CO 2 -dependent opening of Connexin 43 hemichannels

CO2-dependent opening of Connexin 43 hemichannels | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results CO 2 -dependent opening of Connexin 43 hemichannels Valentin-Mihai Dospinescu , Alexander Mascarenhas , Jack Butler , Sarbjit Nijjar , Kyara de Oliveira Taborda , Sean Connors , Lumei Huang , View ORCID Profile Nicholas Dale doi: https://doi.org/10.1101/2025.01.13.632711 Valentin-Mihai Dospinescu 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexander Mascarenhas 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jack Butler 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sarbjit Nijjar 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kyara de Oliveira Taborda 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sean Connors 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lumei Huang 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicholas Dale 1 School of Life Sciences, University of Warwick , Coventry, CV4 7AL, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicholas Dale For correspondence: N.E.Dale{at}warwick.ac.uk Abstract Full Text Info/History Metrics Preview PDF Abstract Sequence and structure comparisons between alpha and beta connexins, Cx43 and Cx26, revealed that Cx43 has a motif, the carbamylation motif, that confers CO 2 -sensitivity on a subset of beta connexins. By using a fluorescent dye loading assay, whole cell patch clamp recordings and real time measurement of ATP release via GRAB ATP we have demonstrated that Cx43 hemichannels open in a highly CO 2 sensitive manner over the range 20 to 70 mmHg. Mutational analysis confirms that the equivalent residues to those in Cx26 that have been shown to be involved in mediating the effects of CO 2 on gating of hemichannels and gap junction channels, also mediate Cx43 hemichannel gating. Our data predicts that Cx43 will be partially open and able to release ATP at resting physiological levels of PCO 2 . We have tested this prediction in acute hippocampal slices, by showing that CO 2 -dependent enhancement of synaptic transmission can be blocked by the Cx43-selective mimetic peptide Gap26. Our data resolves an inconsistency in the literature between in vivo studies suggesting that Cx43 hemichannels are at least partially open at rest, and in vitro studies, performed in the absence of HCO 3- /CO 2 buffering that show Cx43 hemichannels are shut. Our evidence suggests that the ancestral gene that duplicated to give the alpha and beta connexin clades must have possessed the carbamylation motif. CO 2 sensitivity is thus a fundamental ancient characteristic of several connexins that has been lost in more recently derived members of this gene family. Introduction There are 21 connexin genes encoded in the human genome. Connexins are transmembrane proteins that can form hemichannels and gap junction channels. The existence of 21 isoforms suggests functional specialisation across the gene family ( Sohl & Willecke, 2004 ). On the basis of molecular phylogeny, the connexins have been divided into 4 broad clades - the alpha, beta, and gamma and delta groupings ( Mikalsen et al ., 2021 ). Certain members of the β-clade, Cx26, Cx30 and Cx32, are directly sensitive to CO 2 ( Huckstepp et al ., 2010 ; Dospinescu et al ., 2019 ). The mechanism of CO 2 sensitivity has best been studied in Cx26. While Cx26 hemichannels are opened in response to CO 2 ( Huckstepp et al ., 2010 ), CO 2 closes Cx26 gap junction channels ( Nijjar et al ., 2021 ). The CO 2 -dependent gating of Cx26 hemichannels and gap junction channels involves carbamylation of a specific lysine, K125, which is part of a critical carbamylation motif, 125 KVRIEGS 131 ( Meigh et al ., 2013 ). This motif is essential for the CO 2 -sensitivity of Cx26, as it allows the formation of a salt bridge between the carbamylated K125 and R104 of the neighbouring subunit ( Meigh et al ., 2013 ). The formation of this “carbamate” bridge induces conformational changes, ultimately leading to the opening of the hemichannel or closing of the gap junction channel ( Meigh et al ., 2013 ; Nijjar et al ., 2021 ; Brotherton et al ., 2022 ; Brotherton et al ., 2024 ). Mutation of K125 to arginine, an amino acid that cannot be carbamylated, or R104 to alanine so that it cannot form a carbamate bridge, removes the ability of CO 2 to open Cx26 hemichannels ( Meigh et al ., 2013 ) or close Cx26 gap junction channels ( Nijjar et al ., 2021 ; Nijjar et al ., 2025 ). Furthermore, the introduction of the carbamylation motif into a CO 2 -insensitive connexin, Cx31, results in CO 2 -dependent hemichannel opening of Cx31 ( Meigh et al ., 2013 ). Other members of the β-clade, Cx30 and Cx32, are also CO 2 -sensitive, but have different sensitivity profiles ( Huckstepp et al ., 2010 ; Dospinescu et al ., 2019 ). Evolutionary comparisons show that the carbamylation motif has been conserved over 400 million years ( Dospinescu et al ., 2019 ) and appears to be a necessary and sufficient feature for the CO 2 -sensitivity of connexins. Sequence comparisons of the β-connexins and α-connexins, revealed the presence of a potential carbamylation motif within the α-connexin, Cx43. Cx43 has a motif ( 144 KVKMRGG 150 ) homologous to the motif in Cx26 ( 125 KVRIEGS 131 ) suggesting that it might also be sensitive to CO 2 . In this study we show that Cx43 hemichannels are indeed directly sensitive to changes in the partial pressure of CO 2 (PCO 2 ) over the physiological concentration range. Unlike Cx26, the CO 2 -dependent opening mechanism of Cx43 is more complex, involving additional residues beyond the homologs of K125 and R104 in Cx26. Our discovery that Cx43 hemichannels are directly CO 2 sensitive has major physiological implications and may explain why Cx43 hemichannels appear to be open under physiological conditions but closed in vitro where they are usually studied in the absence of CO 2 /HCO 3- buffers. Results Identification of a potential carbamylation motif in Cx43 Cx43, an alpha-connexin, possesses a carbamylation motif homologous to that of the beta-connexins ( Fig 1A ). AlphaFold3 (AF3) predicts a structure very close to the experimentally determined structures for Cx43 ( Lee et al ., 2023a ; Qi et al ., 2023 ) but has the advantage of including portions of the structure that have not been experimentally resolved. This is a valid approach, as the de novo predictions by AF3 for the cytoplasmic loop of Cx26 have been supported by a recent experimentally-derived structure ( Brotherton et al ., 2024 ). By performing alignments of the Cx43 and Cx26 AF3 structures ( Fig 1B ), we found that two residues in Cx43, K144 and K105, aligned with the residues in Cx26 that are essential for hemichannel opening to CO 2 , K125 and R104 ( Fig 1C ). The predicted 3D orientation of the alpha-helices involved are positioned in a similar manner to allow potential interaction between K144 and K105 following carbamylation. Cx43 also presents another feature that is similar to Cx26: the high incidence of Lys residues. In Cx26, 5 lysine residues in the cytoplasmic loop can be carbamylated ( Nijjar et al ., 2025 ), suggesting an environment favourable for this to happen. The presence of multiple Lys residues in the cytoplasmic loop of Cx43 implies that the local environment may be conserved to facilitate the required pKa for CO 2 binding and carbamate bridge formation. Download figure Open in new tab Figure 1: Structures of Cx43 and Cx26 predicted by AF3. ( A ) α- and β-connexin sequence alignment with the known CO 2 -sensitive connexins at the top, Cx26, Cx30 and Cx32, followed by the α-connexin Cx43. The carbamylation motif is highlighted by the green box. ( B ) Cx26 and Cx43 AlphaFold3 structural alignments. ( C ) Cx26 and Cx43 hemichannel structural prediction. Cx43 hemichannels are opened by CO 2 We used an established dye-loading protocol, previously used for CO 2 connexin studies ( Huckstepp et al ., 2010 ; Meigh et al ., 2013 ; Meigh et al ., 2014 ; de Wolf et al ., 2016 ; de Wolf et al ., 2017 ; Cook et al ., 2019 ; Dospinescu et al ., 2019 ; van de Wiel et al ., 2020 ), to test whether Cx43 hemichannels are CO 2 sensitive. We observed significant dye-loading in Cx43-expressing cells when exposed to a high CO 2 stimulus (70 mmHg) when starting from a baseline of 20 mmHg (MW test, PCO 2 20 vs 70 mmHg: p = 0.008, n=5, Fig 2A ). We used a zero Ca 2+ aCSF to unblock hemichannels as a positive control to demonstrate that for WT Cx43, the zero Ca 2+ stimulus and 70 mmHg PCO 2 gave a similar amount of dye loading (MW test: p-value = 0.726, n=5). Download figure Open in new tab Figure 2: Cx43 hemichannels can be opened by an increase in PCO 2 . The CO 2 sensitivity of Cx43 was assayed using three different methods. (A) Dye-loading with CBF from 20 (left) to 70 mmHg PCO 2 (right). The inset represents the 0 Ca 2+ control, white scale bar is 20 μm. Cumulative probability graph represents all the data points measured with more than 200 cells per condition, box plot shows the medians from each independent transfection. (B) Whole cell patch recordings performed on untransfected parental HeLa cells and HeLa cells expressing Cx43 at a holding potential of −50 mV with steps to −40 mV to measure whole cell conductance. The baseline was set to 35 mmHg PCO 2 and three different concentrations were used (20, 55, 70 mmHg). The absolute conductance change evoked by a change in PCO 2 was measured and plotted as a dose response curve (right side, median with interquartile range) with 20 mmHg being assigned to zero and the absolute value of all conductance changes plotted relative to this (see Methods for details). The points were fitted by the Hill equation H=6, EC 50 =43 mmHg (blue points, orange curve). The data for untransfected parental HeLa cells was plotted the same way (grey). (C) The genetically-encoded ATP sensor GRAB ATP was co-transfected alongside Cx43 into HeLa cells (n=22). Representative images at different PCO 2 concentrations of the cells are shown, grey scale bar is 20 μm. Traces from the recording the images were selected from are shown; each line represent a different cell, the fluorescence was normalised by dividing all values by the baseline median pixel intensity before plotting. Box plot shows the µM ATP release as determined by normalisation to the 3 µM control solution applied. (D) Parental HeLa cells transfected only with GRAB ATP do not exhibit CO 2 dependent ATP release (n=14), summary box plot shows the µM ATP release as determined by normalisation to 3 µM ATP calibration. To further support these findings, we next used whole cell patch clamp recordings from HeLa cells expressing Cx43 ( Fig 2B ). We set the holding potential to −50 mV and gave regular steps to −40 mV to measure whole cell conductance. From a baseline of 35 mmHg PCO 2 , an increase in an outward conductance was observed when switching to 55 or 70 mmHg aCSF ( Fig 2B ). On switching to 20 mmHg aCSF, a small conductance decrease and reduction of outward current was observed ( Fig 2B ). Untransfected parental HeLa cells did not display changes in outward current to changes in PCO 2 . However, some parental HeLa cells exhibited a slow increase in an inward current on transfer from 35 to 70 mmHg ( Fig 2B ). This was quite distinct from the outward current observed in Cx43 expressing cells ( Fig 2B ). Overall, the patch clamp data predicted a dose response that could be fitted by the Hill equation with a Hill coefficient of 6 and an EC 50 of 43 mmHg. This is similar to Cx26 hemichannels which also display a steep CO 2 dose response curve with a Hill coefficient of >4 ( Huckstepp et al ., 2010 ; de Wolf et al ., 2017 ; van de Wiel et al ., 2020 ). To assess whether the CO 2 -dependent opening of Cx43 resulted in ATP release, we co-transfected HeLa cells with the genetically encoded ATP sensor - GRAB ATP ( Wu et al ., 2022 ). CO 2 dependent increase in ATP release was indeed observed (n=22, Fig 2C ). We observed an increase in GRAB ATP fluorescence on going from 20 to 35 mmHg PCO 2 and further to 55 mmHg PCO 2 ( Fig 2C ). While ATP release at 70 mmHg was slightly less than that at 55 mmHg, it remained markedly higher than at 35 mmHg PCO 2 ( Fig 2C ). By contrast, HeLa cells that were not transfected with Cx43 showed no responses to any applied CO 2 concentration as concluded from GRAB ATP experiments (n=14, Fig 2D ). Overall, our results show through three independent assays that Cx43 hemichannels are opened by modest changes in PCO 2 around the physiological norm and will be partially open at a PCO 2 of 35 mmHg ( Fig 2 ). Once opened by CO 2 , Cx43 hemichannels display permeability to ATP ( Fig 2C ). Cx43 displays sensitivity to intracellular pH via a sensor located in the C-terminus ( Ek-Vitorín et al ., 1996 ). We assessed the effect of truncations of the C-terminus of Cx43 (truncated after residue 256, Cx43 1-256 ) that remove this pH sensor. Both CO 2 and high K + induced ATP release from HeLa cells that expressed Cx43 1-256 ( Fig 2, figure supplement 1 ). We also measured the effect of changing PCO 2 on intracellular pH (pH i ) of HeLa cells. We found that transferring from 35 to 55 or 70 mmHg respectively induced median changes in pH i of −0.02 (95% CI −0.02, −0.03) and −0.13 (CI −0.10, −0.15; Fig 2, figure supplement 2 ). These results show that the effects of PCO 2 on Cx43 gating are independent of those involved in its pH sensitivity. Download figure Open in new tab Figure 2, figure supplement 1: Cx43 hemichannels with a truncated C-terminus (Cx43 1-256 ) retain their sensitivity to CO 2 and depolarisation. (A) Changes in GRAB ATP fluorescence induced by changing PCO 2 from 20 to 55 mmHg or KCl from 3 to 50 mM. (B) Summary graph showing the concentration of ATP release in response to 55 mmHg PCO 2 and 50 mM KCl, n=41 cells from 3 independent transfections. Download figure Open in new tab Figure 2, figure supplement 2: Effect of hypercapnic solutions on intracellular pH (pH i ) of parental HeLa cells measured by BCECF fluorescence. (A) Images showing BCECF fluorescence at 35, 55 and 70 mmHg, and then calibration to pH 7.0, 7.2 and 7.4 of the same cells after treatment with nigericin. (B) Quantification of the changes in fluorescence. C) Summary plot of the change in pH i induced by 55 and 70 mmHg PCO 2 . 3 independent seeds of cells, n=30. Gap Junction Channels (GJCs) of Cx26 are closed by CO 2 also acting via the carbamylation motif. We therefore tested whether Cx43 GJCs might also be CO 2 sensitive by using a dye transfer assay ( Nijjar et al ., 2021 ). Cx43 GJCs allowed equally fast permeation of dye at all levels of PCO 2 and therefore, unlike the hemichannel, were insensitive to CO 2 ( Fig 2, figure supplement 3 ). Download figure Open in new tab Figure 2, figure supplement 3: Cx43 gap junction channels are insensitive to changes in PCO 2 . (A) DIC image with mCherry fluorescence superimposed. Gap junctions are evident as red stripes between cells (yellow arrows). The fluorescence images show the patch pipette filled with NBDG its loading into the recorded cell and the neighbouring cells coupled via gap junctions. The numbers in each bottom left corner are the time of the recording from whole cell breakthrough in minutes. Scale bar 20 µm. (B) Summary graphs showing the time for the acceptor cell to reach 10% of the fluorescence of the donor cell at three different levels of PCO 2 (n=6 for each condition). Identifying the residues required for CO 2 -sensitivity of Cx43 Having established the dose-response characteristics of Cx43 to CO 2 , we next examined the potential underlying mechanisms for CO 2 -dependent gating via targeted mutagenesis of possible key residues, starting with the equivalents of those known to be involved in the CO 2 -sensitivity of Cx26 ( Fig 1 ). Mutation of K144 to glutamine (K144Q), an amino acid that cannot be carbamylated, partially reduced the CO 2 -sensitivity of the channel: the 70 mmHg stimulus resulted in significantly less increase in dye loading than the zero Ca 2+ positive control ( Fig 3A , MW test: p = 0.048, n=5). However, mutation of Lys105 to a non-charged residue (K105Q) resulted in hemichannels that gave a similar increase in dye loading to the zero Ca 2+ positive control ( Fig 3B , MW test: p = 0.111, n=5). In Cx26, mutation of the residues involved in carbamylation (K125 and R104) results in complete loss of CO 2 sensitivity ( Meigh et al ., 2013 ; Nijjar et al ., 2021 ). The lack of such a complete effect on the CO 2 sensitivity of Cx43 implies that the mechanism is more complex and may involve further residues. Interestingly, mutation of Lys144 to Arg (K144R) completely abolished the CO 2 sensitivity of Cx43 hemichannels as measured by dye loading ( Fig 3, figure supplement 1 ). The difference between the effect of mutating Lys144 to Arg and Gln on CO 2 sensitivity may arise because the substitution to Arg introduces a positive charge. Since K144 appears to be carbamylated, its primary amine must remain unprotonated so that the unpaired electrons of the nitrogen can mount a nucleophilic attack on the carbon of CO 2 to form the carbamate bond. Thus, the K144Q mutation may preserve this charge and be a better substitute that removes the capacity for carbamylation while preserving the overall charge within this region of the protein. Consequently, we have predominantly used the Lys to Gln substitution in this study. Download figure Open in new tab Figure 3, figure supplement 1: Cx43 K144R hemichannels are insensitive to changes in PCO 2 . (A) Cumulative probability plot from 5 independent transfections showing dye loading in response to 20 nd 70 mmHg and the zero Ca 2+ stimulus. (B) Summary graph showing the change in median fluorescence intensity caused by 70 mmHg and zero Ca 2+ in Cx43 WT (data recalculated from Fig 2 ) and Cx43 K144R . Download figure Open in new tab Figure 3: The effect of single Lys mutations on the CO 2 sensitivity of Cx43. HeLa cells transfected with the mutant variant of Cx43 were subjected to the dye-loading protocol. Images of dye loading in response to CO 2 and the 0 Ca 2+ positive control (insets), together with the cumulative probability plots are shown for K105Q ( A ), K109Q ( B ), K144Q ( C ), K234Q ( D ). (E) AlphaFold3 prediction of Cx43 with hypothesised carbamylation bridge residues highlighted – K234 in orange and K109 in red. (F) Summary box plots for dye-loading showing the change in median pixel intensity from 20 mmHg PCO 2 for each condition (70 mmHg PCO 2 – green and 0 Ca 2+ blue, n=5) for WT (recalculated data from Fig 2A for comparison) and each mutation. Scale bars are 20 µm. We further inspected the AF3 structure prediction and identified two further lysine residues of interest ( Fig 3E ). K109 in Cx43 aligns with K108 of Cx26, a residue known to be carbamylated and required for Cx26 gap junction closure ( Nijjar et al ., 2025 ). AF3 also suggests that K109 could potentially interact with K234 following any carbamylation ( Fig 3E ). We therefore mutated both residues to Gln to test their possible involvement in CO 2 sensitivity. The dye-loading assay showed that K109Q remained CO 2 sensitive ( Fig 3C MW test vs zero Ca 2+ control, p = 0.274, n=5). However, the mutation K234Q displayed impaired CO 2 sensitivity ( Fig 3D , MW test vs zero Ca 2+ control: p = 0.016, n=5). As the dye loading assay suggested that single mutations were on the whole rather ineffective in abolishing CO 2 sensitivity, we assayed ATP release via GRAB ATP to gain further supporting evidence. We found that CO 2 -evoked ATP release remained dose dependent and very similar to the WT Cx43 in the K105Q (n=23) and K144Q (n=12) mutants ( Fig 4A, C , Figure 4, figure supplement 1 ). Cx43 hemichannels with the K109Q mutation appeared to have their sensitivity to CO 2 shifted to lower values of PCO 2 (n=20), appearing to give maximal ATP release at 35 mmHg ( Fig 4B,E , Fig 4 figure supplement 1 ). K234Q Cx43 hemichannels had greatly reduced sensitivity to CO 2 (n=15, Fig 4D, F ). To check that K234Q did not have some non-specific effect on hemichannel function, we used high K + induced depolarisation as a way of opening the hemichannel independently of changes in PCO 2 as a positive control. This stimulus resulted in robust ATP release. Download figure Open in new tab Figure 4, figure supplement 1: The effect of single Lys mutations on the CO 2 dose-response properties of Cx43. Comparison of Cx43 WT (data replotted from Fig 2C ), Cx43 K105Q , Cx43 K109Q (data replotted from Fig 4E ) and Cx43 K144Q (data replotted from Fig 4F ). Data is plotted as medians with lower and upper quartiles. For the WT, K105Q and K144Q the fitted curve is drawn according to a modified Hill equation: [ATP]=Max ATP .[(PCO 2 /K) H /(1+(PCO 2 /K) H )].[1−(PCO 2 /K i ) Hi /(1+(PCO 2 /K i ) Hi )] Where K and H are the affinity and Hill coefficient of the channel for opening by CO 2 , K i and H i are the affinity and Hill coefficient for inhibition of the channel by CO 2 , and Max ATP is the asymptotically maximum release of ATP to CO 2 . For K109Q which did not exhibit CO 2 -dependent inhibition at high levels of PCO 2 , the Hill equation was used. The parameters for the curves are: Download figure Open in new tab Figure 4: The effect of single Lys mutations on CO 2 -dependent ATP release from Cx43. The genetically-encoded ATP sensor GRAB ATP was co-transfected alongside Cx43 mutant variations, K105Q ( A ) n=23, K109Q ( B ) n=20, K144Q ( C ) n=12, K234Q ( D ) n=15. Representative trace measurements were selected; each line represents a different cell; the fluorescence was normalised by dividing all values by the baseline median pixel intensity before plotting. Box plot on the right ( E ) and ( F ) represent the µM ATP released as determined through normalisation to the 3 µM ATP application. CO 2 sensitivity of Cx43 involves multiple Lys residues As single Lys mutations mostly had partial effects on the CO 2 sensitivity of Cx43 hemichannels, we next mutated pairs of Lys residues. Using the dye loading assay we found that all combinations of paired mutations abolished CO 2 sensitivity relative to the zero Ca 2+ control ( Fig 5 ): K109Q, K144Q (MW test p = 0.008, n=5); K105Q, K144Q (MW test p = 0.004, n=5); K105Q, K109Q (MW test p = 0.004, n=5); K105Q, K234Q (MW test p = 0.004, n=5); and K144Q, K234Q (MW test p = 0.004, n=5). Using the GRAB ATP assay we confirmed that all of these double mutations also abolished CO 2 -dependent ATP release (MW test for all mutations, CO 2 vs high K + positive control p < 10 −6 , Fig 6 ). Download figure Open in new tab Figure 5: Paired Lys mutations are required to abolish the CO 2 sensitivity of Cx43. Representative images for all combinations tried are shown – K109Q K144Q ( A ), K105Q K144Q ( B ), K105Q K109Q ( C ), K105Q K234Q ( D ), K144Q K234Q ( E ). Insets represent the 0 Ca 2+ positive control. For each construct, the pixel intensity in expressing cells was measured from 5 individual transfections, with at least 40 cells per condition. Cumulative probability plots display all measured data points. ( F) The box plots shows the change in median pixel intensity from 20 mmHg PCO 2 for each transfection for 70 mmHg PCO 2 (green) and 0 Ca 2+ (blue). Download figure Open in new tab Figure 6: Paired Lys mutations abolish CO 2 dependent ATP release via Cx43. HeLa cells co-expressing Cx43 and GRAB ATP were subjected to changing PCO 2 -levels and fluorescence was recorded. Representative traces ( A-E ) for each of the Cx43 mutations – K105Q K144Q ( A ) n=23, K109Q K144Q ( B ) n=19, K105Q K234Q ( C ) n=17, K109Q K234Q ( D ) n=16, K144Q K234Q ( E ) n=13. The traces indicate normalised fluorescence changes (ΔF/F 0 ). 3 µM ATP was applied at the end of each experiment to confirm sensor functionality. Furthermore, as a positive control, 50 mM KCl was applied to depolarise the cells and confirm channel function. ( F-G ) Box plots summarising the total ATP release in µM for each double mutant in 55 mmHg PCO 2 and 50 mM KCl. Data points represent individual measured cells. The effect of introducing negatively charged residues into the carbamylation motif We next sought to mimic carbamylation by mutating Lys to Glu. This introduces into the motif the negative charge that would occur upon carbamylation of Lys. In the dye loading assay the mutation K144E resulted in a complete loss of CO 2 sensitivity while dye loading to zero Ca 2+ was retained (MW test, 70 mmHg vs zero Ca 2+ : p = 0.008, n=5, Fig 7B ). The mutation K105E seemed to disrupt both the CO 2 -dependent and the zero Ca 2+ evoked dye loading ( Fig 7A ). K234E displayed impaired CO 2 -dependent dye loading compared to that evoked by zero Ca 2+ (MW test, 70 mmHg vs zero Ca 2+ : p = 0.048, n=5, Fig 7C ). Download figure Open in new tab Figure 7: Introduction of negative charge into the carbamylation motif via Lys to Glu mutations has mixed effects on CO 2 sensitivity of Cx43 hemichannels. Expressing cells pixel intensity for each construct was measured from 5 individual transfections, with at least 40 cells per condition. ( A-C ) Representative cell images for 20 (left), 70 (right) with insets displaying the 0 Ca 2+ control for each mutant K105E ( A ), K144E ( B ), K234E ( C ). Scale bar represents 20 µm. Cumulative probability graphs of pixel intensities are shown on the right for each mutant with three conditions 20 mmHg PCO 2 (blue line), 70 mmHg PCO 2 (orange) and 0 Ca 2+ (grey line). The box plot shows change in median pixel intensity from 20 mmHg PCO 2 for each transfection for 70 mmHg PCO 2 (green boxes) and 0 Ca 2+ (blue boxes). Using the GRAB ATP assay, we found that each of these single Lys to Glu mutations disrupted CO 2 -dependent ATP release, but did not prevent depolarisation evoked ATP release ( Fig 8 ). This disruptive effect on CO 2 dependence might arise because introduction of the negative charge changes the local environment and makes it harder to carbamylate the other Lys residues. Download figure Open in new tab Figure 8: Lys to Glu mutations abolish CO 2 -dependent ATP Release. GRAB ATP fluorescence traces of ATP release from cells expressing single mutant connexins: K105E ( A ) n=18, K144E ( B ) n=17, K234E ( C ) n=13, in response to 55 mmHg PCO 2 (red bars), 50 mM KCl depolarisation control (orange bars) and a 3 µM ATP control application at the end of all experiments to confirm sensor functionality. Traces show changes in normalised fluorescence over time (ΔF/F), indicating ATP release. On the right, the box plots display the quantified ATP release in µM for each mutant under 55 mmHg PCO 2 and 50 mM KCl. Each data point represents a measurement from an individual cell. As a change in PCO 2 would likely carbamylate more than one Lys residue simultaneously, we made the double mutation K105E and K109E. With the dye loading assay we found that even at a PCO 2 of 20 mmHg, the cells loaded with dye suggesting that the mutated hemichannels were constitutively open ( Fig 9A, D ). Despite this cells that expressed this double mutant had normal morphology ( Fig 9 , movie supplement 1) and their cultures did not exhibit noticeably higher levels of cell death. Presumably the HeLa cells were able to adapt to the presence of these open channels. Download figure Open in new tab Figure 9: The Cx43 double mutant, K105E K109E, is constitutively open. ( A ) Representative fluorescence images of cells expressing the Cx43 K105E K109E mutant under low (20 mmHg PCO 2 , left) and high CO 2 (70 mmHg PCO 2 , right), inset shows the 0 Ca 2+ control. Scale bar is 20 µm. Cumulative probability plot of pixel intensity for each condition is shown on the right, overall indicating higher baseline fluorescence and perpetually open hemichannels. ( B ) Dye-loading with 20 mmHg PCO 2 (left) and 20mmHg PCO 2 with 100 µm LaCl 3 . Cumulative probability plot for pixel intensity under these conditions is shown on the right. ( C ) Fluorescence traces of ATP release from cells co-expressing GRAB ATP and Cx43 K105E K109E (n=16) under 20 mmHg PCO 2 and 20 mmHg PCO 2 with 100 µM LaCl 3 (black bar). Fluorescence is normalised to baseline. ( D ) Fluorescence traces of ATP release from cells co-expressing GRAB ATP and Cx43 WT under 20 mmHg PCO 2 and 20 mmHg PCO 2 with 100 µM LaCl 3 (black bar). ( E ) Box plots summarizing median pixel intensity under the different conditions, showing a significant reduction in intensity in the presence of LaCl 3 (p = 0.03). ( F ) Box plot shows normalised fluorescence changes values for the difference between – 20 mmHg PCO 2 (representing baseline normalisation) and the application of LaCl 3 for both the K105E K109E and WT Cx43 constructs. Figure 9 , movie supplement 1: GRAB ATP fluorescence recorded from HeLa cells expressing Cx43 K105E K109E during application of 100 µM LaCl 3 in 20 mmHg PCO 2 aCSF. Increasing PCO 2 to 70 mmHg gave no further dye loading. To test whether the channels were open even at a PCO 2 of 20 mmHg, we used the general hemichannel blocker La 3+ (100 µM) to show that this reduced the dye loading at 20 mmHg ( Fig 9B, D , MW test: p = 0.016, n=5). We used the GRAB ATP assay to demonstrate that at a PCO 2 of 20 mmHg, the K105E, K109E mutation caused continual release of ATP that could be blocked by La 3+ ( Fig 9C, D , MW test 20 mmHg vs 20 mmHg + La 3+ , p=7.7×10 −7 , n=16; Fig 9 , movie supplement 1). This continual release of ATP at a PCO 2 of 20 mmHg was not evident from cells expressing WT Cx43, as application of 100 µM La 3+ had no effect on the GRAB ATP fluorescence ( Fig 9C, D ). Physiological role for CO 2 -dependent ATP release via Cx43 from astrocytes Cx43 hemichannels are CO 2 sensitive and are likely to be partially open at typical levels of PCO 2 in mammalian tissue. They could thus be able to release ATP under physiological resting conditions (35 mmHg PCO 2 , Fig 2 ). Considering the ubiquitous distribution of Cx43 in astrocytes ( de Ceglia et al ., 2023 ) and the likely resting PCO 2 in the brain ( Hogg et al ., 1984 ), we postulated that, barring external modulatory influences, Cx43 hemichannels could continually release low levels of ATP in the hippocampus. Indeed, there exists compelling evidence for astrocytic Cx43 hemichannels being partially open under resting physiological conditions in vitro in the hippocampus ( Chever et al ., 2014 ). In this study when Cx43 hemichannels were blocked by the mimetic peptide, Gap26, there was a decrease synaptic strength of about 30% that was occluded by targetted knock out of astrocytic Cx43 expression ( Chever et al ., 2014 ). As many investigators state that Cx43 hemichannels are shut under physiological conditions, we tested whether the results of Chever et al (2014) could be explained by the CO 2 sensitivity of Cx43. We predicted that if PCO 2 were lowered sufficiently to largely close Cx43, when PCO 2 was returned to the physiological norm, Cx43 should re-open and result in an increase of synaptic strength, presumably downstream of Cx43-dependent ATP release ( Chever et al ., 2014 ). Because ATP can be rapidly broken down to adenosine in the extracellular space ( Frenguelli et al ., 2007 ; Wall & Dale, 2013 ), which could then act at A1 receptors to inhibit transmission and give confounding effects, we used 8-CPT (8-cyclopentyltheophylline) to selectively block A1 receptors. We preincubated hippocampal slices in 20 mmHg PCO 2 aCSF for 30 mins prior to recording field EPSPs (fEPSPs) in area CA1 evoked by stimulation of the Schaffer colaterals. At constant extracellular pH, in the presence of 8-CPT, elevation of PCO 2 from 20 to 35 mmHg resulted in a 26% increase in the magnitude of the fEPSP (n=6, Fig 10A ). To test whether this effect was mediated via CO 2 -dependent opening of Cx43, we used 100 μM Gap26 to see whether this blocked the increase in fEPSP induced by 35 mmHg PCO 2 . Once again the fEPSP increased by about 30% on transfer to 35 mmHg aCSF ( Fig 10B ), and application of Gap26 reduced the fEPSP to below its baseline value ( Fig 10B, D , Friedmann 2-way ANOVA: p=0.006, n=6). This was an effect specific to the Gap26 peptide as 100 μM of the scrambled peptide did not have significant effects on the amplitude of the fEPSP ( Fig 10C, D , Friedmann 2-way ANOVA: p=0.55, n=5). Download figure Open in new tab Figure 10: PCO 2 -dependent modulation of amplitude of fEPSPs in hippocampus is mediated via Cx43. ( A-C ) Time-course plots showing the average amplitude of the normalised fEPSP (± SEM) amplitude in response to different conditions. Inserts display representative fEPSPs. ( A ) Control condition showing an increase in fEPSP amplitude in response to a modest change to PCO 2 (20 to 35 mmHg), red bar represent the application of 35 mmHg PCO 2 , baseline conditions – 20 mmHg PCO 2 . ( B ) EPSP responses in the presence of Gap26 (black bar) and subsequent wash. ( C ) EPSP responses with scrambled Gap26 peptide applied (black bar). ( D ) Box plots representing the normalised fEPSP size difference (baseline was subtracted) with colour coded conditions: orange – control 35 mmHg PCO 2 , green – Gap26 with pre (35G), after (35S Wash). Pathological mutations of Cx43 cause loss of CO 2 sensitivity A prominent condition associated with mutations of Cx43 is occulodental digital dysplasia (ODDD) ( Laird, 2014 ). This syndrome involves a range of conditions such as soft tissue fusion of the digits, abnormal craniofacial bone development, small eyes and loss of tooth enamel. Later in age conditions such as glaucoma, skin disease and neuropathies may become evident. As pathological mutations of other CO 2 sensitive connexins alter their CO 2 sensitivity ( Meigh et al ., 2014 ; de Wolf et al ., 2016 ; Cook et al ., 2019 ; Butler & Dale, 2023 ), we studied two pathological mutations of Cx43. The mutation L90V causes ODDD ( Shibayama et al ., 2005 ; Lai et al ., 2006 ) and A44V causes the skin condition, erythrokeratodermia variabilis et progressiva (EKVP) ( Cocozzelli & White, 2019 ; Srinivas et al ., 2019 ). We found that the L90V mutation removed CO 2 sensitivity from Cx43 by both the dye loading assay and the GRAB ATP assay ( Fig 11 ). The mutation A44V blocked CO 2 dependent ATP release ( Fig 11 ). Download figure Open in new tab Figure 11: Pathological mutations of Cx43 remove its sensitivity to CO 2 . A , B) L90V prevents CO 2 -dependent dye loading. The dye loading assay shows no change in fluorescence between 20 and 70 mmHg, yet functional channels are expressed as shown by the zero Ca 2+ positive control (inset). Box plot shows the change in median pixel intensity from 20 mmHg PCO 2 to 70 mmHg PCO 2 (green boxes) and 0 Ca 2+ (blue boxes) for each transfection. C-E) GRAB ATP recordings shown that L90V and A44V also abolish CO 2 -dependent ATP release (baseline 20 mmHg PCO 2 , red bar 70 mmHg PCO 2 ). Discussion Hemichannels of Cx43 are CO 2 sensitive In contrast to Cx26 expression, which is restricted to specific, small regions of the brain ( Nagy et al ., 2001 ; Rash et al ., 2001 ; Nagy et al ., 2011 ), Cx43 serves as the principal astrocytic connexin, exhibiting widespread presence throughout the brain ( Schulz et al ., 2015 ; Yin et al ., 2018 ; de Ceglia et al ., 2023 ). This widespread distribution, combined with our results, suggests that Cx43 gives the capacity for CO 2 sensitivity across the entire brain. Our data from three independent assays (dye loading, measurement of ATP release and whole cell recordings) show that hemichannels of Cx43 open to modest changes in PCO 2 . However, Cx43 gap junction channels are insensitive to these same changes in PCO 2 . At physiological levels of PCO 2 Cx43 hemichannels are partially open and able to release ATP. This observation potentially resolves a puzzling inconsistency in the literature. Many biophysical recordings of Cx43 hemichannels, performed in simplified buffered salines without appreciable dissolved CO 2 or HCO 3- , document Cx43 hemichannels as being shut ( Contreras et al ., 2003 ; Schalper et al ., 2010 ). By contrast, under physiological conditions both in vitro and in vivo , which necessarily involve CO 2 -HCO 3- buffering, the evidence suggests that Cx43 hemichannels remain open to some extent ( Chever et al ., 2014 ; Guillebaud et al ., 2020 ; Turovsky et al ., 2020 ). Continual ATP release from astrocytes via Cx43 has been shown to give a tone of ATP that enhances the strength of synaptic transmission in the hippocampus by about 20% ( Chever et al ., 2014 ). Our data is highly complementary to these observations as we find an enhancement of fEPSP amplitude of about 30% when increasing PCO 2 from 20 to 35 mmHg. It is important to note that our experiments were performed under isohydric conditions and differ from a prior study that examined hypocapnia with simultaneous alkalinisation. This manipulation enhanced fEPSP strength through a reduction in extracellular adenosine and consequently less inhibition via A1 receptors ( Dulla et al ., 2005 ). While our data suggests that Cx43 hemichannels are sensitive to PCO 2 over the range 20-70 mmHg, there is an indication that there may be some CO 2 -dependent inhibition of hemichannel opening above 55 mmHg. Slightly less ATP release was consistently seen at a PCO 2 of 70 mmHg compared to 55 mmHg. Furthermore, we cannot be sure that Cx43 hemichannels are completely shut at a PCO 2 of 20 mmHg, as it is not possible to reduce the PCO 2 further without changing to HEPES buffered salines. That Gap26 reduced synaptic transmission below the baseline amplitude recorded in slices bathed in 20 mmHg aCSF suggests that even under this condition there may still be a low level of Cx43 hemichannel gating at least in neural tissue which will continually generate CO 2 via oxidative phosphorylation. Cx43 gap junction channels and hemichannels are closed by intracellular acidification. This involves interaction between the C-terminus and the cytoplasmic loop ( Duffy et al ., 2002 ; Hirst-Jensen et al ., 2007 ). Similar interactions between the C-terminus and cytoplasmic loop are involved in regulating hemichannel activity stimulated by increases in intracellular Ca 2+ ( Iyyathurai et al ., 2018 ). However, the CO 2 dependent opening of Cx43 is independent of these mechanisms as it persists when the C-terminus has been deleted. The residues involved in CO 2 sensitivity and possible mechanisms By exploiting the new structural data for connexins ( Myers et al ., 2018 ; Flores et al ., 2020 ; Lee et al ., 2020 ; Yue et al ., 2021 ; Brotherton et al ., 2022 ; Qi et al ., 2022 ; Lee et al ., 2023a ; Lee et al ., 2023b ; Qi et al ., 2023 ), the predictive power of AF3 and our understanding of carbamylation and CO 2 -dependent gating of Cx26 hemichannels and GJCs ( Nijjar et al ., 2021 ; Brotherton et al ., 2024 ), we identified a series of residues that could plausibly be involved in mediating the CO 2 sensitivity of Cx43: K144, the equivalent of K125 in Cx26; K105, the analogue of R104 in Cx26; K109, the equivalent of K108 in Cx26; and K234, an equivalent of R216 in Cx26. Both K125 and K108 in Cx26 are known to be carbamylated ( Nijjar et al ., 2025 ). Carbamylation of K125 is essential for hemichannel opening to CO 2 , whereas carbamylation of both K125 and K108 is required for GJC closure to CO 2 ( Nijjar et al ., 2025 ). R216 in Cx26 has been tentatively identified as a possible interacting partner for K108 but this could be indirect ( Nijjar et al ., 2025 ). Crucially the spatial arrangements of K144, K105, K109 and K234 in AF3 predictions indicate that these residues are sufficiently close to interact across the subunit boundary. In Cx26, K125 is proposed to interact with R104 following carbamylation, and interestingly mutation of either K125 or R104 is sufficient to abolish CO 2 dependent hemichannel opening and GJC closure. Mutation of K108 is also sufficient by itself to abolish GJC closure to CO 2 ( Nijjar et al ., 2025 ). By contrast single mutations to Gln of any of the 4 Lys residues identified in Cx43 as the equivalents to those involved in CO 2 -dependent gating of Cx26 were unable to completely abolish CO 2 sensitivity. Similarly the mutations K125E and R104E in Cx26 create consitutively open hemichannels. However single Lys to Glu mutations in Cx43 did not have this effect. Our data show that in Cx43 at least 2 of the 4 identified residues must be mutated to completely abolish CO 2 sensitivity or to give constitutively open hemichannels. For example mutation of any two Lys residues to Gln, completely abolishes CO 2 dependent ATP release and dye loading. Conversely the double mutation, K105E and K109E, is required for constitutively open hemichannels. This suggests that in Cx43 there is some redundancy in the effect of CO 2 . One possible interpretation is that two carbamate bridges are formed: one being between K144 and K105 (the direct equivalent of K125-R104 in Cx26); and the second being between K109 and K234. There is certainly some data that support this: the double mutations K109Q-K144Q, K105Q-K234Q and K144Q-K234Q being completely insensitive to CO 2 . These double mutations would remove both of the proposed bridges. However our observation that the mutations K105Q-K144Q, K109Q-K234Q also completely remove CO 2 sensitivity is inconsistent with a simple two bridge hypothesis as these double mutations would only affect one of the bridges and should therefore leave the other intact to give some degree of CO 2 sensitivity. Instead, we favour a hypothesis in which any of these four residues could be carbamylated but that they all have potential to interact. Depending on which residues become carbamylated a bridge could form between K105 and K234 or between K109 and K144, and possibly even K144 and K234. There is some support for this idea from AF3 as K105 and K109 are only one turn distant in an alpha helix and both could therefore interact with either K144 or K234 following carbamylation. The residues in Cx43 that are involved in CO 2 sensitivity are equivalent to those identified in Cx26, where they participate in both hemichannel and gap junction channel gating to CO 2 . However in Cx43 these residues are only involved in mediating the sensitivity of the hemichannel to CO 2 . In this respect it is interesting that mutations of the different Lys residues alter the dose-sensitivity of Cx43 to CO 2 in different ways ( Fig 4, figure supplement 1 ). New cryo-EM studies of Cx43 hemichannels at vitrified different levels of PCO 2 would shed more light on the mechanism of opening, Physiological implications of the CO 2 sensitivity of Cx43 Cx43 is the most widely expressed connexin in the human body, being present in every organ system ( Lucaciu et al ., 2023 ). In metabolically highly active organs such as liver, kidney and brain, our data suggest that the physiological PCO 2 will be sufficient to substantially open Cx43 hemichannels ( Hogg et al ., 1984 ). In the context of the brain, Cx43 is the main astrocytic connexin and is expressed in all subtypes of astrocytes ( de Ceglia et al ., 2023 ). This would imply that CO 2 -sensitivity mediated via Cx43 will extend to potentially all brain regions. As astrocytes are non-excitable, they are unlikely to become sufficiently depolarised for Cx43 hemichannels to open via voltage dependent gating. We suggest therefore, in non-excitable cells such as astrocytes, that variations in PCO 2 may be the most important physiological regulator of Cx43 hemichannel gating. For astrocytes, CO 2 -dependent opening of Cx43 hemichannels is likely to result in the release of a mix of small, neurochemically significant molecules such as ATP, glutamate, D-serine and lactate, depending on their intracellular concentration. Cx43 is also highly expressed in the heart, if a proportion of the population were to be in hemichannel form as opposed to GJCs ( De Smet et al ., 2021 ), the newly-discovered CO 2 sensitivity of hemichannels may have profound implications for heart function given that cardiomyocytes are highly active, and will thus generate high levels of CO 2 through oxidative phosphorylation. There is a long-established link between CO 2 and cardiac function with effects such as increased coronary blood flow in response to CO 2 as well as changes in myocardial contractile function ( Crystal, 2015 ). CO 2 can even cause arrythmias ( Zhang et al ., 2019 ). The effects of CO 2 may be mediated through changes in intracellular pH, which is well known to alter the function of cardiac myocytes ( Spitzer et al ., 2002 ; Vaughan-Jones et al ., 2009 ; Orlowski et al ., 2025 ). However, given that Cx43 is directly sensitive to CO 2 , an investigation as to whether CO 2 also has direct effects on cardiac function that are independent of consequent changes in intracellular pH seems warranted. NF-kappa B and innate immunity exhibit sensitivity to CO 2 ( Cummins et al ., 2010 ; Keogh et al ., 2017 ). Macrophages are CO 2 -sensitive, although at least some of this is indirect and via pH changes mediated by carbonic anhydrase activity ( Strowitzki et al ., 2022 ). However, a direct effect of CO 2 on components of the immune system cannot be excluded. In cell culture, when carbonic anhydrase activity is blocked in THP-1 monocytes, the effects of CO 2 are significantly diminished but not abolished, suggesting the existence of a pH-independent pathway ( Strowitzki et al ., 2022 ). As Cx43 is expressed in monocytes it is a plausible candidate to mediate direct CO 2 sensitivity in these cells. Furthermore, Cx43 function in macrophages is deemed critical for various physiological and pathophysiological processes ( Rodjakovic et al ., 2021 ). The role of CO 2 on such pathways remains to be explored. It is interesting that pathological mutations of Cx43 abrogate CO 2 sensitivity. This follows a pattern seen for Cx26, where a range of mutations linked to syndromic and non-syndromic hearing loss ( Meigh et al ., 2014 ; de Wolf et al ., 2016 ; Cook et al ., 2019 ), and for Cx32, where several mutations that cause X-linked Charcot Marie Tooth Disease ( Butler & Dale, 2023 ) alter or completely block CO 2 sensitivity. Further studies are needed to see whether loss of CO 2 sensitivity from Cx43 does indeed contribute to pathology. Evolution of CO 2 sensitivity in connexins Our previous findings showed that a common carbamylation motif is prevalent across the beta-connexin clade and confers CO 2 -sensitivity on the connexins that possess it ( Dospinescu et al ., 2019 ). This motif for example is present in shark Cx32 (which is CO 2 sensitive). Humans and sharks last shared a common ancestor about 400-450 MYA. Our discovery that a very similar carbamylation motif is present in Cx43 and confers CO 2 sensitivity onto their hemichannels. Examining the sequences of other human and non-human alpha connexins reveals that the carbamylation motif with appropriately oriented residues is present in human Cx50, Cx59 and Cx62 as well as Cx40, Cx46 and Cx57 of non-human species ( Fig 12 , Fig 12 Table Supplement). We have shown Cx50 hemichannels are CO 2 sensitive and this also depends on the carbamylation motif ( Lovatt et al ., 2025 ). These findings suggest that the carbamylation motif and CO 2 sensitivity must have been present in the ancestral connexin gene that predated the establishment of the alpha and beta connexin clades. Further work is needed to understand the evolution of the carbamylation motif and CO 2 sensitivity, but our observations indicate that it is an ancient feature within the molecular phylogeny of the connexins, rather than being a more recently derived feature restricted to the beta connexins. Download figure Open in new tab Figure 12: Occurrence of the carbamylation motif in the alpha connexin clade. Names in red indicate the presence of the motif. The sequences were aligned in TCoffee to check for the presence of the motif. The molecular phylogenetic tree was constructed from the aligned traces by the average distance using Blosum62 in JalView ( Waterhouse et al ., 2009 ). Accession numbers for the protein sequences in the tree are given in the accompanying table supplement. Methods Recording Solutions Hypocapnic (20 mmHg PCO 2 ): 140 mM NaCl, 10 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 3mM KCl, 1 mM MgSO 4 , 10 mM D-glucose and 2 mM CaCl 2 . This was bubbled with a mix of 95%O 2 /5%CO 2 and balanced with sufficient pure O 2 from an oxygen concentrator to give a final pH of ~7.3. Control (35 mmHg PCO 2 ): 124 mM NaCl, 26 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 3 mM KCl, 10 mM D-glucose, 1 mM MgSO 4 , 2 mM CaCl 2 . This was bubbled with 95%O 2 /5% CO 2 and had a final pH of ~7.3. Hypercapnic (55 mmHg PCO 2 ): 100 mM NaCl, 50 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 3 mM KCl, 10 mM D-glucose, 1 mM MgSO 4 , 2 mM CaCl 2 . This was bubbled with sufficient CO 2 (~9%, balance O 2 ) to give a final pH of ~7.3. Hypercapnic (70 mmHg PCO 2 ): 73 mM NaCl, 80 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 3 mM KCl, 10 mM D-glucose, 1 mM MgSO 4 , 2 mM CaCl 2 . This was bubbled with sufficient CO 2 (approximately 12%, balance O 2 ) to give a final pH of ~7.3. Zero Ca 2+ : 140 mM NaCl, 10 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 3mM KCl, 1 mM MgSO 4 . On the day of recording, 10 mM D-glucose, 1 mM EGTA (Ethyleneglycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid) and 2 mM MgCl 2 was added, and the solution bubbled with 95%O 2 /5%CO 2 and balanced with sufficient pure O 2 from an oxygen concentrator to give a final pH of ~7.3. Cloning and mutagenesis Plasmids containing mutated versions of Cx43 were generated using the Gibson Assembly method ( Gibson et al ., 2009 ). Overlapping fragments both containing the desired mutation were PCR amplified with primers. Double mutations were cloned using successive Gibson assemblies. PCR fragments were amplified using Q5® High-Fidelity DNA Polymerase (New England Biolabs) (normal PCR or site-directed mutagenesis). The overlap region generated through PCR for Gibson Assembly was 20 base pairs, and the pCAG vector was used as the backbone. All Cx43 constructs were inserted upstream of an mCherry tag, linked via a 12 AA linker (GVPRARDPPVAT). All PCR primers were purchased from Integrated DNA Technologies (IDT). For double mutations, an additional round of cloning was performed using a single mutant as the template, to introduce the additional mutation. For the truncation (Cx43 1-256 ), we created a PCR product of the truncated version of Cx43 and fused it to the same AA linker and mCherry as previously stated, to allow visualisation of cellular localisation. All constructs were confirmed by DNA sequencing (Eurofins GATC sequencing or Plasmidasaurus). Cell culture HeLa DH cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum, 50 μg/mL penicillin/streptomycin. HeLa DH cells were used for patch clamp studies on Cx43 and for the dye loading of all Cx43 variants. For dye loading experiments, cells were seeded onto coverslips at a density of 7.5 × 10 4 cells per well and transiently transfected with the Cx43 constructs following the PEI Transfection Reagent protocol. Dye-loading Cell expressing the construct (48-72h) were given an initial 5-minute wash with baseline solution (artificial cerebrospinal fluid – aCSF, 20 mmHg PCO 2 ). Following this, the cells were exposed to either control solution, hypercapnic (70 mmHg PCO 2 ), or a zero Ca 2+ positive control (20 mmHg PCO 2 ) solution, all of which contained 200 µM 5(6)-carboxyfluorescein (CBF) for 10 min. Next, cells were put into 20 mmHg PCO 2 solution with 200 µM CBF for 5 minutes to close any open channels and prevent dye loss in the next step. Then the coverslip was washed with control solution (without CBF) for another 30 min to remove extracellular dye. For each condition a different coverslip with HeLa cells was used. Cx43 was tagged with mCherry on the C-terminus using a short linker. Expression was verified using mCherry fluorescence. The experiments were replicated using independent transfections 5 times. Following dye loading, HeLa cells were imaged by epifluorescence (Scientifica Slice Scope, Cairn Research OptoLED illumination, 60x water Olympus immersion objective, NA 1.0, Hamamatsu ImagEM EM-CCD camera, Metafluor software). CBFwas excited by a 470 nm LED, with emission captured between 504-543 nm. Cx43 constructs had a C-terminal mCherry tag, which was excited by a 535 nm LED and emission captured between 570-640 nm. Following acquisition analysis was performed by a person blind to the experimental condition. ImageJ (Wayne Rasband, National Institutes of Health, USA) was used to measure the extent of dye loading by drawing a region of interest (ROI) around each cell, and subsequently, the mean pixel intensity of the ROI was determined. The mean pixel intensity of a representative background ROI for each image was subtracted from each cell measurement from the same image. At least 40 cells were measured for each condition per experiment, and five repetitions of independently transfected HeLa cells were completed. The mean pixel intensities were plotted as cumulative probability distributions, and these graphs show every data point measured. To assess the effect of the 70 mmHg and zero Ca 2+ solutions on dye loading, the difference in the median pixel intensities between the 70mmHg and 20 mmHg conditions, and the zero Ca 2+ and 20 mmHg conditions was calculated for each transfection ( Figs 3 , 5 , 7 and 11 ). GRAB ATP recordings Cells were transiently transfected with the pCAG-Cx43-mCherry construct and pDisplay-GRAB_ATP1.0-IRES-mCherry-CAAX (Addgene plasmid # 167582; RRID:Addgene_167582) ( Wu et al ., 2022 ) 48 hours prior to imaging. Cells were perfused with control aCSF until a stable baseline was reached, before perfusion with either hypercapnic or high K + aCSF (positive control). Once a stable baseline was reached after solution change, cells were again perfused with control aCSF and when a stable baseline reached, recordings were calibrated by direct application of 3 μM of the corresponding analyte. All cells were imaged by epifluorescence as above. The cpGFP in GRAB ATP was excited by a 470 nm LED, with emission captured between 504-543 nm. As the Cx43 constructs were mCherry tagged, we selected only cells that expressed both cpGFP and mCherry for recording. GRAB ATP fluorescence images were acquired every 4 seconds. For each condition, at least 3 independent transfections were performed with at least 2 coverslips per transfection. Analysis of all experiments was carried out in ImageJ. Images were opened as a stack and stabilised. ROIs were drawn around cells co-expressing both sensor and connexin. Median pixel intensity was plotted as normalised fluorescence change (ΔF/F 0 ) over time to give traces of fluorescence change. Amount of ATP release was quantified as concentration by normalising to the ΔF/F 0 caused by application of 3 μM ATP. Intracellular pH measurement BCECF-AM dissolved in DMSO and Pluronic f127 and diluted in 35 mmHg aCSF for a final concentration of 2 μM. Coverslips seeded with parental HeLa DH cells were incubated in BCECF for 20 minutes, before being washed in 35 mmHg aCSF for 10 minutes. Cells were perfused with control aCSF until a stable baseline was reached, before perfusion with either 55 mmHg or 70 mmHg aCSF. Once a stable baseline was reached after solution change, cells were again perfused with control aCSF. pH i was then calibrated following the method in ( James-Kracke, 1992 ). All cells were imaged by epifluorescence with the BCECF excited by a 470 nm LED and emission captured between 504-543 nm. Measurements were obtained from 3 independent transfections were performed with at least 2 coverslips per transfection. Patch clamp recordings Coverslips containing non-confluent HeLa cells were placed into a perfusion chamber at room temperature and superfused with control aCSF. An Axopatch 200B amplifier was used to make whole-cell recordings from single HeLa cells. The intracellular fluid in the patch pipettes contained: K-gluconate 60 mM, Cs-gluconate 50 mM, CsCl 10 mM, TEACl 10 mM, EGTA 10 mM, Na 2 ATP 3mM, MgCl 2 3 mM, CaCl 2 1 mM, HEPES 10 mM, sterile filtered, pH adjusted to 7.2 with KOH. An agarose salt bridge was used to avoid solution changes altering the potential of the Ag/AgCl reference electrode. All whole-cell recordings were performed at a holding potential of −50 mV. Whole-cell conductance was measured by repeated steps to −40 mV. To allow for any drift in whole-cell conductance unrelated to the CO 2 stimulus, the maximal conductance during the CO 2 test stimulus was compared to the average of the conductance before the stimulus and after the stimulus had been fully washed off. Each whole cell recording was considered to be an independent statistical replicate. Control (35 mmHg) aCSF was used as the baseline and switched to 20 mmHg, 55 mmHg or 70 mmHg aCSF to measure the conductance responses to differing levels of PCO 2 . To convert these responses to a PCO 2 dose response curve, assigned a value of zero to 20 mmHg and plotted all changes relative to this. Thus, the values at 35 mmHg were the absolute values of the changes evoked by going from 35 to 20 mmHg, and the absolute value of median change from 35 to 20 mmHg was added to the changes observed going from 35 to 55 and 35 to 70 mmHg. Imaging assay of gap junction transfer 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, NBDG, was included at 200 µM in the patch recording fluid, which contained: K-gluconate 130 mM; KCl 10 mM; EGTA 5 mM; CaCl 2 2 mM, HEPES 10 mM, pH was adjusted to 7.3 with KOH to give a resulting final osmolarity of 295 mOsm. Cells were imaged on a Cleverscope (MCI Neuroscience) with a Photometrics Prime camera under the control of Micromanager 1.4 software. LED illumination (Cairn Research) and an image splitter (Optosplit, Cairn Research) allowed simultaneous imaging of the mCherry-tagged Cx43 subunits and the diffusion of the NBDG into and between cells. Coupled cells for intercellular dye transfer experiments were initially selected based on tagged Cx43 protein expression and the presence of a gap junctional plaque ( Figure 4, figure supplement 1 ). Dye permeation between cells was measured at PCO 2 levels of 20, 55 and 70 mmHg. After establishing the whole cell mode of recording, images were collected every 10 seconds. The assay was performed as described by Nijjar et al ( Nijjar et al ., 2021 ). The start of recording was taken to be the first image following the establishment of a stable whole cell recording. For each gap junction recording (considered as an independent statistical replicate), analysis of the cell images was performed in ImageJ using an ROI drawn on each cell to measure the median pixel intensity of the donor and acceptor cells. The time for the median fluorescence of the acceptor cell to reach 10% of the donor cell was then determined and used as the value for statistical comparisons. Hippocampal slice preparation Mice of either sex, 3-5 weeks old, were killed by cervical dislocation and decapitated in accordance with United Kingdom Animals (Scientific Procedures) Act (1986). The brain was dissected and kept on ice both the cerebellum and the rostral section of the brain were removed. 400 µm thick sagittal slices were cut with a vibratome (Microm HM 650V microsliver) in ice-cold cutting solution composed of (in mM: 85 NaCl, 2.5 KCl, 0.5 CaCl 2 , 1.25 NaH 2 PO 4 , 24 NaHCO 3 , 25 glucose, and 75 sucrose; pH was adjusted to 7.4, bubbled with 95% O 2 + 5% CO 2 ). Then slices were incubated in aCSF composed of (in mM: 127 NaCl, 1.9 KCl, 2 MgCl 2 , 2 CaCl 2 , 1.2 KH 2 PO 4 , 26 NaHCO 3 , 10 D-glucose, pH 7.4, when bubbled with 95% O 2 and 5% CO 2 ) at 33 °C. For fEPSP recordings an individual slice was pre-incubated in 20 mmHg PCO 2 aCSF for 30 mins, following which the slice was transferred to a recording chamber submerged in aCSF at 32°C. For stimulation, pulses were delivered by a stimulator via concentric bipolar metal stimulating electrode placed on the surface of CA3. For extracellular recordings, an aCSF-filled microelectrode was placed in CA1. Statistics Data is plotted as either cumulative probabilities (showing every data point) or box and whisker plots where the box is interquartile rage, bar is median, and whisker extends to most extreme data point that is no more than 1.5 times the interquartile range. All individual data points are superimposed on the plots. In the case of dye loading one transfection was considered one replicate. In the case of patch clamping and GRAB ATP experiments, one cell was considered one replicate. For dye-loading, to assess the effect a mutation would have on the protein, the difference in dye loading evoked by 70 mmHg PCO 2 or 0 Ca 2+ conditions (from the 20 mmHg baseline) for each construct were compared, using the median value for each condition from each transfection. The Mann-Whitney U one-sided test was used for the comparison on the basis that a priori we expected mutations to either reduce CO 2 sensitivity or have no effect. To compare the fEPSP size across different conditions, the non-parametric Friedman two-way ANOVA was used. Statistical analysis was performed using the python language and SciPy library. Reagents used View this table: View inline View popup Conflict of Interest The authors declare that there are no conflicts of interest Funding We thank the BBSRC (BB/T013346/1, ND) for support. JB was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) and University of Warwick funded Midlands Integrative Biosciences Training Partnership (MIBTP) grant number BB/T00746X/1. VMD was funded by the Medical Research Council through the University of Warwick Doctoral Training Partnership, grant number MR/N014294/1. Data availability All data generated in the paper is available as supplements to the figures. Acknowledgements We thank Prof Alexander Cameron for commenting on a draft of this paper. Funder Information Declared Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982 , BB/T013346/1 , BB/T00746X/1 Medical Research Council , MR/N014294/1 Footnotes ↵ * Co-first authors This revision takes into account the points raised in the public reviews. References ↵ Brotherton , D.H. , Nijjar , S. , Savva , C.G. , Dale , N. & Cameron , A.D. 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Share CO 2 -dependent opening of Connexin 43 hemichannels Valentin-Mihai Dospinescu , Alexander Mascarenhas , Jack Butler , Sarbjit Nijjar , Kyara de Oliveira Taborda , Sean Connors , Lumei Huang , Nicholas Dale bioRxiv 2025.01.13.632711; doi: https://doi.org/10.1101/2025.01.13.632711 Share This Article: Copy Citation Tools CO 2 -dependent opening of Connexin 43 hemichannels Valentin-Mihai Dospinescu , Alexander Mascarenhas , Jack Butler , Sarbjit Nijjar , Kyara de Oliveira Taborda , Sean Connors , Lumei Huang , Nicholas Dale bioRxiv 2025.01.13.632711; doi: https://doi.org/10.1101/2025.01.13.632711 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13871) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18561) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22475) Immunology (17713) Microbiology (40328) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)