all have the carbamylation motif that \nis required for CO2-mediated opening. Their CO2 sensitivity lies close to the physiological range \nof PCO2; Cx26, Cx43 and Cx50 are maximally open at ~55 mmHg PCO2, and Cx32 at ~70 mmHg.  \nTo permit the study of the permeation of connexin hemichannels, for physiological molecules \nunder their normal electrochemical gradients and with physiological gating stimuli (voltage and \nPCO2), we have developed an assay to allow real-time imaging of analyte release at single cell \nresolution. We utilised the genetically encoded sensors  GRABATP (Wu et al., 2022) , iGluSnFR \n(Marvin et al., 2013)  and eLACCO1.1 (Nasu et al., 2021)  to measure the release of ATP, \nglutamate and lactate respectively via coexpressed  connexin hemichannels . We find that \nconnexins can be divided essentially into relatively non-selective and highly selective categories. \nConnexins such as Cx26, Cx32 and Cx43 fall into the relatively non-selective category, whereas \nCx36, Cx46 and Cx50 are highly selective. By comparing the highly homologous Cx46 and Cx50, \nwe have shown that key residues in the N -terminus and  in the  interacting portion of TM2 \ndetermine the permeability profile of the hemichannel. \nResults \nWe first transfected HeLa cells with the genetically encoded sensors on their own to ensure that \nthe sensors had no intrinsic responses to CO2 or high KCl solutions, or that parental HeLa cells \nexhibited ATP, glutamate or lactate release in the absence of connexin expression. The median \nchange in normalised fluorescence (∆F/F0) for GRABATP was -0.007 (95% CI: 0.0043, -0.013) and \n-0.004 (0.0017, -0.0064) for 55 mmHg and 50 mM KCl respectively. GRABATP was functional as it \ngave a median response of 0.2 (0.25, 0.16) to 3 µM ATP (Fig.1). The median change in ∆F/F 0 for \niGluSnFR with 55 mmHg was 0 (0.0008, -0.0026), for 50 mM KCl it was -0.014 (-0.011, -0.030), \nand for 3 µM glutamate was 0.1 (0.13, 0.05). Finally, the median ∆F/F0 for eLACCO1.1 (modified \nby insertion into iGluSnFR backbone) was 0.005 (0.0073, -0.0046), -0.002 (0.0048, -0.0069) and \n0.04 (0.046, 0.032) for 55 mmHg, 50 mM KCl and 3 µM lactate, respectively. The n egative \nrecorded values are an artefact of photobleaching. As there were no responses of any of the \ngenetically sensors to a change in PCO2 or membrane depolarisation, we conclude that parental \nHeLa cells do not express any channels capable of releasing ATP, glutamate or lactate to these \nstimuli.  \nCx26, Cx32,Cx43 and Cx31.3 are permeable to ATP, glutamate, lactate \nWe next co-transfected HeLa cells with Cx26 , Cx32, Cx43 or Cx31.3 and one of the genetically \nencoded fluorescent sensors. Throughout all of the assays, we selected cells that co-expressed \nthe connexin and the sensor for measurement and analysis (Fig. 2 figure supplement 1) . Cx26 \nhemichannels are highly permeable  to ATP, glutamate and lactate  (Fig. 2). The median ATP \nrelease to hypercapnic stimuli was 1.5 µM (1.91, 1.32) and with a depolarising stimulus was 2.5 \nµM (3.67, 2.30). This was significantly less than for glutamate and lactate: the median glutamate \nrelease was 5.2 µM (5.71, 4.36) and 1.1 µM (6.10, 0.74) when opened with hypercapnia and a \ndepolarising stimulus, respectively. Lactate release evoked by hypercapnia was comparable to \nglutamate: median release of 4.7 µM (6.26, 2.52). However when stimulated by depolarisation \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nno lactate release was evident : median -0.1 µM (0.23, -1.04). For voltage -gated glutamate \nrelease it appeared that  in some cells the depolarising stimulus was ineffective at evoking \nrelease. This suggests that KCl evoked depolarisation (estimated to be about 70 mV from the \nNernst equation) is a less reliable gating mechanism than CO2 for Cx26.  \nWe determined that Cx32 was also permeable to all three tested analytes . Significantly more \nglutamate and lactate was released compared to  ATP regardless of the stimulus  (Fig. 3). \nHowever the nature of the stimulus did alter the relative amounts of release of the three \nmetabolites. The median release of ATP was 1. 3 (1.51, 1.16) and 3.0 (3.12, 2.86)  µM whe n \nstimulated by hyper capnia and voltage, respectively.  Glutamate releas ed via Cx32  by \nhypercapnia and depolarisation was respectively  3.9 (4.64, 3.41)  µM  and 5.2 (7.44, 4.62)  µM. \nLactate release via Cx32 by hypercapnia and depolarisation was respectively 7.2 (8.32, 5.31) µM \nand 5.5 (6.84, 4.52)  µM.  With hypercapnia, the relative release of both glutamate and lactate \ncompared to ATP was considerably more than might be expected from the calculated \nelectrochemical driving force  (Tables 1 and 2) . This suggests that  Cx32 may have enhanced \npermeability for these molecules when opened by hypercapnia. \nWith a hypercapnic stimulus Cx43 was permeable to ATP, glutamate an d lactate (Fig. 4). The \nmedian ATP, glutamate and lactate release were repsectively 2.2 (2.34, 1.49) µM, 5.3 (5.71, 4.36) \nµM and 4. 5 (5.84, 2.46)  µM (Fig. 4). This amount of release is roughly proportional to the \nelectrochemical driving force on these molecules ( Tables 1 and 2 ) suggesting no selectivity. \nHowever during membrane depolarisation evoked by 50 mM KCl,  the permeation profile was \ndifferent. Whereas median ATP release  was 2.5 (2.88, 2.22) µM (similar to that evoked by CO2), \nthe release of glutamate was reduced, median 1.0 (6.10, 0.74) µM, and lactate was not released \nat all during this stumulus, median -1.0 (-0.069, -1.49) µM. The gating mechanism of Cx43 thus \nalters the permeability profile of the hemichannel to small molecules.  \nWhile permeability of Cx43 hemichannels to ATP has been elegantly demonstrated (Kang et al., \n2008), previous reports suggest that Cx43  hemichannels are  apparently not permeable to \nglutamate or lactate (Hansen et al., 2014; Nielsen et al., 2019). However these previous studies \nused removal of extracellular Ca 2+ to unblock the channel, and the channel might thus have  a \ndifferent permeability profile. Whereas zero [Ca2+]ext  is often achieved by the use of chelators \nsuch as EGTA,  the genetically encoded fluorescent sensors have some degree of Ca2+-\ndependency. To ensure compatibility with the sensors, we omitted EGTA and Ca 2+ to lower but \nnot eliminate extracellular Ca2+. To ensure this was still sufficient to open the hemichannels, we \nemployed a dye loading assay using the hemichannel-permeable dye , FITC. Under control \nconditions (PCO2 20 mmHg, 2 mM [Ca2+]ext) the median pixel intensity was 15.7 (24.72, 14.04). In \nlow [Ca2+]ext (PCO2 20 mmHg), the median pixel intensity was 54.3 (86,74, 51.82)  (Fig. 4, figure \nsupplement 1). For a comparison to a reliable opening stimulus, we use a PCO2 of 55 mmHg to \nopen the hemichannels  and permit loading with FITC . This yielded a median pixel intensity of \n48.0 (54.83, 40.66) and demonstrates that the low [Ca2+]ext solution was an effective stimulus to \nopen Cx43 hemichannels. \nHaving established the efficacy of low [Ca2+]ext at opening Cx43 hemichannels, we assessed the \npermeation of ATP, glutamate and lactate during this stimulus  and compared it to permeation \nin response to hypercapnia in the same cells.  Consistent with our previous findings, \nhypercapnia evoked the release of ATP, glutamate and lactate. However, low [Ca 2+]ext \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nsignificantly reduced the analyte release through Cx43 hemichannels, with a median release of \n0.1 (0.86, 0.08) µM ATP, 0.2 (0.76, 0.004) µM glutamate and 0. 5 (2.11, -0.25) µM lactate (Fig. 4, \nfigure supplement 2 ). The nature of the gating mechanism appears to change channel \npermeability and may account for why our results differ from previous release studies.  \nCx31.3 (also called Cx29) is mainly expressed in myelinating cells (Cisterna et al., 2019). There \nhas been some suggestion that Cx31.3 predominantly forms hemichannels  and mediates ATP \nefflux from cells that expressing this isoform (Sargiannidou et al., 2008; Liang et al., 2011) . We \nwere able to demonstrate a median release of 2.6 µM (95% CI 2.91, 2.26) ATP, 2.3 µM (2.58, 1.91) \nglutamate and 2.8 µM lactate (95% CI 2.92, 2.24) (Fig. 5). While relatively non-selective, Cx31.3 \nnevertheless shows some preference for ATP as the relative permeation of glutamate and \nlactate is less than that predicted by the electrochemical driving force on these molecules  \n(Tables 1 and 2).  \nCx36, Cx46 and Cx50 hemichannels have highly specific permeability profiles \nCx36 acts predominantly as a major neuronal connexin (Condorelli et al., 1998)  and forms the \ngap junctions or electrical synapses that facilitate fast synaptic transmission and synchronous \nneuronal firing  (Srinivas et al., 1999; Deans et al., 2001; Buhl et al., 2003) . We assayed the \nrelease of ATP, glutamate and lactate from HeLa cells transfected with Cx36. Because Cx36 is \ninsensitive to CO 2 (Huckstepp et al., 2010a) , we used the depolarising stimulus to gate the \nchannel. Surprisingly, we found that of the three analytes, Cx36 was only permeable to ATP (Fig. \n6). The median release of ATP evoked by 50 mM KCl was 2.6 (3.27, 2.41) µM, compared to -0.3 \n(0.22, -0.68) and -0.2 ( -0.05, -0.21) µM glutamate and lactate respectively.  As t he \nelectrochemical driving force for release of glutamate and lactate is about double that of ATP  \n(Tables 1 and 2), and these molecules are smaller than ATP, the differential permeability of Cx36 \nsuggests the existence of a selectivity filter within the pore. \nHuman Cx46 also lacks the carbamylation motif and is not sensitive to CO2. We therefore used \nthe high K + stimulus to open Cx46 hemichannels. Like Cx36, Cx46 was only permeable to ATP  \ngiving a median release of 2.6 µM (2.74, 2.34) (Fig. 7). No glutamate or lactate was released via \nCx46 (median release -0.07 µM (0.11, -0.28) and 0.2 µM (0.41, -0.28) for glutamate and lactate \nrespectively, Fig. 7). By contrast, Cx50 was readily permeable to glutamate (median release 1.5 \nµM (1.57, 1.32) ) when stimulated by hypercapnia  and lactate when stimulated by either \nhypercapnia or depolarisation (median release  5.8 µM (7.36, 2.55)  and 3. 4 µM (3.78, -0.55) \nrespectively, Fig 7). However, no ATP could permeate Cx50 (Fig. 7). \nMutational analysis of the differential permeability of Cx46 and Cx50 \nCx46 and Cx5 0 are structurally quite similar yet  have markedly different permeability profiles  \n(Myers et al., 2018; Flores et al., 2020; Yue et al., 2021). These two connexins therefore offer an \nopportunity to explore the mechanistic basis of differential permeability. The N-termini fold into \nthe gap junction pore to form the narrowest point, with hydrophobic residues anchoring the helix \nto transmembrane regions 1 and 2 (TM1/2) to stabilise the open state (Myers et al., 2018). \nAligning the N -termini sequences of Cx46 and Cx50 shows that they have a difference in the \noverall net charge (respectively 0 and -2 for Cx46 and  Cx50, Fig. 8). The first divergence is at \nposition 9, where Cx46 has a positively charged arginine and Cx50 has an asparagine. At position \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n13, Cx50 has a negatively charged glutamate residue where Cx46 has a neutrally charged \nasparagine.  \nTo explore the possible roles of the difference in net charge, we introduced the single mutations \nN9R, E13N and N9 K into Cx50 to make it more like Cx46. These mutations gave a gain of ATP \npermeability to Cx50, though this was still significantly less than that of Cx46 WT (Fig. 8). The \ndouble mutation, Cx50 N9R E13N , gave an  increase in ATP release that matched that of Cx46 WT \nrelease (for a depolarising stimulus) , with a median  release of 2.4  (3.37, 2.07) µM from the \nmutant connexin compared to 2.5 (2.74, 2.34) for Cx46WT. We have therefore shown that the net \ncharge of the N -terminus, and the specific residues N9 and E13,  act to regulate the ATP \npermeability of Cx50.  \nWe next examined whether, if we made the N-terminus of Cx46 more like that of Cx50, we could \nchange the permeability profile of Cx46 to match that of Cx50.  We therefore made the double \nmutation R9N ,N13E in Cx46. However, this d id not  diminish ATP permeation  via Cx46 \nhemichannels (Fig. 9). The median release from Cx46R9N,N13E was 2.8 (3.10,2.47), compared with \n2.5 (2.74, 2.34) µM for Cx46WT. This indicates that the regulation of permeability of Cx46 to ATP \nis more complex than just the net charge of the N-terminus. \n \nWe considered the possibility that interactions between the N -terminus and TM2 may differ \nbetween Cx46 and Cx50 and this could permit ATP permeation even if the net charge of t he N-\ntermini were negative. Both Cx46 and Cx50 have hydrophobic residues at position 14: Cx50 has \na valine, but Cx46 has an alanine. We therefore used experimentally determined structures of \nthese connexins (Jaradat et al., 2022)  to identify  possible interacting residues in TM2 . This \nhighlighted residue 89 as potentially important: in Cx50 this is serine, but in Cx46 it is threonine. \nWe sought to make these interactions in Cx46 more similar to those occurring in Cx50 by \nintroducing the double mutation A14V,T89S . We note that simple introduction of the Cx50 N -\nterminal helix into Cx46 has been reported as resulting in non-functional gap junction channels \ndue to a steric clash between V14 and T89 in the chimaeric channel (Yue et al., 2021). By making \ntwo mutations in Cx46, A14V and T89S, we have avoided this clash. The double mutation A14V, \nT89S did not by itself alter ATP permeation (Fig. 9), indicating that the Cx46A14V,T89S hemichannel \ngated normally to voltage.  However, when A14V and T89S were  then combined with R9N and \nN13E, the quadruply mutated Cx46R9N,N13E,A14V,T89S hemichannels (Cx46QM) exhibited significantly \nreduced ATP permeation compared to the wild-type, with a median release of 0.54 (1.53, 0.34) \nµM (Fig. 9).  \n \nOne possible interpretation of this result is that the quadruple mutation simply reduces overall \nhemichannel permeability rather than having a specific effect on that of ATP . To evaluate this , \nwe examined depolarisation dependent loading of FITC into HeLa cells expressing either Cx46WT \nor  Cx46QM (Fig. 9, figure supplement 1). We found that FITC still permeated into cells expressing \nCx46QMand was not significantly different from the permeation observed in  those expressing \nCx46WT. This suggests that the  quadruple mutant has a somewhat selective effect on  ATP \npermeation. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nDiscussion \nThis study explored  whether there is selectivity to the release of small molecules from connexin \nhemichannels expressed in HeLa cells . To assess their relative permeability via the different \nconnexins, we need to understand the electrochemical driving force on the three metabolites . \nAs these metabolites are charged , we can use the Nernst equation , along with typical \nconcentrations of these molecules in HeLa cells (Piva and McEvoy-Bowe, 1998; Imamura et al., \n2009; San Martin et al., 2013) to calculate the equilibrium potential (Table 1). If we assume that \nthe concentrations of these metabolites are scattered around a mean value that is consistent \nacross all HeLa cells , sufficient recordings of release should statistically reflect these \ntransmembrane concentrations. Based on this analysis, if release through connexins were to be \nnon-selective, the relative proportions of release should corre spond to the relative \nelectrochemical driving forces. Thus, for a completely non-selective connexin we would expect \nto see ATP, glutamate and lactate released approximately in the proportion 1:1.8:1.4 (assuming \na resting potential of -60 mV) to hypercapnia and in the proportion 1:2.1:1.6, for the depolarising \nstimulus. Any notable deviation from these proportions would indicate that there is some \nselectivity to release of small molecules through connexin hemichannels (Table 2).  \n \nOur study is the first to produce a comprehensive permeability profile of a wide range of \nconnexin isoforms for release of physiological metabolites with physiological opening stimuli . \nConnexin hemichannels fall into two b road categories : relatively non-selective (Cx26, Cx32, \nCx43, Cx31.3); and highly selective (Cx36, Cx46 and Cx50). For Cx26 and Cx32 , the release of \nglutamate and lactate relative to ATP  is more than  predicted by the electrochemical driving \nforce, suggesting that the smaller molecules may permeate more readily than ATP  (Table 2) . \nCx32 in particular shows enhanced permeability to lactate  (nearly 4  times that predicted by \ndriving force). Interestingly, when Cx32 was opened by depolarisation , the relative release of \nanalytes followed that predicted by the electrochemical driving force  more closely. For Cx43, \nwhen opened by hypercapnia, the relative release of ATP, glutamate and lactate follows very \nclosely the pattern predicted by the electrochemical driving force (Table 2). Cx31.3 shows some \npreference for ATP over glutamate and lactate but is nevertheless permeable to these smaller \nmolecules (Table 2). \n \nRather surprisingly, Cx50 hemichannels are impermeable to ATP . To our knowledge this is the \nonly connexin hemichannel t hat ATP cannot permeate. Traditionally, connexin permeability \nstudies have studied selectivity by increasing the size of fluorescent dyes (Weber et al., 2004; \nHarris, 2007), with the idea being that any molecule that was below the limiting pore diameter \n(around 12 Å) should permeate. This would suggest minimal selectivity between permeants, \nleaving the major driving force as intracellular concentration. Our resul ts modify this idea . \nAlthough the smaller molecules glutamate and lactate do permeate the relatively non-selective \nchannels more easily, lactate being the smallest molecule should permeate all connexins : but \nit cannot permeate Cx36 or Cx46, whereas ATP, a much larger molecule can. \n \nWe also find that the permeability profile of the hemichannel can alter with the nature of the \ngating stimulus. For those connexins that are directly CO 2 sensitive, opening the hemichannel \nby hypercapnia seems to give greater release than depolarisation (summarized in Table 2).  One \npossibility is that 50 mM KCl , while predicted to depolarise the cell by about 70 mV , may not \nsufficiently depolarise the membrane to obtain full channel opening. We also find that lowered \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n[Ca2+]ext seems to be the least effective stimulus for release. While this manipulation may \nunblock the channel (by removing the ring of bound Ca 2+ ions), the N-termini may still partially \nblock the channel and alter the permeation pathway in a way that may not happen with a more \nphysiological stimulus. \n \nThe selective permeability of connexins may match their physiological roles \n \nThat there are 21 connexin genes in the human genome, indicates distinct fundamental roles in \nphysiological processes.  The need for so many isoforms suggest functional specialisation -\ncombinations of properties that match connexin to function.  Sensitivity to gating stimuli, and \npermeability to small molecules are two important properties that may determine  which \nfunctional roles connexins are suited to. \n \nThe relatively non -selective connexins, Cx26, Cx32 and Cx43, have different CO 2 sensitivity \nprofiles. Cx26 is suited to detection of systemic PCO 2 and has a role in the control of breathing \n(Huckstepp et al., 2010b; van de Wiel et al., 2020; Dale, 2021). Cx32 requires much higher levels \nof PCO2 to open and may be more suited to detecting local CO 2 production (Huckstepp et al., \n2010a; Dospinescu et al., 2019; Butler and Dale, 2023) . It is interesting the Cx32 is highly \npermeable to lactate when opened by hypercapnia. This lends support to an attractive \nhypothesis that Cx32 may detect hotspots of metabolic activity and, by opening and permitting \nrelease of lactate, could provide metabolic support for highly active cells  (Barros, 2013). Cx43 \nwith its extensive C-terminus interacts with many other proteins  (Iacobas et al., 2003; Iacobas \net al., 2007) , yet is also CO 2 sensitive (Dospinsecu et al., 2025)  and is partially open under \nphysiological conditions (Chever et al., 2014; Turovsky et al., 2020).  \n \nWe also discovered a group of connexins with highly selective permeability profiles: Cx36, Cx46 \nand Cx50. Cx36 is expressed predominantly in neurons and is impermeable to glutamate and \nlactate. The lack of glutamate permeability may be functionally significant  as most excitatory \nneurons use this as their main neurotransmitter. This lack of permeability may ensure that \nglutamate release is tightly regulated under ph ysiological conditions and occurs mainly via \nvesicular exocytosis at synaptic sites.  As lactate is an effective metabolite for oxidative \nphosphorylation in neurons , the lack of  permeability of Cx36  hemichannels to lactate would \nprevent unregulated efflux of lactate from neurons.  \n \nCx46 and Cx50 are expressed almost exclusively in the lens  of the eye   (Mathias et al., 2010; \nBerthoud and Ngezahayo, 2017). Cx46 and Cx50 form gap junctions between the lens epithelial \nand fibre cells, and between lens fibre cells. Cx50 is also present as hemichannels in lens fibre \ncells. As lens fibre cells mature , they lose their intracellular organelles including mitochondria \n(Bassnett, 2002)  and thus the ability to make ATP via oxidative phosphorylation.  One can \nspeculate that having ATP permeable C x46 gap junction channels may be valuable in allowing \ndiffusion of this key metabolite from the metabolically active cells in the lens into the relatively \ninactive lens fibre cells. Equally, the lack of ATP permeability of Cx50 hemichannels may permit \npreservation of this scarce resource in the lens fibre cells. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nThe N-terminus is a fundamental determinant of selectivity \n \nConsiderable evid ence suggest that t he N -terminus is part of the gating mechanism of \nconnexins. Depending on the isoform , the N -terminus projects into the pore to form a n \noccluding plug or forms a cap at the cytoplasmic vestibule also to close it. In the open state the \nN-termini line the pore and thus experience the membrane electric field. This  may explain why \nincreasing the charge of the Cx50 N -terminus appears to enhance the voltage -gating of the \nmutants (Figs 7 & 8 ) (Kalmatsky et al., 2009) . As the N-terminus lines the entrance to the pore \n(Maeda et al., 2009; Myers et al., 2018; Flores et al., 2020; Yue et al., 2021; Brotherton et al., \n2022; Jaradat et al., 2022; Brotherton et al., 2024) it may regulate selectivity to small molecules. \nOur evidence suggests that this is the case: by manipulating the charge on the N -terminus of \nCx50 to make it more like Cx46,  we were able to create a mutant channel that gained ATP \nsensitivity over the wild type Cx50 hemichannel. We note that the mutation Cx50N9R increase the \npermeability to ATP through the hemichannel, despite this mutation being reported to decrease \nthe conductance of the Cx50 gap junction channel (Yue et al., 2021) . However, the reverse set \nof mutations -changing charge on the N -terminus of Cx46 to make it more Cx50 -like did not \ndiminish ATP permeability of the mutant Cx46 hemichannel. This shows that other mechanisms \ncan be important.  \n \nOur data suggests that residues that alter the interaction between the N-terminus and the lining \nof the pore -notably TM2 – play a role in selectivity  (Brotherton et al., 2022; Brotherton et al., \n2024). We identified Ala14 and Thr89 of Cx46 as residues that might be important in this \ninteraction and mutated those to their equivalent in Cx50 ( A14V,T89S). Thr89 in Cx46  is \nequivalent to Ala88 of Cx26, which interacts with Val13 on the N terminus  (Brotherton et al., \n2022). Substitution of larger residues (A88V) change Cx26 channel function and are pathological \n(Koval, 2013; Meigh et al., 2014) . However, the double mutation in which a smaller residue is \nsubstituted (T89S) avoids the steric clash between V14 and T89 previously reported in Cx46-50 \nchimaeric channels which were non -functional (Yue et al., 2021) . The double mutation s A14V \nand T89S or R9N and N13E by themselves did not alter ATP permeability. However, when all four \nmutations were combined to create Cx46QM there was a considerable reduction of ATP release \ncompared to Cx46WT. The effect of the quadruple mutation FITC permeation was not statistically \nsignificant. This suggests that rather than simply resulting in a poorly permeable channel, the \nquadruple mutation has a somewhat selective effect on the ATP permeation pathway. Our study \nnot only highlights the importance of the N -terminus in determining selectivity of connexin \nhemichannels to small molecules, but also that even in two very closely related connexins there \nare other structural elements that control permselectivity.  \n \nMaterials and Methods \nConnexin mutagenesis \ncDNAs for the  Cx26 and Cx32 genes were synthesised by Genscript  and for the Cx46, Cx50, \nCx36, Cx43 and Cx 31.3 genes by IDT. These were subsequently subcloned into pCAG -GS-\nmCherry vector prior to transfection. Point mutations were introduced using Gibson assembly. \nOverlapping fragments both containing the desired mutation were PCR amplified with primers \n(IDT). Successful mutagenesis was confirmed using Sanger sequencing (GATC Biotech). Double \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nmutants (Cx50 N9R E13N , Cx46R9N N13E ) were cloned using successive Gibson assemblies. All Cx \nconstructs were inserted upstream of an mCherry tag, linked via a 12 AA linker \n(GVPRARDPPVAT). \nCell culture \nParental HeLa DH cells (ECACC 96112022 RRID:CVCL_2483) were cultured with low-glucose \nDMEM (Merck Life Sciences UK Ltd, CAT# D6046) supplemented with 10% foetal bovine serum \n(Labtech.com, CAT# FCS-SA) and 5% penicillin/streptomycin. Cells were seeded onto \ncoverslips at a density of 4x104 cells per well. Cells were transiently transfected to co-express \none connexin isoform and one genetically encoded fluorescent sensor: \npDisplay-GRAB_ATP1.0-IRES-mCherry-CAAX was a gift from Yulong Li (Addgene plasmid # \n167582 ; http://n2t.net/addgene:167582 ; RRID:Addgene_167582) (Wu et al., 2022). \npAEMXT-eLACCO1.1 was a gift from Robert Campbell (Addgene plasmid # 167946 ; \nhttp://n2t.net/addgene:167946 ; RRID:Addgene_167946)  (Nasu et al., 2021) . To improve \nexpression of eLACCO1.1, this construct was subcloned into the iGluSnFR exxpression vector \nbackbone. Sequences were verified with Sanger sequencing (GATC). \npCMV(MinDis).iGluSnFR was a gift from Loren Looger (Addgene plasmid # 41732 ; \nhttp://n2t.net/addgene:41732 ; RRID:Addgene_41732) (Marvin et al., 2013). \nA mixture of 1 µg of DNA from pCAG -Cx-mCherry construct and 1 µg sensor with 3 µg PEI was \nadded to cells for 4-8h. Cells were imaged 48 hours after transfection.  \naCSF \nControl (20 mmHg pCO2) – 140 mM NaCl, 10 mM NaHCO3, 1.25 mM NaH2PO4, 3mM KCl, 1 mM \nMgSO4.   \nControl (35 mmHg pCO2) – 124 mM NaCl, 26 mM NaHCO 3, 1.25 mM NaH2PO4, 3mM KCl, 1 mM \nMgSO4. \nHypercapnic (55 mmHg pCO2) - 100 mM NaCl, 50 mM NaHCO3, 1.25 mM NaH2PO4, 3 mM KCl, 1 \nmM MgSO4.  \nHypercapnic (70 mmHg pCO2) - 70 mM NaCl, 80 mM NaHCO 3, 1.25 mM NaH2PO4, 3 mM KCl, 1 \nmM MgSO4. \nHigh K+ (20 mmHg) – 47 mM NaCl, 10 mM NaHCO3, 1.25 mM NaH2PO4, 50mM KCl, 1 mM MgSO4. \nFor 35 mmHg, the recipe is the same but with 77 mM NaCl. \nAll solutions ha d 10 mM D -glucose and 2 mM CaCl 2 (or MgCl2 where [Ca2+]0 solution desired) \nadded just before  use and saturated with 98% O2/2% CO 2 (20 mmHg), 95% O 2/5% CO 2 \n(carbogen) (35 mmHg), or carbogen plus CO2 (55 mmHg and 70 mmHg). CO2 in all solutions was \nadjusted to give a pH of ~7.4. \nLive cell fluorescence imaging and analysis \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nCells were transiently transfected with one pCAG -Cx-mCherry construct and one genetically \nencoded fluorescent sensor 48 hours prior to imaging. Cells were perfused with control aCSF \nuntil a stable baseline was reached, before perfusion with either hypercapnic or high  K+ aCSF. \nOnce a stable baseline was reached after solution change, cells were again perfused with \ncontrol aCSF and when a stable baseline reached, recordings were calibrated by direct \napplication of 3 µM of the corresponding analyte. \nAll cells were imaged by epifluorescence ( Scientifica Slice Scope, Cairn Research OptoLED \nillumination, 60x water Olympus immersion objective, NA 1.0, Hamamatsu ImagEM EM -SSC \ncamera, Metafluor software). cpGFP in the sensors were excited by a 470 nm LED, with emission \ncaptured between 504 -543 nm. Connexin constructs have a C -terminal mCherry tag, which is \nexcited by a 535 nm LED and emission captured between 570 -640 nm. Only cells expressing \nboth cpGFP and mCherry were selected for recording, with cpGFP images acquired ev ery 4 \nseconds. For each condition, at least 3 independent transfections were performed with at least \n2 coverslips per transfection.   \nAnalysis of all experiments was carried out in ImageJ. Images were opened as a stack and \nstabilised (Li, 2008). ROIs were drawn around cells co -expressing both sensor and connexin. \nMedian pixel intensity was plotted as normalised fluorescence change (∆F/F 0) versus time to \ngive traces of fluorescence change. The a mount of analyte release was quantified as \nconcentration by normalising to the ∆F/F 0 caused by application of 3 µM of analyte, which was \nwithin the linear portion of the dose response curve for each sensor . Release froma single cell \nwas considered to be a statistical replicate. \nDye loading \nCells were transiently transfected with pCAG-Cx43-mCherry, pCAG-Cx46WT-mCherry or pCAG-\nCx43QM-mCherry constructs 48 hours prior to experiments. After washing in 20 mmHg aCSF , \ncells were perfused with one of the following solutions: 20 mmHg aCSF, 55 mmHg aCSF (Cx43 \nonly), Ca2+-free aCSF (Cx43 only) or 50 mM KCl aCSF (Cx46WT and Cx46QM only) each containing \n50 µM fluor escein isothiocyanate (FITC) for 10 mins. The cells were then washed, first by \nperfusion with 20 mmHg in the absence of FITC, before being placed in serial washes of 20 \nmmHg aCSF. Cells were fixed in 4% paraformaldehyde for 30 mins before being washed 3 times \nwith PBS. Coverslips were mounted inverted on a microscope slide using Fluorshield™ with DAPI \nmounting medium (Sigma-Aldrich, Cat# F6057). Images were taken using the Zeiss-880 or Zeiss-\n980 confocal LSM, specifically using the 488 and 561 nm lasers. Subsequent analysis was done \nusing the FIJI software.  For Figure 9, figure supplement 1 , the median pixel intensity for each  \ntransfection was considered to be a statistical replicate. \nStatistical analysis \nAll quantitative data is presented as box and whisker plots, where the line represents the \nmedian, the box is the interquartile range, and the whiskers are the range, with all individual data \npoints included. Pairwise Mann Whitney U-tests were carried out with corrections for multiple \ncomparisons using the false discovery method, with a maximum false discovery set to 0.05 \n(Curran-Everett, 2000). In text, all data is presented as median (95% CI, upper, lower limit). All \ncalculations were performed on GraphPad Prism.  \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nAuthor Contributions:  AL: Conceptualisation, data curation, investigation, writing – original \ndraft, review and editing, formal analysis . JB: Investigation (dye loading and confocal \nmicroscopy, sensor optimisation). ND: Conceptualisation, supervision, writing – review and \nediting. \n \nFunding: JB was supported by the Biotechnology and Biological Sciences Research Council \n(BBSRC) and University of Warwick funded Midlands Integrative Biosciences Training \nPartnership (MIBTP) grant number BB/T00746X/1.  AL was funded by the Medical Research \nCouncil through the University of Warwick Doctoral Training Partnership, grant number \nMR/N014294/1 \n \nConflicts of interest: The authors declare that there are no conflicts of interest. \n \nAcknowledgements: We thank Prof Alexander Cameron for reading a draft of this paper. \n \nReferences \nBarros LF (2013) Metabolic signaling by lactate in the brain. Trends Neurosci 36:396-404. \nBassnett S (2002) Lens organelle degradation. Experimental eye research 74:1-6. \nBennett BC, Purdy MD, Baker KA, Acharya C, McIntire WE, Stevens RC, Zhang Q, Harris AL, \nAbagyan R, Yeager M (2016) An electrostatic mechanism for Ca(2+)-mediated regulation \nof gap junction channels. Nat Commun 7:8770. \nBerthoud VM, Ngezahayo A (2017) Focus on lens connexins. BMC cell biology 18:6. \nBrotherton DH, Savva CG, Ragan TJ, Dale N, Cameron AD (2022) Conformational changes and \nCO2-induced channel gating in connexin26. 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Neuron \n110:770-782 e775. \nYue B, Haddad BG, Khan U, Chen H, Atalla M, Zhang Z, Zuckerman DM, Reichow SL, Bai D (2021) \nConnexin 46 and connexin 50 gap junction channel properties are shaped by structural \nand dynamic features of their N-terminal domains. J Physiol 599:3313-3335. \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nFigures and Legends \nFigure 1. HeLa cells expressing the g enetically encoded fluorescent sensors GRAB ATP, \niGluSnFr and eLACCO1.1 alone do not respond to connexin gating stimuli. A. Representative \nimages of cells at 55 mmHg PCO2 (inset is 20 mmHg control), 50 mM KCl, and after application \nof 3 µM corresponding analyte. Scale bar represents 20 µm. B. Representative traces showing \nsensor responses to 50 mM KCl (green bar), 55 mmHg (blue bar), and 3 µM corresponding \nanalyte (red bar). C. Summary data showing median ∆F/F0 for ATP (n = 16 cells), glutamate (n = \n8) and lactate (n = 8).  \n  \n200s\n10% \nΔF/F0\n100s\n10% \nΔF/F0\n200s\nATPGlutamateLactate\n55 mmHg 50 mM KCl20 mmHg 3 µM\nA B C\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n200s\n200s\n200s\nCO\n2\n50 mM KCl\n3 µMCO\n2\n50 mM KCl\n3 µMCO\n2\n50 mM KCl\n3 µM\n-0.1\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\nΔF/F0\nATP LactateGlutamate\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 2. Cx26 mediates release of  ATP, glutamate and lactate. A. Representative images \nshowing cells from each sensor under each condition. Scale bar represents 20 µm. B. \nRepresentative traces of normalised fluorescence changes in response to 55 mmHg PCO2 (blue \nbar), 50 mM KCl (green bar) and a 3 µM calibration of the corresponding analyte (red bar). C. \nSummary data showing the median release for ATP (n = 15 cells), glutamate (n = 19) and lactate \n(n = 27) through Cx26 hemichannels stimulated either by 55 mmHg (filled circles), or 50 mM KCl \n(open circles). Mann Whitney U-test, p < 0.0001 (55 mmHg ATP vs glutamate, 55 mmHg ATP vs \nlactate). The order of stimulus (CO2 or KCl) was regularly reversed between recordings to avoid \nany potential depletion of ATP release.  Data present is from at least 3 independent \ntransfections. \n  \n55 mmHg 50 mM KCl20 mmHgATPGlutamateLactate\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n100s\n200s\n100s\nA B C\n-5\n0\n5\n10Concentration (µM)\nATP Glutamate Lactate\n<0.0001\n<0.0001\n10% \nΔF/F0\n200s\n-5\n0\n5\n10Concentration (µM)\nATP Glutamate Lactate\n<0.0001\n<0.0001\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \n \nFigure 2, figure supplement 1. Co-expression of Cx-mCherry and sensor (GFP). Using Cx36 \nas an example, the images demonstrate that cells were selected only if they co-expressed both \nthe connexin construct, which is tagged with mCherry (top row), and the sensor, which is tagged \nwith GFP (bottom row). Scale bar represents 20 µm.  \n  \nCx36-mCherry\nGRABATP iGluSnFR eLACCO1.1\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 3. Cx32 mediates release of ATP, glutamate and lactate. A. Representative images \nshowing cells from each sensor under each condition. Scale bar represents 20 µm. B. \nRepresentative traces of normalised fluorescence changes in response to 70 mmHg PCO2 (blue \nbar), 50 mM KCl (green bar) and a 3 µM calibration of the corresponding analyte (red bar). C. \nSummary data showing the median release of: ATP (n = 18), glutamate (n = 27 for CO2, n = 14 for \nKCl), and lactate (n = 15), through Cx32 showing changes in release depending on whether the \nchannel was opened by CO 2 (open circles), or a depolarising stimulus (closed circles). Data \npresented is from at least 3 independent transfections. Mann Whitney U-test, p < 0.0001 (CO 2 \nATP vs glutamate), p < 0.0001 (CO2 ATP vs lactate), p < 0.0001 (50 mM KCl ATP vs glutamate), p \n< 0.0001 (50 mM KCl ATP vs lactate). \n  \n70 mmHg 50 mM KCl35 mmHg\nATPGlutamateLactate\n10% \nΔF/F0\n200s\n20% \nΔF/F0\n200s\n10% \nΔF/F0\n100s\n10% \nΔF/F0\n200s\n20% \nΔF/F0\n200s\nA B C\n0\n4\n8\n12Concentration (µM)\nATP Glutamate Lactate\n<0.0001\n<0.0001\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 4. Cx43 mediates release of ATP, glutamate and lactate. A. Representative images \nshowing cells from each sensor under each condition. Scale bar represents 20 µm. B. \nRepresentative traces of normalised fluorescence changes in response to 55 mmHg PCO2 (blue \nbar), 50 mM KCl (green bar) and a 3 µM calibration of the corresponding analyte (red bar). C. \nSummary data showing the median release of analytes through Cx43: ATP (n = 33 (CO 2), n = 17 \n(high KCl)), glutamate (n = 19), lactate (n = 18). Data shows CO 2-dependent release (open \ncircles), and release to a depolarising stimulus (closed circles). Data presented is from at least \n3 independent transfections. Mann Whitney U-test, p < 0.0001 (CO 2 ATP vs glutamate), p < \n0.0001 (CO2 ATP vs lactate). \n \n  \n200s\n10% ΔF/F0\n200s\n20% ΔF/F0\n200s\n55 mmHg 50 mM KCl20 mmHgATPGlutamateLactate\n10% \nΔF/F0\n200s\n100s\n20% \nΔF/F0\n20% \nΔF/F0\n200s\nA B C\n-5\n0\n5\n10Concentration (µM)\n<0.0001\n<0.0001\nATP Glutamate Lactate\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 4 , Figure supplement 1. Low [Ca 2+]ext is sufficient to open Cx43 hemichannels. A. \nRepresentative images of FITC loaded into mCherry-tagged Cx43 positive cells. Scale bar 20 µm. \nB. Summary data showing the median pixel intensity under control (20 mmHg) (n = 19), \nhypercapnia (55 mmHg) (n = 18) and low [Ca2+]ext (n = 17). Mann Whitney U-test, p < 0.0001 (low \n[Ca2]ext vs 20 mmHg).  \n \n  \n20 mmHg 55 mmHg Low [Ca2+]ext\n20 mmHg55 mmHg\nlow [Ca\n2 ]ext \n0\n30\n60\n90\nPixel Intensity\n<0.0001\n20mmHg55 mmHglow [Ca\n2+]ext\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 4, figure supplement 2 . Removal of extracellular Ca 2+ changes the permeability of \nCx43 to physiological molecules . A. Representative images showing cells from each sensor \nunder each condition. Scale bar represents 20 µm. B. Representative traces of normalised \nfluorescence changes in response to 55 mmHg pCO 2 (blue bar), low [Ca2+]ext (yellow bar) and a \n3 µM calibration of the corresponding analyte (red bar). C. Summary data showing the median \nrelease of analytes through Cx43 and how it differs depending on the stimulus: ATP (n = 33 for \nCO2, n = 17 for high KCl), glutamate (n = 19), lactate (n = 18). Data presented is from a t least 3 \nindependent transfections. For 55 mmHg vs low [Ca2+]ext for ATP, glutamate and lactate release, \nMann Whitney U-test was performed, p < 0.0001. \n \n  \n55 mmHg Low [Ca2+]ext\nATP\nGlutamateLactate\n20 mmHg\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n200s\nA B C\nATP CO2ATP 0 Ca\nGlutamate CO2Glutamate 0 Ca\nLactate CO2Lactate 0 Ca\n-5\n0\n5\n10\n15Concentration (µM)\nATP GlutamateLactate\n+55 mmHg\nLow [Ca2+]ext +\n+\n+\n+\n+\n<0.0001\n<0.0001 <0.0001\nATP\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \n \nFigure 5. Cx31.3 mediates release of ATP, glutamate and lactate. A. Representative images \nshowing cells from each sensor under each condition. Scale bar represents 20 µm. B. \nRepresentative traces of normalised fluorescence changes in response to 50 mM KCl (green \nbar), and 3 µM analyte (red bar). C. Summary data showing the median ATP (n = 32) , glutamate \n(n = 19) and lactate (n= 50) release from Cx31.3 hemichannels in response to a depolarising \nstimulus. Data presented is from at least 3 independent transfections. Mann Whitney U-test, p \n= 0.0371 (ATP vs glutamate) and p=0.004 (lactate vs glutamate). \n \n  \n20% \nΔF/F0\n10% \nΔF/F0\n100s\n200s\n20 mmHg 50 mM KCl\nATPGlutamate\nA B C\nLactate\n10% \nΔF/F0\n100s ATP \nGlutamate\nLactate\n0\n1\n2\n3\n4Concentration (µM)\n0.03\n0.004\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \nFigure 6. Cx36 hemichannels mediate release of ATP but are impermeable to glutamate and \nlactate. A. Representative images showing cells from each sensor under each condition: ATP (n \n= 24), glutamate (n = 22), lactate (n = 15). Scale bar represents 20 µm. B. Representative traces \nof normalised fluorescence changes in response to 50 mM KCl (green bar) and 3 µM analyte (red \nbar). C. Summary data depicting ATP release from Cx36 and no permeability to glutamate or \nlactate. Mann Whitney U-test, p < 0.0001 (ATP vs glutamate, ATP vs lactate). \n \n  \nATPGlutamateLactate 10% \nΔF/F0\n100s\n10% \nΔF/F0\n100s\n10% \nΔF/F0\n100s\n100s\n100s\n10% \nΔF/F0\n10% \nΔF/F0\nA B C\n3 mM KCl50 mM KCl\nATP\nGlutamate\nLactate\n-4\n-2\n0\n2\n4\n6\nCx36\nConcentration (µM)\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \n \nFigure 7. Cx46 and Cx50 have opposing permeability profiles. A. Representative traces \nshowing normalised fluorescence changes in response to 55 mmHg (blue bar), 50 mM KCl \n(green bar) and 3 µM (analyte). B. Summary data depicting analyte release under each stimulus: \nCx46 ATP (n = 40), Cx46 glutamate (n = 25), Cx46 lactate (n = 16), Cx50 ATP (n = 28), Cx50 \nglutamate (n = 12), Cx50 lactate (n = 11). Cx50 is CO 2 sensitive, and thus release is stimulated \nby 55 mmHg pCO 2 (open circles), and 50 mM KCl (closed circles). Data presented is from 3 \nindependent transfections.  \n  \n200s\n20% ΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n10% \nΔF/F0\n5% \nΔF/F0\n100s\n100s\n100s\n200s\n100s\n100s\nCx50 ATP\nCx50 glutamate\nCx50 lactate\nCx46 ATP\nCx46 glutamate\nCx46 lactate\nA B\n-2\n0\n2\n4\n6\n8\nConcentration (µM)\nCx46 Cx50\nATPGlutamateLactate\nATP Glutamate Lactate\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 8. N -terminal mutations make Cx50 permeable to  ATP. A.  Sequence alignments for \nresidues 1-26 of Cx46 and Cx50, to show the residues in Cx50 that have been mutated to those \nin Cx46 (purple boxes). B. Representative images showing cells from each sensor under each \ncondition. Scale bar represents 20 µm. C. Representative traces of normalised fluorescence \nchanges in response to 55 mmHg (blue bar), 50 mM KCl (green bar) and 3 µM ATP (red bar). D. \nSummary data showing ATP release in response to depolarisation from Cx50 mutants: N9R (n = \n26), N9K (n = 13), E13N (n = 18), N9R E13N ( n = 11). Data shows CO 2-dependent release (open \ncircles), and release to a depolarising stimulus (closed circles).  The dotted lines show the \nmedian and 95% confidence limits for ATP release  from Cx46 in response to depolarisation. \nMann Whitney U-test, p = 0.0018, p = 0.0155 and p = 0.0004 (N9R, N9K and E13N respectively, \nwhere release is evoked by 50 mM KCl, compared to Cx46 WT). Mann Whitney U-test, p = 0.96 \n(Cx46WT vs Cx50N9R E13N). E.  Summary data demonstrating CO2-evoked ATP release form the Cx50 \nmutant channels N9R (n = 26), N9K (n = 13), E13N (n = 18), N9R E13N (n = 11). \nMGDWSFLGRLLENAQEHSTVIGKVWL\nMGDWSFLGNILEEVNEHSTVIGRVWLCx50/1-26\nCx46/1-26A\nN9R\n55 mmHg 50 mM KCl\nN9KE13NN9R E13N\n20 mmHg\n200s\n10% \nΔF/F0\n200s\n10% \nΔF/F0\n200s\n10% \nΔF/F0\n100s\n10% \nΔF/F0\n200s\n10% \nΔF/F0\n200s\n10% \nΔF/F0\n200s\n10% \nΔF/F0\n100s\n10% \nΔF/F0\nED\nB C\nN9R N9K E13N\nN9R E13N\n0\n1\n2\n3[ATP] release (µM)\n55 mmHg\nN9R N9K E13N\nN9R E13N\n0\n1\n2\n3\n4[ATP] release (µM)\n50 mM KCl\nN9R N9K E13N\nN9R E13N\n0\n1\n2\n3\n4[ATP] release (µM)\n50 mM KCl\nN9R N9K E13N\nN9R E13N\n0\n1\n2\n3[ATP] release (µM)\n55 mmHg\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 9. The permeability of Cx46 to ATP appears to be reliant on N-terminal charge and  \ninteractions between the N-terminus and TM2. A. Sequence alignments from 1-26 and 80-89 \nfor Cx50 and Cx46, to show the residues  in Cx46 which have been mutated to correspond to \nthose in Cx50 (red). B. Representative images showing cells expressing Cx46WT, Cx46R9N N13E and \nCx46QM under each condition. Scale bar represents 20 µm. C. Representative traces showing \nnormalised change in fluorescence to 50 mM KCl (green bar), and 3 µM ATP (red bar). D. \nSummary data depicting the median ATP release from Cx46WT (the dotted lines show the median \nand 95% confidence interval for Cx46WT), Cx46R9N N13E (n = 7) and Cx46QM (n = 9). Mann Whitney U-\ntest, p < 0.0001 (Cx46WT vs Cx46QM). \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \nFigure 9, figure supplement 1. Cx46QM remains permeable to FITC. A, Representative images \nshowing cells expressing Cx46WT or Cx46QM loaded with FITC under each condition. Scale bar \nrepresents 15 µm. B, Summary data depicting the median change in fluorescence pixel intensity \nbetween 20 mmHg and 50 mM KCl for cells expressing Cx46WT or Cx46QM from 5 independent \ntransfections. There is no significant difference in FITC  loading into the Cx46 QM compared to \nCx46WT (Mann Whitney U Test). For Cx46WT, 43 cells in 20 mmHg and 35 cells in 50 mM KCl were \nanalyzed. For Cx46QM, 79  cells in 20 mmHg and 79 cells for Cx46QM in 50 mM KCl were analyzed. \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \n \nTable 1. Calculations of the Nernstian equilibrium potential for ATP, glutamate and lactate \nin HeLa cells. We assigned the valence of ATP under the assumption that it is chelated to Mg2+. \nDriving force is calculated assuming a resting potential of -60 mV.  The intracellular \nconcentrations were taken from studies that investigated HeLa cells: a, (Imamura et al., 2009); \nb, (Piva and McEvoy-Bowe, 1998); c, (San Martin et al., 2013). \n \n  \nAnalyte Valence [Analyte]I \n(mM) \n[Analyte]o \n(mM) \nVeq (mV) Driving force at rest \nATP -2 10a 10-4 145 205 \nGlutamate -1 20b 10-4 307 367 \nLactate -1 1c 10-4 232 292 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint \n\n \n \n \n \nConnexin ATP Glu Lac \nCx26 1 3.5 (0.44) 3.1 (0) \nCx32 1 3.0 (1.73) 5.5 (1.83) \nCx43 1 2.4 (0.4) 2.0 (0) \nCx31.3 1 0.9 1.1 \nCx36 1 0 0 \nCx46 1 0 0 \nCx50 0 1.5 (0) µM 5.8 (3.4) µM \n \nTable 2. Relative release of ATP, glutamate and lactate of connexin hemichannels \nnormalised to amount of ATP release.  The numbers given are for the hypercapnic stimulus, \nexcept for Cx31.3, Cx36 and Cx46 which are insensitive to CO2. For those channels sensitive to \nCO2, the numbers in brackets are normalised values for the depolarising stimulus. For Cx50, \nwhich is not permeable to ATP, the numbers are absolute concentration in µM.  The ratio of the \nNernstian driving force  for each analyte (normalised to that  of ATP) during the hypercapnic \nstimulus is 1: 1.8:1.4, during the depolarising stimulus it is  (1:2.1:1.6) assuming that the \nmembrane depolarises to 0 mV. \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted March 12, 2025. ; https://doi.org/10.1101/2025.03.12.642803doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}