elife01213.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN"  "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01213</article-id><article-id pub-id-type="doi">10.7554/eLife.01213</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group></article-categories><title-group><article-title>CO<sub>2</sub> directly modulates connexin 26 by formation of carbamate bridges between subunits</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-6587"><name><surname>Meigh</surname><given-names>Louise</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-8166"><name><surname>Greenhalgh</surname><given-names>Sophie A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6588"><name><surname>Rodgers</surname><given-names>Thomas L</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6589"><name><surname>Cann</surname><given-names>Martin J</given-names></name><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-6590"><name><surname>Roper</surname><given-names>David I</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-6438"><name><surname>Dale</surname><given-names>Nicholas</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">School of Life Sciences</institution>, <institution>University of Warwick</institution>, <addr-line><named-content content-type="city">Coventry</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff2"><institution content-type="dept">Biophysical Sciences Institute</institution>, <institution>University of Durham</institution>, <addr-line><named-content content-type="city">Durham</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff3"><institution content-type="dept">Department of Chemistry</institution>, <institution>University of Durham</institution>, <addr-line><named-content content-type="city">Durham</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff4"><institution content-type="dept">School of Biological and Biomedical Sciences</institution>, <institution>University of Durham</institution>, <addr-line><named-content content-type="city">Durham</named-content></addr-line>, <country>United Kingdom</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Aldrich</surname><given-names>Richard</given-names></name><role>Reviewing editor</role><aff><institution>The University of Texas at Austin</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>n.e.dale@warwick.ac.uk</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>12</day><month>11</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e01213</elocation-id><history><date date-type="received"><day>10</day><month>07</month><year>2013</year></date><date date-type="accepted"><day>08</day><month>10</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Meigh et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Meigh et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01213.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01213.001</object-id><p>Homeostatic regulation of the partial pressure of CO<sub>2</sub> (PCO<sub>2</sub>) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO<sub>2</sub> and direct CO<sub>2</sub>-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO<sub>2</sub>. A ‘carbamylation motif’, present in CO<sub>2</sub>-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO<sub>2</sub>-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO<sub>2</sub>. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO<sub>2</sub> sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO<sub>2</sub>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.001">http://dx.doi.org/10.7554/eLife.01213.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01213.002</object-id><title>eLife digest</title><p>A number of gaseous molecules, including nitric oxide and carbon monoxide, play important roles in many cellular processes by acting as signalling molecules. Surprisingly, however, it has long been assumed that carbon dioxide – a gaseous molecule that is produced during cellular metabolism – is not a signalling molecule.</p><p>Controlling the concentration of carbon dioxide (CO<sub>2</sub>) in a biological system is essential to sustain life, and it was thought that the body used pH – which is the concentration of hydrogen ions – as a proxy for the level of CO<sub>2</sub>. The concentration of CO<sub>2</sub> is related to pH because CO<sub>2</sub> reacts with water to form carbonic acid, which quickly breaks down to form hydrogen ions and bicarbonate ions. This close relationship has led many researchers to equate pH-sensing with CO<sub>2</sub>-sensing, and to suggest that a physiological receptor for CO<sub>2</sub> does not exist.</p><p>Recent research into structures called connexin hemichannels has challenged this view. Researchers found that when pH levels were held constant, increasing the level of CO<sub>2</sub> caused the structures to open up, suggesting that CO<sub>2</sub> could be directly detected by the hemichannels. Each hemichannel contains six connexin subunits, but the details of how the CO<sub>2</sub> molecules interact with the individual connexin subunits to open up the hemichannels remained mysterious.</p><p>Now Meigh et al. show that CO<sub>2</sub> molecules bind to a specific amino acid (lysine) at a particular place (residue 125) in one of the connexin subunits to form a carbamate group. This group then interacts with the amino acid (arginine) at residue 104 in a neighbouring connexin subunit to form a carbamate bridge between the two subunits. This leads to structural changes that cause the gap junction hemichannels to open and release signals that can activate other cells. Since connexin hemichannels are found throughout the human body, these results suggest that CO<sub>2</sub> might act as a signalling molecule in processes as diverse as the control of blood flow, breathing, hearing and reproduction.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.002">http://dx.doi.org/10.7554/eLife.01213.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>respiratory chemosensitivity</kwd><kwd>connexins</kwd><kwd>signal transduction</kwd><kwd>membrane channels</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Human</kwd><kwd>Mouse</kwd><kwd>Rat</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Medical Research Council</institution></institution-wrap></funding-source><award-id>G1001259</award-id><principal-award-recipient><name><surname>Dale</surname><given-names>Nicholas</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>Biotechnology and Biological Sciences Research Council</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Meigh</surname><given-names>Louise</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Engineering and Physical Sciences Research Council</institution></institution-wrap></funding-source><award-id>EP/H051759/1</award-id><principal-award-recipient><name><surname>Rodgers</surname><given-names>Thomas L</given-names></name><name><surname>Cann</surname><given-names>Martin J</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A CO<sub>2</sub> receptor and a mechanism for the CO<sub>2</sub>-mediated opening of gap junction hemichannels have been identified.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>CO<sub>2</sub> is the unavoidable by-product of cellular metabolism. Humans produce approximately 20 moles of CO<sub>2</sub> per day (<xref ref-type="bibr" rid="bib16">Marshall and Bangert, 2008</xref>). The dissolved CO<sub>2</sub> can readily combine with water, aided by carbonic anhydrase, to form H<sub>2</sub>CO<sub>3</sub>, which dissociates rapidly to H<sup>+</sup> and HCO<sub>3</sub><sup>−</sup>. In any physiological solution therefore, the partial pressure of CO<sub>2</sub> (PCO<sub>2</sub>) will be in equilibrium with, and inescapably related to, the pH and the concentration of HCO<sub>3</sub><sup>−</sup> of that solution. Regulation of PCO<sub>2</sub> is thus a vital homeostatic function that is linked to acid-base balance.</p><p>As might be expected, chemosensory reflexes regulate the frequency and depth of breathing to ensure homeostatic control of blood gases. The field of respiratory chemosensitivity has been dominated by ‘reaction theory’ which posits that pH is a sufficient signal for detection of changes in PCO<sub>2</sub> (<xref ref-type="bibr" rid="bib13">Loeschcke, 1982</xref>). Many investigators therefore equate pH-sensing with CO<sub>2</sub>-sensing. There are several areas of the medulla oblongata which contain neurons that respond to changes in pH/CO<sub>2</sub>, especially near the highly vascularised ventral surface. For example a population of neurons highly sensitive to pH/CO<sub>2</sub> have been described in the retrotrapezoid nucleus (RTN) (<xref ref-type="bibr" rid="bib19">Mulkey et al., 2004</xref>, <xref ref-type="bibr" rid="bib18">2006</xref>; <xref ref-type="bibr" rid="bib8">Guyenet et al., 2008</xref>) and the medullary raphé nucleus (<xref ref-type="bibr" rid="bib21">Richerson, 2004</xref>; <xref ref-type="bibr" rid="bib20">Ray et al., 2011</xref>). Despite the acceptance of pH-sensing as the predominant mechanism by which PCO<sub>2</sub> is measured, there is substantial evidence for an additional and independent effect of molecular CO<sub>2</sub> (<xref ref-type="bibr" rid="bib4">Eldridge et al., 1985</xref>; <xref ref-type="bibr" rid="bib24">Shams, 1985</xref>; <xref ref-type="bibr" rid="bib9">Huckstepp and Dale, 2011</xref>). For example, if pH is carefully controlled at the medullary surface, an increase in PCO<sub>2</sub> at constant pH will still enhance breathing by as much as a pH change at constant PCO<sub>2</sub> (<xref ref-type="bibr" rid="bib24">Shams, 1985</xref>). We have recently shown that connexin 26 (Cx26) hemichannels, open in response to increases in PCO<sub>2</sub> at constant extracellular pH and are an important conduit for the CO<sub>2</sub>-dependent, as opposed to pH-dependent, release of ATP (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>). Cx26 hemichannels contribute to the chemosensory control of breathing (<xref ref-type="bibr" rid="bib11">Huckstepp et al., 2010b</xref>; <xref ref-type="bibr" rid="bib30">Wenker et al., 2012</xref>). Hemichannels of two closely related connexins, Cx30 and Cx32, also exhibited CO<sub>2</sub>-sensitive opening (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>). Despite this evidence, widespread acceptance of direct sensing of CO<sub>2</sub> requires a detailed molecular explanation of any putative transduction system.</p><p>One possible way that CO<sub>2</sub> can interact with proteins is via carbamylation—the formation of a covalent bond between the carbon of CO<sub>2</sub> and a primary amine group. For example, CO<sub>2</sub> forms carbamate bonds with haemoglobin (<xref ref-type="bibr" rid="bib12">Kilmartin and Rossi-Bernardi, 1971</xref>) and the plant enzyme RuBisCo (<xref ref-type="bibr" rid="bib14">Lundqvist and Schneider, 1991</xref>). Here we document an important new advance—the mechanism by which CO<sub>2</sub> binds directly to Cx26, most probably via carbamylation of a lysine residue, to cause hemichannel opening. Our work establishes a new field of direct CO<sub>2</sub> sensing that can be mediated by CO<sub>2</sub>-dependent carbamylation of certain β connexins. As these are widely distributed in the brain and other tissues, it is likely that direct detection of CO<sub>2</sub> via this mechanism is important in many different physiological processes.</p></sec><sec id="s2" sec-type="results"><title>Results</title><p>We have previously demonstrated that Cx26, and two related β connexins, Cx30 and Cx32, open when exposed to modest increases in PCO<sub>2</sub> at constant pH (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>). This previous study demonstrated, in inside-out and outside-out excised membrane patches at a transmembrane potential of −40 mV, that Cx26 hemichannel gating respectively increased and decreased in response to increases and decreases of PCO<sub>2</sub>. To reconfirm our previous findings that Cx26, and not some other hemichannel senses CO<sub>2</sub> (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>), we demonstrated that the CO<sub>2</sub>-dependent dye loading of HeLa cells expressing Cx26 was blocked by 100 µM carbenoxolone, but unaffected by 1 mM probenecid, a blocker of pannexin-1, (<xref ref-type="bibr" rid="bib26">Silverman et al., 2008</xref>), and 20 µM ruthenium red, a blocker of calhm1 (<xref ref-type="bibr" rid="bib27">Taruno et al., 2013</xref>), (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). Parental HeLa cells do not exhibit CO<sub>2</sub>-dependent dye loading demonstrating that the heterologous expression of Cx26 is essential for this function (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>) (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>).</p><sec id="s2-1"><title>The extent of CO<sub>2</sub> chemosensitivity within the β connexins</title><p>To document the extent to which this sensitivity to CO<sub>2</sub> is limited within the β connexin family (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), and to form the basis of a bioinformatic comparison to identify possible CO<sub>2</sub> binding motifs, we investigated whether another β connexin, Cx31, might also be sensitive to CO<sub>2</sub>. We expressed, in HeLa cells, either rat Cx31 or rat Cx26 tagged at the C-terminal with mCherry and used a previously described dye loading assay (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>) to test whether the cells could load with carboxyfluorescein (CBF) in a CO<sub>2</sub>-dependent manner. As expected from our previous work, HeLa cells expressing the Cx26 readily loaded with CBF when exposed to this dye in the presence of elevated PCO<sub>2</sub> (55 mmHg, at pH 7.5, <xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). However, HeLa cells expressing Cx31 failed to dye load in a CO<sub>2</sub>-dependent manner (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). As the connexins were tagged with mCherry, we could verify the presence of fluorescent puncta in both the Cx26 and Cx31 expressing HeLa cells (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). To check for the existence of functional hemichannels in the Cx31-expressing HeLa cells, we removed extracellular Ca<sup>2+</sup> as a positive control. This manipulation will open all types of connexin hemichannel. Parental HeLa cells do not load with dye when Ca<sup>2+</sup> is removed from the medium (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>); they therefore possess virtually no endogenous hemichannels. The removal of extracellular Ca<sup>2+</sup> readily caused loading of CBF into the Cx31-expressing HeLa cells (<xref ref-type="fig" rid="fig1">Figure 1A</xref>, inset), demonstrating the presence of functional Cx31 hemichannels.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.003</object-id><label>Figure 1.</label><caption><title>Identification of the motif in Cx26 that imparts CO<sub>2</sub> sensitivity.</title><p>(<bold>A</bold>) Dye loading assay demonstrates CO<sub>2</sub>-dependent loading of carboxyfluorescein into HeLa cells expressing Cx26, but not into those expressing Cx31. The inset in Cx31 shows that these hemichannels are expressed and functional in the membrane by utilizing a zero Ca<sup>2+</sup> stimulus to open them and allow dye loading. Scale bars 40 µm. (<bold>B</bold>) Cumulative probability plots of pixel intensity in the control and following exposure to PCO<sub>2</sub> of 55 mmHg. Each curve is comprises the measurements of mean pixel intensity for at least 40 cells. (<bold>C</bold>) Sequences (from mouse) for Cx26, 30, 32 and 31 to show K125 and four following amino acids that are present in Cx26, Cx30 and Cx32, but absent from Cx31. R104 in Cx26 and 30, and K104 in Cx32 and Cx31 are also highlighted. Accession numbers: Cx26, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/protein/NP_032151">NP_032151</ext-link>; Cx30, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/protein/AAH13811">AAH13811</ext-link>; Cx32, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/protein/AAH26833">AAH26833</ext-link>; and Cx31, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/protein/NP_001153484">NP_001153484</ext-link>. (<bold>D</bold>) The structure of Cx26 drawn from the 2zw3 PDB file, cytoplasmic face of the channel upwards. On each subunit K125 and R104 are drawn. (<bold>E</bold>) Detail from the structure of Cx26 (dashed square) showing the orientation of K125 (red) towards R104 (dark grey). The short distance between the two amino acid side chains suggests that this gap could be bridged by carbamylation by CO<sub>2</sub> of K125 and a subsequent salt bridge with R104. (<bold>F</bold>) Phylogenetic tree showing relationship between Cx26 and other β connexins. The three CO<sub>2</sub> sensitive connexins are very closely related to each other while Cx31 is more distant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.003">http://dx.doi.org/10.7554/eLife.01213.003</ext-link></p></caption><graphic xlink:href="elife01213f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01213.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Expression of Cx26 in HeLa cells imparts sensitivity to CO<sub>2</sub>.</title><p>Top, diagram of dye loading protocol, showing the methods for assessing background dye loading under control conditions (no additional CO<sub>2</sub>), dye loading during a hypercapnic challenge (PCO<sub>2</sub> 55 mmHg) and the use of the hemichannel blockers to probe the identity of the hemichannel required for CO<sub>2</sub> sensitive dye loading. Bottom, cumulative probability plots demonstrating that exposure of Cx26-expressing HeLa cells to elevated PCO<sub>2</sub> caused dye loading compared to the background control. This CO<sub>2</sub>-dependent loading was blocked by 100 µM carbenoxolone (Carb), but unaffected by probenecid or ruthenium red (RuRed), demonstrating that the heterologously expressed Cx26 rather than any potential endogenous pannexin-1 or calhm1 was responsible for the CO<sub>2</sub> sensitivity. Minimum of 40 cells measured from three independent repetitions of each treatment. The slight reduction in dye loading caused by carbenoxolone compared to the background control loading is to be expected, as Cx26 will partially gate in response to control aCSF which has a PCO<sub>2</sub> of 35 mmHg. Parental HeLa cells do not exhibit CO<sub>2</sub>-dependent dye loading (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.004">http://dx.doi.org/10.7554/eLife.01213.004</ext-link></p></caption><graphic xlink:href="elife01213fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01213.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Expression of connexin variants in HeLa cells.</title><p>The connexin variants were tagged with mCherry, allowing verification of successful of expression. Examples of wild type Cx26 and Cx31 are shown along with two mutant variants. Scale bar 40 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.005">http://dx.doi.org/10.7554/eLife.01213.005</ext-link></p></caption><graphic xlink:href="elife01213fs002"/></fig></fig-group></p></sec><sec id="s2-2"><title>Identification of a carbamylation motif in CO<sub>2</sub> sensitive β connexins</title><p>The CO<sub>2</sub>-sensitivity in the β connexins therefore appears to be limited to the three closely related connexins, Cx26, Cx30 and Cx32, and Cx31 has no sensitivity to increases in PCO<sub>2</sub> (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). We hypothesized that CO<sub>2</sub> carbamylated a lysine residue in Cx26 to induce conformational change and hence opening of the hemichannel. We therefore compared the sequences of the three CO<sub>2</sub>-sensitive connexins to Cx31 to identify a lysine present in all three CO<sub>2</sub> sensitive connexins but absent from Cx31 (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). This analysis revealed K125 and four further amino acids as forming a motif that was absent from Cx31. The existing crystal structure for Cx26 (<xref ref-type="bibr" rid="bib15">Maeda et al., 2009</xref>), shows that K125 is at the end of an alpha helix and that the sequence KVREI (‘carbamylation motif’) orients K125 towards R104 on the neighbouring subunit (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). The distance from K125 to R104 is only 6.5 Å (<xref ref-type="bibr" rid="bib15">Maeda et al., 2009</xref>), strongly suggesting that if K125 were to be carbamylated it could form a salt bridge between these two residues in adjacent subunits (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Interestingly, R104 is present in Cx30, but conservatively substituted by a lysine residue in Cx32 (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), which has a lower sensitivity to CO<sub>2</sub> than Cx26 (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010a</xref>).</p></sec><sec id="s2-3"><title>Insertion of the carbamylation motif into Cx31 creates a CO<sub>2</sub>-sensitive mutant hemichannel</title><p>Our analysis predicts that if we were to introduce the putative carbamylation-motif of Cx26 into Cx31, the resulting mutant Cx31 (mCx31) should be sensitive to CO<sub>2</sub> as the lysine introduced into the sequence should be able to form a salt bridge with the native residue K104 in mCx31 once carbamylated (<xref ref-type="fig" rid="fig1 fig2">Figure 1C–E and 2A</xref>). We therefore made mCx31 in which the motif TQKVREI was introduced in place of K123H124 of the native connexin (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). This insertion/substitution maintained the correct orientation of the K125 with respect to K104 of Cx31. HeLa cells expressing mCx31 displayed clear CO<sub>2</sub>-dependent dye loading (<xref ref-type="fig" rid="fig2">Figure 2B,C</xref>). We confirmed the CO<sub>2</sub> sensitivity of mCx31 expressing HeLa cells by performing whole cell patch camp recordings. mCx31-expressing cells exhibited a conductance change of 3.3 ± 0.84 nS (mean ± SEM, n = 8, <xref ref-type="supplementary-material" rid="SD4-data">Supplementary file 1</xref>) when exposed to elevated PCO<sub>2</sub> (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). Cells, expressing wild type Cx31 showed no CO<sub>2</sub>-dependent changes in their whole cell conductance (mean conductance change −0.002 ± 0.023 nS, n = 6, <xref ref-type="supplementary-material" rid="SD4-data">Supplementary file 1</xref>, <xref ref-type="fig" rid="fig2">Figure 2D</xref>).<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.006</object-id><label>Figure 2.</label><caption><title>Insertion of the identified motif into Cx31 creates a CO<sub>2</sub>-sensitive hemichannel.</title><p>(<bold>A</bold>) Comparison of the WT and mutated Cx31 amino acid sequence to show the insertion of the K125 and surrounding residues. (<bold>B</bold>) The dye loading assay demonstrates gain of CO<sub>2</sub>-sensitivity in mCx31. Scale bar 40 µm. (<bold>C</bold>) Cumulative probability of mean pixel density of 40 cells in five independent replications. (<bold>D</bold>) Whole cell patch clamp recordings from HeLa cells expressing mCx31 and Cx31. Recordings were performed under voltage clamp at a holding potential of −50 mV with a constant 10 mV step to assess whole cell conductance. The cells expressing mCx31 show a clear conductance change on exposure to a change in PCO<sub>2</sub>, whereas cells expressing wild type Cx31 showed no such change (inset).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.006">http://dx.doi.org/10.7554/eLife.01213.006</ext-link></p></caption><graphic xlink:href="elife01213f002"/></fig></p></sec><sec id="s2-4"><title>K125 and R104 are essential for CO<sub>2</sub> sensitivity</title><p>To demonstrate that K125 is the key residue for interaction with CO<sub>2</sub>, we made mCx31<sup>K125R</sup>, by inserting TQRVREI into Cx31 in place of K123H124. Unlike lysine, the arginine side chain cannot be carbamylated by CO<sub>2</sub> as its pKa (12.5) is much higher than that of lysine (10.5), therefore this variant should have no sensitivity to CO<sub>2</sub>. mCx31<sup>K125R</sup> did indeed lack sensitivity to CO<sub>2</sub> (<xref ref-type="fig" rid="fig3">Figure 3A–C</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 3—source data 1</xref>). This was not because the mutant channel failed to assemble correctly, as the positive control of zero Ca<sup>2+</sup> dye loading demonstrated the presence of functional hemichannels (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Next we investigated the relevant residues in Cx26 itself. The carbamate bridge that we propose must involve K125 (being carbamylated) and R104 (forming the salt bridge with the carbamylated lysine). We therefore made mutations that individually disrupted both of these functions: K125R to prevent carbamylation, and R104A to disrupt formation of the salt bridge. Neither Cx26<sup>K125R</sup> nor Cx26<sup>R104A</sup> exhibited sensitivity to CO<sub>2</sub> sensitivity. Nevertheless the positive controls demonstrated the presence of functional mutant hemichannels in the expressing HeLa cells (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.007</object-id><label>Figure 3.</label><caption><title>K125 and R104 are essential residues for CO<sub>2</sub> sensitivity.</title><p>(<bold>A</bold>) Insertion of the motif (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) from Cx26 but with the mutation K125R into Cx31 (mCx31<sup>K125R</sup>) does not give a gain of CO<sub>2</sub> sensitivity indicating that this is an essential residue. Introducing the mutations K125R or R104A into Cx26 destroys the CO<sub>2</sub>-sensitivity of Cx26. Insets show the zero Ca<sup>2+</sup> positive controls to demonstrate the presence of functional hemichannels in the cells. Scale bars, 40 µm. (<bold>B</bold>) Cumulative probability distributions demonstrate that none of these mutant channels are sensitive to CO<sub>2</sub>. (<bold>C</bold>) Summary data demonstrating: gain of function in the mCx31 hemichannel and subsequent loss in mCx31<sup>K125R</sup>; and loss of function in the Cx26<sup>K125R</sup> and Cx26<sup>R104A</sup> mutants. The graphs shown the median of the median change in pixel intensity from five independent replications for each type of hemichannel. KW: Kruskal-Wallis ANOVA, pairwise comparisons by the Mann-Whitney U-test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.007">http://dx.doi.org/10.7554/eLife.01213.007</ext-link></p><p><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.01213.008</object-id><label>Figure 3—source data 1.</label><caption><title>Median differences in pixel intensity between CO<sub>2</sub> and control dye loading experiments for the various connexin hemichannel variants in the histograms of <xref ref-type="fig" rid="fig3">Figure 3C</xref> and statistical analysis: Kruskal-Wallis anova, pairwise Mann-Whitney tests and false discovery rate procedure.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.008">http://dx.doi.org/10.7554/eLife.01213.008</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01213s001.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01213f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01213.009</object-id><label>Figure 3—figure supplement 1.</label><caption><title>All connexin variants form functional hemichannels capable of opening in response to zero Ca<sup>2+</sup>.</title><p>Dye loading of wild type HeLa cells and HeLa cells expressing Cx26, Cx31 and the mutated versions used in this study. In all cases zero Ca<sup>2+</sup> (red trace) increases the amount of dye loading in connexin expressing cells over the control (light grey trace). For reference the dye loading caused by CO<sub>2</sub> is also shown (dark grey trace). Neither CO<sub>2</sub> nor zero Ca<sup>2+</sup> altered dye loading in parental HeLa cells devoid of heterologous connexin expression, demonstrating that these cells have virtually no endogenous hemichannels. For Cx26, the amount of dye loading caused by CO<sub>2</sub> and zero Ca<sup>2+</sup> was similar. In all other connexin variants zero Ca<sup>2+</sup> caused dye loading but CO<sub>2</sub> had no effect. The cumulative probability distributions comprise all measurements from five independent replications of every set of hemichannel variants that is the entire dataset arising from the image analysis of these connexin variants.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.009">http://dx.doi.org/10.7554/eLife.01213.009</ext-link></p></caption><graphic xlink:href="elife01213fs003"/></fig></fig-group></p></sec><sec id="s2-5"><title>Engineering an analogue of the carbamylated lysine into Cx26 makes it constitutively open</title><p>To test further our prediction that the carbamylated K125 forms a salt bridge with R104, we made the mutation K125E in Cx26. The insertion of glutamate in place of the lysine has the potential to act as an analogue of the carbamylated K125. If our hypothesis is correct, this mutated channel should be constitutively open, as the carboxyl group of the E125 should be able to form a salt bridge with R104. We found that HeLa cells expressing Cx26<sup>K125E</sup> readily loaded with dye under control conditions and exhibited no sensitivity to CO<sub>2</sub> (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The Cx26<sup>K125E</sup>-expressing HeLa cells showed much greater loading under control conditions than parental HeLa cells (<xref ref-type="fig" rid="fig4">Figure 4</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 4—source data 1</xref>). To further confirm that the constitutive dye loading occurred via the misexpressed connexin, we demonstrated that carbenoxolone (100 µM) completely blocked CO<sub>2</sub>-dependent dye loading in HeLa cells expressing Cx26<sup>K125E</sup> (<xref ref-type="fig" rid="fig4">Figure 4</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 4—source data 1</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.010</object-id><label>Figure 4.</label><caption><title>Engineering an analogue of the carbamylated lysine residue, Cx26<sup>K125E</sup>, creates a constitutively open hemichannel that no longer responds to CO<sub>2</sub>.</title><p>(<bold>A</bold>) HeLa cells expressing Cx26<sup>K125E</sup> readily load with dye under control conditions. Increasing the PCO<sub>2</sub> does not give a further increase in dye loading. This dye loading was blocked by 100 µM carbenoxolone (Carb), indicating that it occurred through the heterologously expressed connexin. Scale bar 40 µm. (<bold>B</bold>) Cumulative probability plots comparing the median pixel intensities of at least 40 cells per experiment and five independent repetitions for the control, hypercapnic and carbenoxolone treatments with that of parental HeLa cells (four independent repetitions). (<bold>C</bold>) Summary data showing the median of the median pixel intensity for the three conditions for Cx26<sup>K125E</sup> and the background loading for parental HeLa cells. Pairwise comparisons by the Mann-Whitney U-test; KW Kruskall-Wallis Anova. Neither the difference between control and CO<sub>2</sub> nor the difference between Cx26<sup>K125E</sup> treated with carbenoxolone and parental HeLa cells is significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.010">http://dx.doi.org/10.7554/eLife.01213.010</ext-link></p><p><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.01213.011</object-id><label>Figure 4—source data 1.</label><caption><title>Median pixel intensity values for histogram in <xref ref-type="fig" rid="fig4">Figure 4C</xref> and statistical analysis: Kruskal-Wallis anova and pairwise Mann-Whitney tests.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.011">http://dx.doi.org/10.7554/eLife.01213.011</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01213s002.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01213f004"/></fig></p><p>Reasoning that if bridge formation between subunits was key to opening the hemichannel, we also made the further mutation R104E. In the mutated channel E104 has the potential to form a salt bridge in the reverse direction with K125 and we predicted that if this were to happen such a mutant hemichannel should also be constitutively open. We found that HeLa cells expressing Cx26<sup>R104E</sup> did indeed load with dye under control conditions and that this enhanced dye loading was blocked with carbenoxolone (<xref ref-type="fig" rid="fig5">Figure 5</xref>, <xref ref-type="supplementary-material" rid="SD3-data">Figure 5—source data 1</xref>).<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.012</object-id><label>Figure 5.</label><caption><title>Bridging in the reverse direction: the mutation R104E forms a salt bridge with K125 in Cx26<sup>R104E</sup> to create a constitutively open hemichannel that no longer responds to CO<sub>2</sub>.</title><p>(<bold>A</bold>) HeLa cells expressing Cx26<sup>R104E</sup> readily load with dye under control conditions. Increasing the PCO<sub>2</sub> does not give a further increase in dye loading. This dye loading was blocked by 100 µM carbenoxolone (Carb), indicating that it occurred through the heterologously expressed connexin. Scale bar 40 µm. (<bold>B</bold>) Cumulative probability plots comparing the median pixel intensities of at least 40 cells per experiment and five independent repetition for the control, hypercapnic and carbenoxolone treatments. (<bold>C</bold>) Summary data showing the median of the median pixel intensity for the three conditions for Cx26<sup>R104E</sup>. Pairwise comparisons by the Mann-Whitney U-test; KW Kruskall-Wallis Anova. The difference between control and CO<sub>2</sub> is not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.012">http://dx.doi.org/10.7554/eLife.01213.012</ext-link></p><p><supplementary-material id="SD3-data"><object-id pub-id-type="doi">10.7554/eLife.01213.013</object-id><label>Figure 5—source data 1.</label><caption><title>Median pixel intensity values for histogram in <xref ref-type="fig" rid="fig5">Figure 5C</xref> and statistical analysis: Kruskal-Wallis anova and pairwise Mann-Whitney tests.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.013">http://dx.doi.org/10.7554/eLife.01213.013</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01213s003.xlsx"/></supplementary-material></p></caption><graphic xlink:href="elife01213f005"/></fig></p></sec><sec id="s2-6"><title>Elastic network model of Cx26 shows that carbamylation leads to hemichannel opening</title><p>Although our experimental data point to the importance of carbamylation of K125 and the formation of a salt bridge to R104 in the adjacent subunit, it is not clear how this would lead to opening of the Cx26 hemichannel. Course-grained modelling reduces protein atomistic complexity for more efficient computational studies of harmonic protein dynamics and is particularly suited to examining hemichannel opening over millisecond time scales (<xref ref-type="bibr" rid="bib25">Sherwood et al., 2008</xref>). We therefore built coarse-grained elastic network models (ENM) to gain insight into the mechanism by which CO<sub>2</sub> maintains Cx26 in the open state. In an ENM the Cα-atom co-ordinates of an atomic resolution structure are used to represent a protein structure. The total global protein motion within the ENM consists of a defined number of modes, each of a characteristic frequency and representing a particular harmonic motion within the protein. ENMs are known to reproduce the global low frequency modes of protein motion well in comparison to experimental data (<xref ref-type="bibr" rid="bib3">Delarue and Sanejouand, 2002</xref>; <xref ref-type="bibr" rid="bib29">Valadie et al., 2003</xref>). We used the co-ordinates from a high-resolution crystal structure to construct an ENM (<xref ref-type="bibr" rid="bib28">Tirion, 1996</xref>) for the Cx26 hexamer in the unbound and CO<sub>2</sub>-bound states. CO<sub>2</sub> was represented in the ENM by the inclusion of six additional Hookean springs between residues K125 and R104 of neighbouring monomers (<xref ref-type="fig" rid="fig6">Figure 6A</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.014</object-id><label>Figure 6.</label><caption><title>Elastic network model (ENM) of Cx26 demonstrating that CO<sub>2</sub> binding restricts the motion of the hemichannel and biases it to the open state.</title><p>(<bold>A</bold>) Left, diagram of Cx26 from the 2zw3 structure, indicating the ENM (black lines) superimposed on the tertiary structure of Cx26 and showing the position of the hookean springs (white lines) introduced to simulate binding of CO<sub>2</sub> to K125 and bridging to R104. Right, ENM of Cx26 seen end on from the cytoplasmic side of the channel showing the six springs (white lines) that represent CO<sub>2</sub> binding. (<bold>B</bold>) Frequency modes of channel motion plotted for CO<sub>2</sub> bound against those without CO<sub>2</sub> bound. The grey scale on the right indicates the similarity of the modes between the CO<sub>2</sub> bound and unbound states. Note that there is a high degree of similarity between most modes in the bound and unbound state, indicating that binding of CO<sub>2</sub> reorders the modes of motion. In the graph, the modes that fall on the dotted line (x = y) have not changed between the two states. Mode 1 without CO<sub>2</sub> bound (closing of hemichannel) moves to Mode 9 with CO<sub>2</sub> bound (dashed upward arrow) indicating that it contributes much less to the total channel motions when CO<sub>2</sub> is bound. Most of the other modes fall below the dotted line, indicating that they become more frequent when CO<sub>2</sub> is bound. (<bold>C</bold>) Vectors indicating the Mode 1 motions of the α helices without CO<sub>2</sub> bound (left) and with CO<sub>2</sub> bound (right). The binding of CO<sub>2</sub> and creation of the carbamylation bridge between subunits greatly restricts hemichannel motion.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.014">http://dx.doi.org/10.7554/eLife.01213.014</ext-link></p></caption><graphic xlink:href="elife01213f006"/></fig></p><p>Analysis of the model revealed that in the absence of CO<sub>2</sub> the lowest frequency mode (mode 1) represented an opening/closing motion that was able to fully occlude the hemichannel central pore (<xref ref-type="other" rid="video1">Video 1</xref>). Addition of springs representing CO<sub>2</sub>-binding to the ENM restricted the closing motions of the hemichannel and thus connexin 26 was maintained in the open state (<xref ref-type="other" rid="video2">Video 2</xref>). We examined the overlap in the ordering of the modes in the unbound and CO<sub>2</sub> bound states to gain insight into how this occurs. A significant reordering of the lowest frequency modes to higher frequencies was observed in the presence of CO<sub>2</sub> rather than the removal of any modes from the total protein motion (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Mode 1, the lowest frequency mode that represents the opening/closing mode, represented about 40% of the total protein motion in the absence of CO<sub>2</sub>. In the presence of CO<sub>2</sub> this closing mode is reordered through a change in its frequency as mode 9, which accounts for only about 2% of the total motion (<xref ref-type="fig" rid="fig6">Figure 6B,C</xref>). CO<sub>2</sub> therefore opens Cx26 through a reordering of the normal modes of global protein motion such that the normal closing motion of Cx26 no longer significantly contributes to the total motion of the hemichannel. We infer from this that the carbamate bridge formed between Cx26 monomers represents a constraining force that hinders hemichannel closure.<media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="avi" mimetype="video" xlink:href="elife01213v001.avi"><object-id pub-id-type="doi">10.7554/eLife.01213.015</object-id><label>Video 1.</label><caption><title>Hemichannel mode 1 motions in absence of CO<sub>2</sub>.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.015">http://dx.doi.org/10.7554/eLife.01213.015</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="avi" mimetype="video" xlink:href="elife01213v002.avi"><object-id pub-id-type="doi">10.7554/eLife.01213.016</object-id><label>Video 2.</label><caption><title>Hemichannel mode 1 motions in presence of CO<sub>2</sub>.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.016">http://dx.doi.org/10.7554/eLife.01213.016</ext-link></p></caption></media></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Evidence from six different experimental tests supports our hypothesis that CO<sub>2</sub> forms a carbamate bridge between K125 and R104 on the adjacent subunit to open the Cx26 hemichannel. Firstly, we demonstrated the sufficiency of the carbamylation motif to confer CO<sub>2</sub> sensitivity by inserting it into a CO<sub>2</sub>-insensitive connexin, Cx31. Secondly, we showed that K125 of the carbamylation motif was essential for this motif to confer CO<sub>2</sub> sensitivity on Cx31. Thirdly and fourthly, we demonstrated that the mutations K125R and R104A in Cx26 (to prevent carbamate bridging at either end of the bridge) destroyed the CO<sub>2</sub> sensitivity of this connexin. Fifthly, by exploiting glutamate as an analogue of the carbamylated K125 (in Cx26<sup>K125E</sup>), we demonstrated a gain of function—Cx26<sup>K125E</sup> was constitutively open, yet had lost sensitivity to CO<sub>2</sub>. Finally, we further tested the bridging concept by demonstrating that the bridge is in effect bidirectional: the mutated hemichannel Cx26<sup>R104E</sup>, in which E104 can bridge to K125 in the reverse direction, was also constitutively open, but had no sensitivity to CO<sub>2</sub>.</p><p>Although we have not directly demonstrated CO<sub>2</sub> binding to Cx26, our extensive testing of this hypothesis through selective mutations leads us to conclude that CO<sub>2</sub> interacts with Cx26 directly and that no other protein is required for CO<sub>2</sub> sensitivity. This interaction is most probably via carbamylation of K125. Interestingly, the mutations Cx26<sup>K125E</sup> and Cx26<sup>K125R</sup> can be considered respectively as open-state and closed-state analogues of the wild type channel. Collectively, our data strongly suggests that CO<sub>2</sub> binds to the intracellular surface of Cx26 and must therefore cross the membrane to reach this site. This could occur either direct diffusion through the membrane bilayer, potentially via Cx26 itself, or via other CO<sub>2</sub> permeable channels (<xref ref-type="bibr" rid="bib1">Boron et al., 2011</xref>). Amongst its many other functions, Cx26 can therefore be regarded as a receptor for CO<sub>2</sub>. Interestingly, this mechanism of modulation applies to both Cx30 and Cx32, which can both potentially form a carbamate bridge at equivalent residues to Cx26. In the case of Cx32 this would involve bridging to K104 rather than R104 (in Cx26 and 30). Cx26 can co-assemble with both Cx30 and Cx32 to form heteromeric hemichannels (<xref ref-type="bibr" rid="bib6">Forge et al., 2003</xref>; <xref ref-type="bibr" rid="bib32">Yum et al., 2007</xref>). Our structural studies predict that, as Cx30 and Cx32 have K125 and either R104 or K104, carbamate bridges could form in such heteromeric hemichannels and that they should also therefore be CO<sub>2</sub>-sensitive.</p><p>Carbamylation involves formation of a labile covalent bond between the carbon of CO<sub>2</sub> and a primary amine. For this to occur the amine must be in a restricted hydration space so that it is not protonated. Some examples of physiologically significant carbamylation are known. The carbamylation of the N-terminal amines of haemoglobin contributes to the Bohr effect (<xref ref-type="bibr" rid="bib12">Kilmartin and Rossi-Bernardi, 1971</xref>), whereby the affinity of haemoglobin for O<sub>2</sub> is reduced in the presence of elevated CO<sub>2</sub>. However in mammalian systems no other examples of carbamylation by CO<sub>2</sub> have been described. In C3 photosynthetic plants, the enzyme RuBisCo, that participates in the Calvin cycle and carbon fixation is activated by carbamylation of a specific lysine residue (<xref ref-type="bibr" rid="bib14">Lundqvist and Schneider, 1991</xref>). Several microbial enzymes are also carbamylated (<xref ref-type="bibr" rid="bib17">Maveyraud et al., 2000</xref>; <xref ref-type="bibr" rid="bib7">Golemi et al., 2001</xref>; <xref ref-type="bibr" rid="bib31">Young et al., 2008</xref>).</p><p>Despite this precedent, the functional significance of CO<sub>2</sub>-carbamylation and its potential as a transduction principle for the measurement of CO<sub>2</sub> has been almost completely overlooked in vertebrate physiology. The mechanism of formation of a salt bridge between a carbamylated lysine and an appropriately oriented arginine on the neighbouring subunit is a unique mechanism for modulation of an ion channel and establishes carbamylation as a mechanistic basis for the direct signalling of PCO<sub>2</sub> in mammalian physiology. This carbamylation of a lysine to transduce the concentration of CO<sub>2</sub> into a biological signal is somewhat equivalent to the nitrosylation of a cysteine residue by NO/nitrite. It establishes a CO<sub>2</sub>-dependent signalling paradigm in which the concentration of CO<sub>2</sub> is signalled by ATP release via Cx26 from the chemosensory cell and consequent activation of neighbouring cells, or potentially by a Ca<sup>2+</sup> influx through the Cx26 hemichannel (<xref ref-type="bibr" rid="bib5">Fiori et al., 2012</xref>) to initiate a Ca<sup>2+</sup> wave within the chemosensory cell itself and further Ca<sup>2+</sup>-dependent signalling processes.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Hemichannel expression and mutagenesis</title><p>All connexin genes except Cx26<sup>R104A</sup>, Cx26<sup>K125E</sup> and Cx26<sup>R104E</sup> were synthesised by Genscript USA and subcloned into the pCAG-GS mCherry vector. The sequence for wild type Cx26 and Cx31 genes were respectively take from accession numbers NM_001004099.1 and NM_019240.1. To produce Cx26<sup>R104A</sup>, Cx26<sup>K125E</sup> and Cx26<sup>R104E</sup> site directed mutagenesis was performed using Quikchange II site directed mutagenesis kit. All wild type and mutant genes were sequenced to verify that the correct sequence was present. Hela cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich Company Ltd, Gillingham, UK), 10% FCS (Biosera Europe, Labtech International Ltd, Uckfield, UK), 1:1000 pen/strep and supplemented with 3 mM CaCl<sub>2</sub>. Cells were grown in a humidified 5% CO<sub>2</sub> incubator at 37°C. The connexin proteins were expressed via transient transfection. Cells were plated in six-well plates at 1 × 10<sup>5</sup> cells per well for Cx26 and its mutants and 5 × 10<sup>4</sup> cells per well for Cx31 and its mutants. Following the GeneJuice transfection reagent (Merck-Millipore UK, Merck KGaA, Darmstadt, Germany) user protocol, cells were transfected with 1 µg of the appropriate DNA. Experiments were performed when the connexin proteins had reached the cell membrane. This was found to be approximately 2 days for Cx26 and its mutants and approximately 3 days for Cx31 and its mutants.</p></sec><sec id="s4-2"><title>Solutions used</title><sec id="s4-2-1"><title>Standard artificial cerebrospinal fluid (aCSF, normocapnic)</title><p>124 mM NaCl, 3 mM KCl, 2 mM CaCl<sub>2</sub>, 26 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 1 mM MgSO<sub>4</sub>, 10 mM D-glucose saturated with 95% O<sub>2</sub>/5% CO<sub>2</sub>, pH 7.5, PCO<sub>2</sub> 35 mmHg.</p></sec><sec id="s4-2-2"><title>50 mM HCO<sub>3</sub><sup>−</sup> aCSF (isohydric hypercapnic)</title><p>100 mM NaCl, 3 mM KCl, 2 mM CaCl<sub>2</sub>, 50 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 1 mM MgSO<sub>4</sub>, 10 mM D-glucose, saturated with 9% CO<sub>2</sub> (with the balance being O<sub>2</sub>) to give a pH of 7.5 and a PCO<sub>2</sub> of 55 mmHg respectively.</p></sec></sec><sec id="s4-3"><title>Dye loading assay and image analysis</title><p>Connexin expressing HeLa cells were plated on cover slips. A coverslip was placed in a small flow chamber and the cells were exposed to either: control aCSF with 200 µM carboxyfluorescein for 10 min; isohydric hypercapnic aCSF with 200 µM carboxyfluorescein for 10 min; or zero Ca<sup>2+</sup>, 1 mM EGTA-containing aCSF plus 200 µM carboxyfluorescein for 10 min. This was followed by control aCSF plus 200 µM carboxyfluorescein for 5 min and then thorough washing for 30 min with control aCSF. These protocols are summarized in <xref ref-type="fig" rid="fig7">Figure 7</xref>.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01213.017</object-id><label>Figure 7.</label><caption><title>Dye loading protocols.</title><p>The control background loading tests for any potential CO<sub>2</sub>-insensitive pathways of dye loading that are constitutively active in the HeLa cells. Hypercapnic dye loading uses the 50 mM HCO<sub>3</sub><sup>−</sup> aCSF to test CO<sub>2</sub>-sensitive loading under conditions of isohydric hypercapnia (PCO<sub>2</sub> 55 mmHg). The zero Ca<sup>2+</sup> positive control tests for the presence of functional hemichannels in those cases where the misexpressed hemichannels exhibit no sensitivity to CO<sub>2</sub>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.017">http://dx.doi.org/10.7554/eLife.01213.017</ext-link></p></caption><graphic xlink:href="elife01213f007"/></fig></p><p>The cells were then imaged by epifluorescence (Scientifica Slice Scope (Scientifica Ltd, Uckfield, UK), Cairn Research OptoLED illumination (Cairn Research Limited, Faversham, UK), 60x water Olympus immersion objective, NA 1.0 (Scientifica), Hamamatsu ImageEM EMCCD camera (Hamamatsu Photonics K.K., Japan), Metafluor software (Cairn Research)). Using ImageJ, the extent of dye loading was measured by drawing a region of interest (ROI) around individual cells and calculating the mean pixel intensity for the ROI. The mean pixel intensity of the background fluorescence was also measured in a representative ROI, and this value was subtracted from the measures obtained from the cells. All of the images displayed in the figures reflect this procedure in that the mean intensity of the pixels in a representative background ROI has been subtracted from every pixel of the image. At least 40 cells were measured in each condition, and the mean pixel intensities plotted as cumulative probability distributions.</p><p>For the dye loading experiments, the median pixel intensities of the control and CO<sub>2</sub> dye loading conditions (minimum of five independent repetitions) were compared by a Kruskal Wallace ANOVA and pairwise comparions by the Mann-Whitney test. The false discovery rate procedure (<xref ref-type="bibr" rid="bib2">Curran-Everett, 2000</xref>) was used to determine whether the multiple pairwise comparisons remained significant.</p></sec><sec id="s4-4"><title>Patch clamp recordings</title><p>Cover slips containing non-confluent cells were placed into a perfusion chamber at 28°C in sterile filtered standard aCSF. Standard patch clamp techniques were used to make whole-cell recordings. The intracellular fluid in the patch pipette contained: K-gluconate 120 mM, CsCl 10 mM, TEACl 10 mM, EGTA 10 mM, ATP 3 mM, MgCl<sub>2</sub> 1 mM, CaCl<sub>2</sub> 1 mM, sterile filtered, pH adjusted to 7.2 with KOH. All whole-cell recordings were performed at a holding potential of −40 mV with regular steps of 5 s to −50 mV to assess whole-cell conductance.</p></sec><sec id="s4-5"><title>Elastic network model–course-grained simulations</title><p>Elastic network model (ENM) simulations were performed based on its regular implementation using pdb file 2ZW3, where all the Cα atoms in the protein within a given cut-off radius are joined with simple Hookean spring (<xref ref-type="bibr" rid="bib28">Tirion, 1996</xref>; <xref ref-type="bibr" rid="bib22">Rodgers et al., 2013a</xref>). The spring constants were set to a constant value of 1 kcal mol<sup>−1</sup> Å<sup>−2</sup> with a cut-off radius of 8 Å. The presence of CO<sub>2</sub> molecules were represented in the ENM by the inclusion of an additional Hookean spring between residues K125 and R104 of each set of neighbouring monomers (<xref ref-type="bibr" rid="bib23">Rodgers et al., 2013b</xref>). The first six modes, that is the lowest frequency modes, represent the solid body translational and rotational motions of the protein and are thus ignored from the analysis.</p></sec></sec></body><back><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>LM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>ND, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>SAG, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>TLR, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>MJC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>DIR, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD4-data"><object-id pub-id-type="doi">10.7554/eLife.01213.018</object-id><label>Supplementary file 1.</label><caption><title>Conductance changes source data.</title><p>Raw values for whole cell conductance changes (nS) in response to an isohydric CO<sub>2</sub> challenge (PCO<sub>2</sub> 55 mmHg) in Cx31 and mCx31 expressing HeLa cells.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01213.018">http://dx.doi.org/10.7554/eLife.01213.018</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01213s004.xlsx"/></supplementary-material><sec sec-type="datasets"><title>Major dataset</title><p>The following previously published dataset was used:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Maeda</surname><given-names>S</given-names></name>, <name><surname>Nakagawa</surname><given-names>S</given-names></name>, <name><surname>Suga</surname><given-names>M</given-names></name>, <name><surname>Yamashita</surname><given-names>E</given-names></name>, <name><surname>Oshima</surname><given-names>A</given-names></name>, <name><surname>Fujiyoshi</surname><given-names>Y</given-names></name>, <name><surname>Tsukihara</surname><given-names>T</given-names></name>, <year>2009</year><x>, </x><source>Structure of the connexin 26 gap junction channel at 3.5 A resolution</source><x>, </x><object-id pub-id-type="art-access-id">2ZW3</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/explore/explore.do?structureId=2ZW3">http://www.rcsb.org/pdb/explore/explore.do?structureId=2ZW3</ext-link><x>, </x><comment>Publicly available at RCSB Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org</ext-link>).</comment></related-object></p></sec></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Boron</surname><given-names>WF</given-names></name><name><surname>Endeward</surname><given-names>V</given-names></name><name><surname>Gros</surname><given-names>G</given-names></name><name><surname>Musa-Aziz</surname><given-names>R</given-names></name><name><surname>Pohl</surname><given-names>P</given-names></name></person-group><year>2011</year><article-title>Intrinsic CO<sub>2</sub> permeability of cell membranes and potential biological relevance of CO<sub>2</sub> 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letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Aldrich</surname><given-names>Richard</given-names></name><role>Reviewing editor</role><aff><institution>The University of Texas at Austin</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “CO<sub>2</sub> directly modulates connexin 26 by formation of carbamate bridges between subunits” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 2 reviewers, along with a member of our Board of Reviewing Editors.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Michael Marletta (Senior editor); Richard Aldrich (Reviewing editor); Juan Saez (peer reviewer).</p><p>The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The observations in the manuscript by Meigh et al. are highly novel and represent a new way in which we can look at the body's ability to sense CO<sub>2</sub>. The involvement of connexin hemichannels in this process is particularly novel. The data presented to implicate direct carbamylation of the connexin protein (at K125 in Cx26) are compelling, given that CO<sub>2</sub> induced opening of hemichannels is eliminated using mutants at this site, and can be introduced into a non-CO<sub>2</sub> sensitive Cx31 by inserting the target sequence.</p><p>However, the authors conclude strongly that: (1) the carbamylation of Cx26 (and presumably the other CO<sub>2</sub> sensitive connexins) leads directly to opening of hemichannels, and that, (2) this is caused by formation of a salt bridge with R104. The data for both these claims is currently inadequate.</p><p>With respect to (1), the authors do show that the increased membrane conductance and dye uptake are dependent on Cx expression. Thus, Cx hemichannels are a likely candidate to explain the leak, but other channels like Pannexins, or more recently, the CALHM channels, could similarly cause this conductance and dye permeability increase. Ca<sup>2+</sup> block of this conductance is shown, but this is not very specific, and the authors do not show that it is reversible.</p><p>More definitive evidence that connexin hemichannels cause the leak is necessary. This should be easy to obtain by simply doing some single channel patch recordings, where Cx hemichannels are readily distinguished from Pnx1 channels. This would also allow the authors to determine if the changes in conductance really reflect enhanced open probability (as they conclude in the absence of evidence), or possibly as a result of enhanced trafficking or other changes. Different pharmacological blockers like La<sup>+++</sup> that are specific for connexins could also be used to distinguish connexin hemichannels from other candidates.</p><p>With respect to (2), this is based on structural models of Cx26, at regions that are not well resolved in the original diffraction pattern. It is supported by one R104A mutant that disrupts CO<sub>2</sub> gating. But mutants that ablate function are not very instructive, as this could be for many reasons. The authors could also test an R104K mutant that one might expect to retain at least partial function, if it is dependent on a salt bridge as proposed by the authors.</p><p>Carbamylation to control activity is well known. Rubisco is the best example but there are others such the beta lactamases as cited by the authors. This is mentioned in the Discussion. Carbamylation is the central theme of this paper and must be initially brought up in the Introduction.</p><p>In citing their past work where connexins 30 and 32 open in response to CO<sub>2</sub> with constant pH, that constant pH is extracellular. Admittedly the bulk evidence supports a direct role of pCO<sub>2</sub> and not a pH change with the increase in CO<sub>2</sub>, these experiments do not incisively rule out an intracellular pH effect.</p><p>In the opening of the discussion the authors state:</p><p>“Our analysis has demonstrated that CO<sub>2</sub> binds to the intracellular surface of Cx26 and must therefore diffuse through the membrane to reach this site.”</p><p>Direct binding was not demonstrated. This is the most serious weakness in the paper. The experiments as designed are excellent but the authors stop short of the most critical molecular detail.</p><p>The definitive proof of a carbamoylated connexin structure is no doubt what the authors would like and so would we. <sup>14</sup>C-CO<sub>2</sub> binding in WT and mutants should be clear. And easy. The authors need to show binding with <sup>14</sup>CO<sub>2</sub> and/or the unique NMR signal generated with <sup>13</sup>CO<sub>2</sub>.</p><p>Together, the electrophysiological and binding experiments would allow the authors to be more secure in their conclusions, which at this point are more speculative than presented in the manuscript.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01213.020</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The identity of the hemichannel underlying the conductance change and dye permeation – in particular reassurance that this is due to the misexpression of Cx26 rather than some other endogenous conductance (such as pannexin1 and calhm1). There was also a request for single channel recordings</italic>.</p><p>We published data to this effect in Huckstepp et al. 2010 J Physiol 588, 3901 and Huckstepp et al. 2010, J Physiol 588, 3921. In the first of these papers we demonstrated that CO<sub>2</sub>-dependent ATP release from the medulla was blocked by a number of agents active at connexin hemichannels (carbenoxolone at high concentration, proadifen, NPPB and Co<sup>2+</sup>), but not by agents selective for pannexin-1 (carbenoxolone at low concentration and probenecid). In the second of these papers we demonstrated that the whole cell conductance change in responses to changes in PCO<sub>2</sub> were accompanied an increase in current noise (indicative of increased channel gating) and also performed single channel recordings in isolated membrane patches from Cx26-epxressing HeLa cells in the inside-out and outside-out configurations. These recordings demonstrated that increasing and decreasing PCO<sub>2</sub> respectively increased and decreased single channel gating in both configurations of the isolated patches at a membrane potential of -40 mV (which would by itself rule out panx-1, as this hemichannel requires membrane depolarization to open).</p><p>However at the time we were unaware of the existence of calhm1, so did not specifically test whether this hemichannel could be involved. We therefore provide additional data, in a new supplement to <xref ref-type="fig" rid="fig1">Figure 1</xref>, to demonstrate the block of CO<sub>2</sub>-dependent dye loading by carbenoxolone (not active at calhm1, <xref ref-type="bibr" rid="bib27">Taruno et al. 2013</xref> Nature doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.1038/nature11906">10.1038/nature11906</ext-link>), and a corresponding lack of effect of probenecid (to repeat earlier findings ruling out Panx-1) and ruthenium red (active at calhm1). These data show that only carbenoxolone prevents the CO<sub>2</sub>-dependent dye loading of the HeLa cells and thus rule out these alternative possibilities. We have also made more complete reference to our earlier work in the initial section of the results to address the issues of hemichannel identity and single channel conductances.</p><p>We also thought it would be helpful to show in and additional supplement to <xref ref-type="fig" rid="fig1">Figure 1</xref> examples of the mCherry expression patterns for selected mutant and WT connexins.</p><p><italic>2) Direct binding of CO</italic><sub><italic>2</italic></sub> <italic>to the hemichannel has not been demonstrated, and the evidence supporting the bridge is weak</italic>.</p><p>It is true that we have not directly shown the binding of CO<sub>2</sub> to Cx26, and the demonstration of such would be highly desirable. Unfortunately we do not think that either alternative proposed by the reviewers is practicable owing to the large amount of protein required.</p><p><sup>14</sup>CO<sub>2</sub> binding typically requires 80 nmol of protein per single technical replicate (J Biol Chem [1979] 254, 5599-601) corresponding to just over 2 mg of Cx26 released from membranes (typically up to 10% of total expressed and purified Cx26 – J Cell Biol [1991] 115, 141-50). This corresponds to about 20 mg of protein for 3 biological replicates with 3 technical replicates each. The total protein required would be considerably more with method development. Given that HeLa cells express only low amounts of connexin protein, this approach is therefore unfeasible for Cx26 with the expression systems we have currently available to us. Similarly, large amounts of protein are required for <sup>13</sup>C-NMR (62.5 mg/ml protein BBRC [1983] 111, 544; 0.5 mM protein J Biol Chem (1976) 251, 477 and (1977) 252, 2234; 70 mg/ml protein Proc Natl Acad Sci [1979] 76, 673), even allowing for the increased sensitivity modern machines.</p><p>However, we have given further thought as to how we could provide further evidence to support both the concept of CO<sub>2</sub> binding and the concept of the carbamate bridge that the reviewers correctly point out is supported by only a single mutation. We reasoned that we could in effect “engineer in” the binding of CO<sub>2</sub> by replacing K125 with a glutamate residue. The carboxy side chain of glutamate has the potential to provide the same functionality as the carbamylated lysine and could thus form a salt bridge with R104. We would predict that such a channel (Cx26<sup>K125E</sup>) should be both constitutively open and no longer sensitive to CO<sub>2</sub>. We now present evidence that both these predictions are true and that Cx26<sup>125E</sup> can be regarded as an analogue of the open, carbamylated hemichannel.</p><p>The suggestion that we test the mutant R104K is a good one. However in our gain of function mutant, mCx31, the other end of the bridge is K104 and the mutation R104K in Cx26 would give relatively little further information as we already know that a lysine at this position can substitute for the arginine. As an alternative approach, it struck us that we could provide further support for the idea of the bridge between K125 and R104 simply by reversing the direction of the bridge. The mutation R104E provides the required carboxy functionality from residue 104 and it should, on the basis of our hypothesis, be able to form a salt bridge in the reverse direction with K125 and thus also give a constitutively open hemichannel. We now provide evidence that Cx26<sup>R104E</sup> is also constitutively open, and no longer sensitive to CO<sub>2</sub>.</p><p>We summarize these new data for Cx26<sup>K125E</sup> and Cx26<sup>R104E</sup> in <xref ref-type="fig" rid="fig4 fig5">Figures 4 and 5</xref>. We have amended the Discussion to the effect that we have not demonstrated direct binding of CO<sub>2</sub>, but that 6 experimental tests derived from the hypothesis of CO<sub>2</sub> binding (gain of function in mCx31, loss of function in mCx31<sup>K125R</sup>, loss of function in Cx26<sup>K125R</sup>, loss of function in Cx26<sup>R104A</sup>, gain of function in Cx26<sup>K125E</sup>, and gain of function in Cx26<sup>R104E</sup>) provide results that strongly support this hypothesis.</p><p><italic>3) Carbamylation is the central theme of this paper and must be initially brought up in the Introduction</italic>.</p><p>We now introduce the concept of carbamylation in the Introduction.</p><p><italic>4) Intracellular pH – a possible contributor</italic>?</p><p>Our previous work in the 2010 papers leaves very little room for intracellular pH changes as the causation of the hemichannel gating or CO<sub>2</sub>-dependent ATP release via Cx26. Perhaps the strongest evidence from this previous work is that isolated membrane patches in both inside out or outside out configuration exhibit changes in channel gating under conditions of constant bath pH (Huckstepp et al. 2010, J Physiol 588, 3921). Our new data, especially the observation that K125E and R104E mutants are both constitutively open and lack CO<sub>2</sub> sensitivity, makes the concept of pH changes underlying the CO<sub>2</sub>-dependent channel gating even less likely. We have opted not to add any discussion of this in the paper.</p></body></sub-article></article>