Permalink
Cannot retrieve contributors at this time
Fetching contributors…
Cannot retrieve contributors at this time
| <?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 xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.1d1"><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">04249</article-id><article-id pub-id-type="doi">10.7554/eLife.04249</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research advance</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Human biology and medicine</subject></subj-group></article-categories><title-group><article-title>Connexin26 hemichannels with a mutation that causes KID syndrome in humans lack sensitivity to CO<sub>2</sub></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">1</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17185"><name><surname>Hussain</surname><given-names>Naveed</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17186"><name><surname>Mulkey</surname><given-names>Daniel K</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con3"/><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">1</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><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"><label>2</label><institution content-type="dept">Division of Neonatal Pediatrics, Connecticut Children's Medical Center NICU</institution>, <institution>University of Connecticut Health Center</institution>, <addr-line><named-content content-type="city">Farmington</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><label>3</label><institution content-type="dept">Department of Physiology and Neurobiology</institution>, <institution>University of Connecticut</institution>, <addr-line><named-content content-type="city">Storrs</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Marletta</surname><given-names>Michael A</given-names></name><role>Reviewing editor</role><aff><institution>The Scripps Research Institute</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 publication-format="electronic" date-type="pub"><day>25</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e04249</elocation-id><history><date date-type="received"><day>05</day><month>08</month><year>2014</year></date><date date-type="accepted"><day>17</day><month>11</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014, Meigh et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Meigh et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">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="elife04249.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="article-reference" xlink:href="10.7554/eLife.01213"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04249.001</object-id><title>Abstract</title><p>Mutations in connexin26 (Cx26) underlie a range of serious human pathologies. Previously we have shown that Cx26 hemichannels are directly opened by CO<sub>2</sub> (<xref ref-type="bibr" rid="bib15">Meigh et al., 2013</xref>). However the effects of human disease-causing mutations on the CO<sub>2</sub> sensitivity of Cx26 are entirely unknown. Here, we report the first connection between the CO<sub>2</sub> sensitivity of Cx26 and human pathology, by demonstrating that Cx26 hemichannels with the mutation A88V, linked to Keratitis-Ichthyosis-Deafness syndrome, are both CO<sub>2</sub> insensitive and associated with disordered breathing in humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04249.001">http://dx.doi.org/10.7554/eLife.04249.001</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>respiratory apnea</kwd><kwd>CO<sub>2</sub> chemosensing</kwd><kwd>gap junctions</kwd><kwd>hemichannels</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>human</kwd><kwd>rat</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000265</institution-id><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><funding-statement>The funder 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>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Building on previous work (Meigh et al., 2013) we show that A88V, a mutation in connexin26 (Cx26) that causes Keratitis-Ichthyosis-Deafness (KID) syndrome in humans, is linked to a lack of sensitivity to CO<sub>2</sub> by Cx26 hemichannels.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p>Connexin26 (Cx26) is one of 21 connexin genes found in humans (<xref ref-type="bibr" rid="bib4">Cruciani and Mikalsen, 2006</xref>). The canonical function of connexins is to form gap junctions in which two hexameric connexons, or hemichannels, in closely apposed membranes dock together to form an intercellular channel. However connexins can also function as hemichannels, thereby providing large conductance channels, which allow passage of small molecules such as ATP into the extracellular space (<xref ref-type="bibr" rid="bib19">Stout et al., 2004</xref>; <xref ref-type="bibr" rid="bib20">Wang et al., 2013</xref>). We have recently shown that Cx26 hemichannels are directly sensitive to CO<sub>2</sub> (<xref ref-type="bibr" rid="bib9">Huckstepp et al., 2010a</xref>; <xref ref-type="bibr" rid="bib15">Meigh et al., 2013</xref>). When CO<sub>2</sub> binds to Cx26, it carbamylates K125, forms a salt bridge to R104 and opens the hemichannel (<xref ref-type="bibr" rid="bib15">Meigh et al., 2013</xref>). Cx26 hemichannels are thus a source of CO<sub>2</sub>-gated ATP release (<xref ref-type="bibr" rid="bib9">Huckstepp et al., 2010a</xref>).</p><p>Mutations of Cx26 are the commonest cause of non-syndromic hearing loss (<xref ref-type="bibr" rid="bib3">Cohn and Kelley, 1999</xref>; <xref ref-type="bibr" rid="bib13">Kelley et al., 2000</xref>; <xref ref-type="bibr" rid="bib22">Xu and Nicholson, 2013</xref>). Some of these mutations cause loss of functional protein, while other mutations result in gap junctions and hemichannels with altered properties. However the effect of these mutations on the CO<sub>2</sub> sensitivity of Cx26 has never been examined. Some missense mutations of Cx26 cause serious pathologies in humans, such as the very rare ectodermal disorder, Keratitis-Ichthyosis-Deafness (KID) syndrome. KID syndrome involves a combination of deafness, visual impairment, and dermatological abnormalities (<xref ref-type="bibr" rid="bib2">Caceres-Rios et al., 1996</xref>). About 100 cases have been reported in the literature, and of these around 70% are caused by de novo mutations in Cx26, with the remainder being inherited in an autosomal dominant manner or via germ line mosaicism (<xref ref-type="bibr" rid="bib18">Sbidian et al., 2010</xref>). To date there are nine missense mutations that can cause KID syndrome (<xref ref-type="bibr" rid="bib22">Xu and Nicholson, 2013</xref>). The severity of the symptoms of KID syndrome depends on the particular mutation in Cx26 (<xref ref-type="bibr" rid="bib11">Janecke et al., 2005</xref>; <xref ref-type="bibr" rid="bib12">Jonard et al., 2008</xref>).</p><p>The mutation, Cx26<sup>A88V</sup>, is linked to a very severe form of KID syndrome, which is fatal in infancy (<xref ref-type="bibr" rid="bib8">Haruna et al., 2010</xref>; <xref ref-type="bibr" rid="bib14">Koppelhus et al., 2010</xref>). In one of the original reports linking Cx26<sup>A88V</sup> to KID syndrome, the patient required mechanical ventilation (<xref ref-type="bibr" rid="bib14">Koppelhus et al., 2010</xref>), suggesting a possible effect of the mutation on the neural control of breathing. In KID syndrome caused by a different missense mutation (G45E), which is fatal within the first year of life, there are also reports of breathing problems. One patient required mechanical ventilation immediately after birth (<xref ref-type="bibr" rid="bib11">Janecke et al., 2005</xref>) and a second died from breathing failure (<xref ref-type="bibr" rid="bib18">Sbidian et al., 2010</xref>). Nevertheless, without detailed recordings of cardiorespiratory activity, it is not possible to know whether these patients experienced inadequate central respiratory drive. For other mutations linked to KID syndrome there are no reports of abnormal breathing in the literature.</p><p>The reason why the A88V and G45E mutations should cause such pervasive and severe pathology remains unclear as subunits of Cx26<sup>A88V</sup> and Cx26<sup>G45E</sup> form both functional gap junctions and hemichannels (<xref ref-type="bibr" rid="bib7">Gerido et al., 2007</xref>; <xref ref-type="bibr" rid="bib16">Mhaske et al., 2013</xref>). Expression of Cx26<sup>A88V</sup> in HeLa cells gives rise to enhanced hemichannel-mediated currents (compared to wild type Cx26, Cx26<sup>WT</sup>) at positive transmembrane potentials and in the absence of extracellular Ca<sup>2+</sup>, leading to the suggestion that this mutation represents a gain of function (<xref ref-type="bibr" rid="bib16">Mhaske et al., 2013</xref>). The G45E mutation, also causes enhanced hemichannel activity in the absence of extracellular Ca<sup>2+</sup>, and increased permeability to Ca<sup>2+</sup> (<xref ref-type="bibr" rid="bib7">Gerido et al., 2007</xref>; <xref ref-type="bibr" rid="bib17">Sanchez et al., 2010</xref>). A gain of function has therefore been suggested as underlying the actions of this mutation too. Although the absence of extracellular Ca<sup>2+</sup> opens connexin hemichannels, this condition is unlikely to occur in physiological systems. Thus the consequences of the A88V and G45E mutations on physiologically relevant gating of Cx26 remain unclear.</p><p>We identified a patient with KID syndrome caused by a heterozygous Cx26 A88V mutation. This patient failed to breathe spontaneously at birth and initially required mechanical ventilation. Later when he started to breathe spontaneously, he continued to demonstrate periods of apnea and bradycardia. A pneumogram performed at a post-menstrual age of 40 weeks showed abnormal persistence of central apnea lasting ≥20 s and accompanied by periods of bradycardia and prolonged oxygen desaturation (<xref ref-type="fig" rid="fig1">Figure 1</xref>). This respiratory pattern is abnormal for the age of the infant and is suggestive of blunted chemosensory control of breathing. Given the previously described role of Cx26 in mediating the CO<sub>2</sub>-dependent drive to breathe (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010b</xref>; <xref ref-type="bibr" rid="bib21">Wenker et al., 2012</xref>), we considered whether the mutation A88V might alter the CO<sub>2</sub>-sensitivity of Cx26.<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04249.002</object-id><label>Figure 1.</label><caption><title>Incidence of central sleep apnea in a patient with Cx26<sup>A88V</sup>.</title><p>Recording of cardiorespiratory activity during sleep from an infant at a post-menstrual age of 40 weeks diagnosed with KID syndrome. Traces of nasal air flow, thoracic movement, electrocardiogram (ECG), heart rate (HR) and arterial O<sub>2</sub> saturation show that this patient exhibited a prolonged period during which no effort was made to breathe and this was followed by pronounced bradycardia and arterial O<sub>2</sub> desaturation, all of which are characteristic of central sleep apnea. Unfortunately, at 2 months of age this patient died from overwhelming sepsis.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04249.002">http://dx.doi.org/10.7554/eLife.04249.002</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04249f001"/></fig></p><p>We introduced the A88V mutation into Cx26 and then tested the CO<sub>2</sub> sensitivity of Cx26<sup>A88V</sup> hemichannels expressed in HeLa cells via an established and sensitive dye-loading protocol (<xref ref-type="bibr" rid="bib9">Huckstepp et al., 2010a</xref>; <xref ref-type="bibr" rid="bib15">Meigh et al., 2013</xref>). Under conditions of normal extracellular Ca<sup>2+</sup>, HeLa cells expressing wild type Cx26 hemichannels readily load with carboxyfluorescein when exposed to a moderately hypercapnic saline (PCO<sub>2</sub> 55 mmHg) (<xref ref-type="bibr" rid="bib9">Huckstepp et al., 2010a</xref>; <xref ref-type="bibr" rid="bib15">Meigh et al., 2013</xref>). However HeLa cells expressing Cx26<sup>A88V</sup> showed no such CO<sub>2</sub>-dependent dye loading even when exposed to higher levels of PCO<sub>2</sub> (70 mmHg, <xref ref-type="fig" rid="fig2">Figure 2</xref>). The failure to exhibit CO<sub>2</sub>-dependent dye loading was not due to a lack of functional hemichannels as the positive control of removing extracellular Ca<sup>2+</sup>, which opens all connexin hemichannels, caused robust dye loading (<xref ref-type="fig" rid="fig2">Figure 2</xref>). HeLa cells transfected with an empty vector do not show any dye loading in response to a CO<sub>2</sub> stimulus or removal of extracellular Ca<sup>2+</sup> (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Surprisingly therefore, the conservative mutation A88V caused Cx26 hemichannels to lose their sensitivity to CO<sub>2</sub>. As this mutation is far from the residues involved in CO<sub>2</sub> binding (K125 and R104), the mechanism for the loss of CO<sub>2</sub> sensitivity is unclear.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04249.004</object-id><label>Figure 2.</label><caption><title>Cx26<sup>A88V</sup> hemichannels are no longer sensitive to CO<sub>2</sub>.</title><p>(Top) Images of HeLa cells expressing Cx26<sup>A88V</sup> under control, hypercapnic and zero Ca<sup>2+</sup> conditions. The cells were exposed to 200 µM carboxyfluorescein (CBF) for 5 min under each condition before being washed. Some low background loading of CBF is seen under control conditions. In presence of CO<sub>2</sub> no loading is seen. The positive control of removal of extracellular Ca<sup>2+</sup> causes robust dye loading demonstrating the presence of functional hemichannels. (Bottom) Cumulative probability distributions of pixel intensity of HeLa cells expressing Cx26<sup>A88V</sup> under control, hypercapnia (two levels of PCO<sub>2</sub>) and zero Ca<sup>2+</sup>. Only the removal of extracellular Ca<sup>2+</sup> causes dye loading as shown by the rightward shift of the curve to higher pixel intensities (p = 0.004, Mann Whitney U test compared to control). These distributions show all of the measurements made (minimum 40 cells each from five independent repetitions).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04249.004">http://dx.doi.org/10.7554/eLife.04249.004</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04249f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04249.003</object-id><label>Figure 2—figure supplement 1.</label><caption><title>HeLa cells transfected with the empty pCAG-GS mCherry vector show no sensitivity to CO<sub>2</sub> and do not dye load when exposed to zero Ca<sup>2+</sup> aCSF.</title><p>(<bold>A</bold>) Cumulative probability distributions of pixel intensity for HeLa cells transfected with pCAG-GS mCherry under control, hypercapnia and zero Ca<sup>2+</sup> conditions. The cells were exposed to 200 µM CBF for 5 min under each condition before being washed. The graphs show all of the measurements from 4 independent repetitions for each condition. (<bold>B</bold>) When transfected with pCAG-GS mCherry, the HeLa cells exhibit diffuse red fluorescence from expression of the mCherry. This contrasts with the punctate fluorescence seen flowing transfection with pCAG-GS Cx26-mCherry (inset). Scale bars 20 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04249.003">http://dx.doi.org/10.7554/eLife.04249.003</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04249fs001"/></fig></fig-group></p><p>As the missense mutations which underlie KID syndrome act in a dominant manner (<xref ref-type="bibr" rid="bib12">Jonard et al., 2008</xref>; <xref ref-type="bibr" rid="bib22">Xu and Nicholson, 2013</xref>), we tested whether the expression of Cx26<sup>A88V</sup> subunits might have a dominant negative action on the CO<sub>2</sub> sensitivity of Cx26<sup>WT</sup>. We transfected HeLa cells that stably expressed Cx26<sup>WT</sup> with the Cx26<sup>A88V</sup> subunit and documented their sensitivity to CO<sub>2</sub> following transfection. 4 days after transfection with Cx26<sup>A88V</sup>, the HeLa cells still exhibited sensitivity to CO<sub>2</sub> (<xref ref-type="fig" rid="fig3">Figure 3A</xref>), but this was reduced compared to the Cx26<sup>WT</sup> HeLa cells that had not been transfected with Cx26<sup>A88V</sup> (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). 5 and 6 days after transfection, the HeLa cells showed no sensitivity to CO<sub>2</sub> (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Nevertheless functional hemichannels were still present as the removal of extracellular Ca<sup>2+</sup> caused dye loading (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). The loss of CO<sub>2</sub> sensitivity was not simply a consequence of days in culture, as Cx26<sup>WT</sup> HeLa cells that had not been transfected with Cx26<sup>A88V</sup> retained their sensitivity to CO<sub>2</sub> over the whole period examined (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). We therefore conclude that Cx26<sup>A88V</sup> subunits are able to act in a dominant negative manner to cause loss of CO<sub>2</sub> sensitivity from wild type Cx26 hemichannels.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04249.005</object-id><label>Figure 3.</label><caption><title>Cx26<sup>A88V</sup> hemichannels act in a dominant negative manner to remove CO<sub>2</sub> sensitivity from Cx26<sup>WT</sup>.</title><p>(<bold>A</bold>) Cumulative probability distributions for CO<sub>2</sub>-dependent dye loading in HeLa cells that stably express Cx26<sup>WT</sup>, which have been transfected with Cx26<sup>A88V</sup>. 4 days after transfection with Cx26<sup>A88V</sup> the cells still exhibit significant sensitivity to 55 mmHg PCO<sub>2</sub> stimulus (p = 0.048 CO<sub>2</sub> compared to control, Mann Whitney U test). 5 and 6 days after transfection the CO<sub>2</sub> sensitivity of the HeLa cells was abolished. On all 3 days, the positive control of zero Ca<sup>2+</sup> caused dye loading, demonstrating the presence of functional hemichannels. The graphs show all of the measurements made from 5 independent repetitions of the experiment. (<bold>B</bold>) Comparison of the sensitivity to CO<sub>2</sub> of HeLa cells stably expressing Cx26<sup>WT</sup> which have been transfected with Cx26<sup>A88V</sup> (Cx26<sup>WT</sup> + Cx26<sup>A88V</sup>, n = 5) with those that have not (Cx26<sup>WT</sup>, n = 7). In the absence of transfection, the Cx26<sup>WT</sup>-expresssing HeLa cells retain sensitivity to CO<sub>2</sub> on all 3 days. By contrast Cx26<sup>A88V</sup> causes significantly depressed CO<sub>2</sub> sensitivity 4 days after transfection (p = 0.001), and loss of sensitivity on days 5 (p = 0.024) and 6 (p = 0.001). Comparisons of Cx26<sup>WT</sup> with Cx26<sup>WT</sup> + Cx26<sup>A88V</sup> via Mann Whitney U test, and False Discovery Rate procedure for multiple comparisons (<xref ref-type="bibr" rid="bib5">Curran-Everett, 2000</xref>). Error bars upper and lower quartiles.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04249.005">http://dx.doi.org/10.7554/eLife.04249.005</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife04249f003"/></fig></p><p>This is the first instance in which a mutation linked to serious human pathologies has been demonstrated to abolish the CO<sub>2</sub> sensitivity of Cx26. This in turn suggests that Cx26-mediated CO<sub>2</sub> sensing may be important for human physiology in the range of contexts that are associated with the diverse pathologies linked to this mutation. In the closely related β connexin, connexin30 (Cx30), the mutation A88V connected to Clouston's Syndrome (<xref ref-type="bibr" rid="bib1">Bosen et al., 2014</xref>), may result in constitutively open Cx30 hemichannels (<xref ref-type="bibr" rid="bib6">Essenfelder et al., 2004</xref>). However Cx30 is also opened by CO<sub>2</sub> (<xref ref-type="bibr" rid="bib9">Huckstepp et al., 2010a</xref>) and the effect of this mutation on the CO<sub>2</sub> sensitivity of Cx30 has not yet been investigated. There are no reports in the literature of disordered breathing in patients with Clouston's syndrome.</p><p>Previous studies suggesting that the A88V mutation gave a gain of function in Cx26, examined hemichannel function in the absence of extracellular Ca<sup>2+</sup> (<xref ref-type="bibr" rid="bib16">Mhaske et al., 2013</xref>). As the CO<sub>2</sub> sensitivity of the mutated hemichannel was not specifically examined in this previous study, it is likely that both sets of findings are correct—an enhancement of macroscopic hemichannel currents (<xref ref-type="bibr" rid="bib16">Mhaske et al., 2013</xref>), and a loss of CO<sub>2</sub> sensitivity. However under physiological conditions of normal extracellular Ca<sup>2+</sup> and in the presence of physiological CO<sub>2</sub>/HCO<sub>3</sub><sup>−</sup> buffering, we suggest that A88V should be considered as a loss-of-function mutation that effectively removes the capacity for CO<sub>2</sub>-evoked ATP release via Cx26 hemichannels.</p><p>Our report is the first to document altered central respiratory drive in a KID syndrome patient. In rodents, CO<sub>2</sub>-sensitivity of Cx26 contributes to the chemosensory control of breathing (<xref ref-type="bibr" rid="bib10">Huckstepp et al., 2010b</xref>; <xref ref-type="bibr" rid="bib21">Wenker et al., 2012</xref>). Although we do not know if the loss of CO<sub>2</sub> sensitivity in Cx26 contributed to the aberrant respiratory drive exhibited by this patient, these results are consistent with this possibility, and represent the first evidence to suggest that Cx26 hemichannels are a requisite component of the drive to breathe in humans. Overall the ability of physiological levels of PCO<sub>2</sub> to permit ATP release via Cx26 hemichannels may be important in the epidermis, cochlea and brain. Investigation of whether the absence of this mechanism of ATP release in patients with Cx26<sup>A88V</sup> contributes to the serious pathological abnormalities that they suffer would seem to be warranted.</p><sec sec-type="materials|methods" id="s1"><title>Materials and methods</title><sec id="s1-1"><title>Case study</title><p>The Institutional Review Board of the Connecticut Children's Medical Center considered this under the category of a case report and thus exempt from formal review.</p></sec><sec id="s1-2"><title>Mutant connexin production</title><p>Puc19 Cx26<sup>A88V</sup> was produced from wild type Cx26 via the Quikchange protocol using the following primers: forward 5′ TGT CCA CGC CGG TCC TCC TGG TAG C 3′ reverse 5′ GCT ACC AGG AGG ACC GGC GTG GAC A 3′. Cx26<sup>A88V</sup> was subcloned into a pCAG-GS mCherry vector for mammalian cell transfection. Successful mutation of Cx26 was confirmed by sequencing which also verified that apart from the desired mutation the sequence was identical to the wild type.</p></sec><sec id="s1-3"><title>HeLa cell culture</title><p>HeLa cells were cultured by standard methods in DMEM, 10% FCS with addition of 3 mM CaCl<sub>2</sub>. For experimentation, cells were plated onto coverslips at a density of 5 × 10<sup>4</sup> cells per well. Transient transfections were performed using the genejuice protocol.</p></sec><sec id="s1-4"><title>Solutions used</title><sec id="s1-4-1"><title>Control aCSF</title><p>124 mM NaCl, 26 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 3 mM KCl, 10 mM D-glucose, 1 mM MgSO<sub>4</sub>, 1 mM CaCl<sub>2</sub>.</p></sec><sec id="s1-4-2"><title>Zero Ca<sup>2+</sup> aCSF</title><p>124 mM NaCl, 26 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 3 mM KCl, 10 mM D-glucose, 1 mM MgSO<sub>4</sub>, 1 mM MgCl<sub>2</sub>, 1 mM EGTA.</p></sec><sec id="s1-4-3"><title>Hypercapnic (55 mmHg CO<sub>2</sub>) aCSF</title><p>100 mM NaCl, 50 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 3 mM KCl, 10 mM D-glucose, 1 mM MgSO<sub>4</sub>, 1 mM CaCl<sub>2</sub>.</p></sec><sec id="s1-4-4"><title>Hypercapnic (70 mmHg CO<sub>2</sub>) aCSF</title><p>70 mM NaCl, 80 mM NaHCO<sub>3</sub>, 1.25 mM NaH<sub>2</sub>PO<sub>4</sub>, 3 mM KCl, 10 mM D-glucose, 1 mM MgSO<sub>4</sub>, 1 mM CaCl<sub>2</sub>.</p><p>Hypercapnic aCSF was saturated with sufficient CO<sub>2</sub> (the remaining balance being O<sub>2</sub>) to adjust the final pH (pH 7.5) to that of the control aCSF removing any potential effects of changes in extracellular pH.</p><p>All other solutions were saturated with 95% O<sub>2</sub>/5% CO<sub>2</sub>.</p></sec></sec><sec id="s1-5"><title>Dye loading protocols</title><p>Coverslips plated with HeLa cells transiently transfected with Cx26<sup>A88V</sup> were exposed to Hypercapnic aCSF (55 mmHg or 70 mmHg) containing 200 µM CBF for 10 min. This was followed by control aCSF with 200 µM CBF for 5 min and a 30 min wash with control aCSF to ensure that all dye is removed from the outside of the cells.</p><p>A control comparison was used to establish any baseline loading occurring in the absence of a stimulus. HeLa cells expressing Cx26<sup>A88V</sup> were exposed to 200 µM CBF in control aCSF for 15 min, followed by 30 min of washing.</p><p>A zero Ca<sup>2+</sup> positive control was also performed to ensure functional connexin hemichannels were being expressed. Cx26<sup>A88V</sup> expressing HeLa cells were exposed to 200 µM CBF in zero Ca<sup>2+</sup> aCSF for 10 min. This was followed by control aCSF with 200 µM CBF for 5 min and 30 min of washing with aCSF.</p></sec><sec id="s1-6"><title>Imaging and analysis</title><p>For each condition cells were imaged by epifluorescence (Scientifica Slice Scope, Cairn Research OptoLED illumination, 60× water Olympus immersion objective, NA 1.0, Hamamatsu ImageEM EMCCD camera, Metafluor software). 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. The analysis of the CO<sub>2</sub> sensitivity of Cx26<sup>A88V</sup> was performed as five independent repetitions in which at least 40 cells were measured in each condition, and the mean pixel intensities plotted as cumulative probability distributions.</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>NH, 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>DKM, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>ND, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Human subjects: The Institutional Review Board of the Connecticut Children's Medical Center considered this under the category of a case report and thus exempt from formal review.</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bosen</surname><given-names>F</given-names></name><name><surname>Schutz</surname><given-names>M</given-names></name><name><surname>Beinhauer</surname><given-names>A</given-names></name><name><surname>Strenzke</surname><given-names>N</given-names></name><name><surname>Franz</surname><given-names>T</given-names></name><name><surname>Willecke</surname><given-names>K</given-names></name></person-group><year>2014</year><article-title>The Clouston syndrome mutation connexin30 A88V leads to hyperproliferation of sebaceous glands and hearing impairments in mice</article-title><source>FEBS Letters</source><volume>588</volume><fpage>1795</fpage><lpage>1801</lpage><pub-id pub-id-type="doi">10.1016/j.febslet.2014.03.040</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Caceres-Rios</surname><given-names>H</given-names></name><name><surname>Tamayo-Sanchez</surname><given-names>L</given-names></name><name><surname>Duran-Mckinster</surname><given-names>C</given-names></name><name><surname>de la Luz Orozco</surname><given-names>M</given-names></name><name><surname>Ruiz-Maldonado</surname><given-names>R</given-names></name></person-group><year>1996</year><article-title>Keratitis, ichthyosis, and deafness (KID syndrome): review of the literature and proposal of a new terminology</article-title><source>Pediatric Dermatology</source><volume>13</volume><fpage>105</fpage><lpage>113</lpage><pub-id pub-id-type="doi">10.1111/j.1525-1470.1996.tb01414.x</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cohn</surname><given-names>ES</given-names></name><name><surname>Kelley</surname><given-names>PM</given-names></name></person-group><year>1999</year><article-title>Clinical phenotype and mutations in connexin 26 (DFNB1/GJB2), the most common cause of childhood hearing loss</article-title><source>American Journal of Medical Genetics</source><volume>89</volume><fpage>130</fpage><lpage>136</lpage><pub-id pub-id-type="doi">10.1002/(SICI)1096-8628(19990924)89:3<130::AID-AJMG3>3.0.CO;2-M</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cruciani</surname><given-names>V</given-names></name><name><surname>Mikalsen</surname><given-names>SO</given-names></name></person-group><year>2006</year><article-title>The vertebrate connexin family</article-title><source>Cellular and Molecular Life Sciences</source><volume>63</volume><fpage>1125</fpage><lpage>1140</lpage><pub-id pub-id-type="doi">10.1007/s00018-005-5571-8</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Curran-Everett</surname><given-names>D</given-names></name></person-group><year>2000</year><article-title>Multiple comparisons: philosophies and illustrations</article-title><source>American Journal of Physiology. Regulatory, Integrative and Comparative Physiology</source><volume>279</volume><fpage>R1</fpage><lpage>R8</lpage></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Essenfelder</surname><given-names>GM</given-names></name><name><surname>Bruzzone</surname><given-names>R</given-names></name><name><surname>Lamartine</surname><given-names>J</given-names></name><name><surname>Charollais</surname><given-names>A</given-names></name><name><surname>Blanchet-Bardon</surname><given-names>C</given-names></name><name><surname>Barbe</surname><given-names>MT</given-names></name><name><surname>Meda</surname><given-names>P</given-names></name><name><surname>Waksman</surname><given-names>G</given-names></name></person-group><year>2004</year><article-title>Connexin30 mutations responsible for hidrotic ectodermal dysplasia cause abnormal hemichannel activity</article-title><source>Human Molecular Genetics</source><volume>13</volume><fpage>1703</fpage><lpage>1714</lpage><pub-id pub-id-type="doi">10.1093/hmg/ddh191</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gerido</surname><given-names>DA</given-names></name><name><surname>DeRosa</surname><given-names>AM</given-names></name><name><surname>Richard</surname><given-names>G</given-names></name><name><surname>White</surname><given-names>TW</given-names></name></person-group><year>2007</year><article-title>Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness</article-title><source>American Journal of Physiology. Cell Physiology</source><volume>293</volume><fpage>C337</fpage><lpage>C345</lpage><pub-id pub-id-type="doi">10.1152/ajpcell.00626.2006</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Haruna</surname><given-names>K</given-names></name><name><surname>Suga</surname><given-names>Y</given-names></name><name><surname>Oizumi</surname><given-names>A</given-names></name><name><surname>Mizuno</surname><given-names>Y</given-names></name><name><surname>Endo</surname><given-names>H</given-names></name><name><surname>Shimizu</surname><given-names>T</given-names></name><name><surname>Hasegawa</surname><given-names>T</given-names></name><name><surname>Ikeda</surname><given-names>S</given-names></name></person-group><year>2010</year><article-title>Severe form of keratitis-ichthyosis-deafness (KID) syndrome associated with septic complications</article-title><source>The Journal of Dermatology</source><volume>37</volume><fpage>680</fpage><lpage>682</lpage><pub-id pub-id-type="doi">10.1111/j.1346-8138.2010.00839.x</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huckstepp</surname><given-names>RT</given-names></name><name><surname>Eason</surname><given-names>R</given-names></name><name><surname>Sachdev</surname><given-names>A</given-names></name><name><surname>Dale</surname><given-names>N</given-names></name></person-group><year>2010a</year><article-title>CO<sub>2</sub>-dependent opening of connexin 26 and related beta connexins</article-title><source>The Journal of Physiology</source><volume>588</volume><fpage>3921</fpage><lpage>3931</lpage><pub-id pub-id-type="doi">10.1113/jphysiol.2010.192096</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huckstepp</surname><given-names>RT</given-names></name><name><surname>Id Bihi</surname><given-names>R</given-names></name><name><surname>Eason</surname><given-names>R</given-names></name><name><surname>Spyer</surname><given-names>KM</given-names></name><name><surname>Dicke</surname><given-names>N</given-names></name><name><surname>Willecke</surname><given-names>K</given-names></name><name><surname>Marina</surname><given-names>N</given-names></name><name><surname>Gourine</surname><given-names>AV</given-names></name><name><surname>Dale</surname><given-names>N</given-names></name></person-group><year>2010b</year><article-title>Connexin hemichannel-mediated CO<sub>2</sub>-dependent release of ATP in the medulla oblongata contributes to central respiratory chemosensitivity</article-title><source>The Journal of Physiology</source><volume>588</volume><fpage>3901</fpage><lpage>3920</lpage><pub-id pub-id-type="doi">10.1113/jphysiol.2010.192088</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Janecke</surname><given-names>AR</given-names></name><name><surname>Hennies</surname><given-names>HC</given-names></name><name><surname>Gunther</surname><given-names>B</given-names></name><name><surname>Gansl</surname><given-names>G</given-names></name><name><surname>Smolle</surname><given-names>J</given-names></name><name><surname>Messmer</surname><given-names>EM</given-names></name><name><surname>Utermann</surname><given-names>G</given-names></name><name><surname>Rittinger</surname><given-names>O</given-names></name></person-group><year>2005</year><article-title>GJB2 mutations in keratitis-ichthyosis-deafness syndrome including its fatal form</article-title><source>American Journal of Medical Genetics. Part A</source><volume>133A</volume><fpage>128</fpage><lpage>131</lpage><pub-id pub-id-type="doi">10.1002/ajmg.a.30515</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jonard</surname><given-names>L</given-names></name><name><surname>Feldmann</surname><given-names>D</given-names></name><name><surname>Parsy</surname><given-names>C</given-names></name><name><surname>Freitag</surname><given-names>S</given-names></name><name><surname>Sinico</surname><given-names>M</given-names></name><name><surname>Koval</surname><given-names>C</given-names></name><name><surname>Grati</surname><given-names>M</given-names></name><name><surname>Couderc</surname><given-names>R</given-names></name><name><surname>Denoyelle</surname><given-names>F</given-names></name><name><surname>Bodemer</surname><given-names>C</given-names></name><name><surname>Marlin</surname><given-names>S</given-names></name><name><surname>Hadj-Rabia</surname><given-names>S</given-names></name></person-group><year>2008</year><article-title>A familial case of Keratitis-Ichthyosis-Deafness (KID) syndrome with the GJB2 mutation G45E</article-title><source>European Journal of Medical Genetics</source><volume>51</volume><fpage>35</fpage><lpage>43</lpage><pub-id pub-id-type="doi">10.1016/j.ejmg.2007.09.005</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kelley</surname><given-names>PM</given-names></name><name><surname>Cohn</surname><given-names>E</given-names></name><name><surname>Kimberling</surname><given-names>WJ</given-names></name></person-group><year>2000</year><article-title>Connexin 26: required for normal auditory function</article-title><source>Brain Research. Brain Research Reviews</source><volume>32</volume><fpage>184</fpage><lpage>188</lpage><pub-id pub-id-type="doi">10.1016/S0165-0173(99)00080-6</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Koppelhus</surname><given-names>U</given-names></name><name><surname>Tranebjaerg</surname><given-names>L</given-names></name><name><surname>Esberg</surname><given-names>G</given-names></name><name><surname>Ramsing</surname><given-names>M</given-names></name><name><surname>Lodahl</surname><given-names>M</given-names></name><name><surname>Rendtorff</surname><given-names>ND</given-names></name><name><surname>Olesen</surname><given-names>HV</given-names></name><name><surname>Sommerlund</surname><given-names>M</given-names></name></person-group><year>2010</year><article-title>A novel mutation in the connexin 26 gene (GJB2) in a child with clinical and histological features of keratitis-ichthyosis-deafness (KID) syndrome</article-title><source>Clinical and Experimental Dermatology</source><volume>36</volume><fpage>142</fpage><lpage>148</lpage><pub-id pub-id-type="doi">10.1111/j.1365-2230.2010.03936.x</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Meigh</surname><given-names>L</given-names></name><name><surname>Greenhalgh</surname><given-names>SA</given-names></name><name><surname>Rodgers</surname><given-names>TL</given-names></name><name><surname>Cann</surname><given-names>MJ</given-names></name><name><surname>Roper</surname><given-names>DI</given-names></name><name><surname>Dale</surname><given-names>N</given-names></name></person-group><year>2013</year><article-title>CO<sub>2</sub> directly modulates connexin 26 by formation of carbamate bridges between subunits</article-title><source>eLife</source><volume>2</volume><fpage>e01213</fpage><pub-id pub-id-type="doi">10.7554/eLife.01213</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mhaske</surname><given-names>PV</given-names></name><name><surname>Levit</surname><given-names>NA</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>HZ</given-names></name><name><surname>Lee</surname><given-names>JR</given-names></name><name><surname>Shuja</surname><given-names>Z</given-names></name><name><surname>Brink</surname><given-names>PR</given-names></name><name><surname>White</surname><given-names>TW</given-names></name></person-group><year>2013</year><article-title>The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity</article-title><source>American Journal of Physiology. Cell Physiology</source><volume>304</volume><fpage>C1150</fpage><lpage>C1158</lpage><pub-id pub-id-type="doi">10.1152/ajpcell.00374.2012</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sanchez</surname><given-names>HA</given-names></name><name><surname>Mese</surname><given-names>G</given-names></name><name><surname>Srinivas</surname><given-names>M</given-names></name><name><surname>White</surname><given-names>TW</given-names></name><name><surname>Verselis</surname><given-names>VK</given-names></name></person-group><year>2010</year><article-title>Differentially altered Ca<sup>2+</sup> regulation and Ca<sup>2+</sup> permeability in Cx26 hemichannels formed by the A40V and G45E mutations that cause keratitis ichthyosis deafness syndrome</article-title><source>The Journal of General Physiology</source><volume>136</volume><fpage>47</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1085/jgp.201010433</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sbidian</surname><given-names>E</given-names></name><name><surname>Feldmann</surname><given-names>D</given-names></name><name><surname>Bengoa</surname><given-names>J</given-names></name><name><surname>Fraitag</surname><given-names>S</given-names></name><name><surname>Abadie</surname><given-names>V</given-names></name><name><surname>de Prost</surname><given-names>Y</given-names></name><name><surname>Bodemer</surname><given-names>C</given-names></name><name><surname>Hadj-Rabia</surname><given-names>S</given-names></name></person-group><year>2010</year><article-title>Germline mosaicism in keratitis-ichthyosis-deafness syndrome: pre-natal diagnosis in a familial lethal form</article-title><source>Clinical Genetics</source><volume>77</volume><fpage>587</fpage><lpage>592</lpage><pub-id pub-id-type="doi">10.1111/j.1399-0004.2009.01339.x</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stout</surname><given-names>C</given-names></name><name><surname>Goodenough</surname><given-names>DA</given-names></name><name><surname>Paul</surname><given-names>DL</given-names></name></person-group><year>2004</year><article-title>Connexins: functions without junctions</article-title><source>Current Opinion in Cell Biology</source><volume>16</volume><fpage>507</fpage><lpage>512</lpage><pub-id pub-id-type="doi">10.1016/j.ceb.2004.07.014</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>N</given-names></name><name><surname>De Bock</surname><given-names>M</given-names></name><name><surname>Decrock</surname><given-names>E</given-names></name><name><surname>Bol</surname><given-names>M</given-names></name><name><surname>Gadicherla</surname><given-names>A</given-names></name><name><surname>Vinken</surname><given-names>M</given-names></name><name><surname>Rogiers</surname><given-names>V</given-names></name><name><surname>Bukauskas</surname><given-names>FF</given-names></name><name><surname>Bultynck</surname><given-names>G</given-names></name><name><surname>Leybaert</surname><given-names>L</given-names></name></person-group><year>2013</year><article-title>Paracrine signaling through plasma membrane hemichannels</article-title><source>Biochimica et Biophysica Acta</source><volume>1828</volume><fpage>35</fpage><lpage>50</lpage><pub-id pub-id-type="doi">10.1016/j.bbamem.2012.07.002</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wenker</surname><given-names>IC</given-names></name><name><surname>Sobrinho</surname><given-names>CR</given-names></name><name><surname>Takakura</surname><given-names>AC</given-names></name><name><surname>Moreira</surname><given-names>TS</given-names></name><name><surname>Mulkey</surname><given-names>DK</given-names></name></person-group><year>2012</year><article-title>Regulation of ventral surface CO<sub>2</sub>/H<sup>+</sup>-sensitive neurons by purinergic signalling</article-title><source>The Journal of Physiology</source><volume>590</volume><fpage>2137</fpage><lpage>2150</lpage><pub-id pub-id-type="doi">10.1113/jphysiol.2012.229666</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xu</surname><given-names>J</given-names></name><name><surname>Nicholson</surname><given-names>BJ</given-names></name></person-group><year>2013</year><article-title>The role of connexins in ear and skin physiology - functional insights from disease-associated mutations</article-title><source>Biochimica et Biophysica Acta</source><volume>1828</volume><fpage>167</fpage><lpage>178</lpage><pub-id pub-id-type="doi">10.1016/j.bbamem.2012.06.024</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04249.006</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Marletta</surname><given-names>Michael A</given-names></name><role>Reviewing editor</role><aff><institution>The Scripps Research Institute</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://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 “Cx26 hemichannels with the A88V mutation causing Keratitis-Ichthyosis-Deafness syndrome in humans lack CO<sub>2</sub> sensitivity” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Michael Marletta (Senior editor) and 2 reviewers, one of whom, Juan Saez, has agreed to reveal his identity.</p><p>The Senior editor has assembled the following comments to help you prepare a revised submission.</p><p>Your findings reported here are very interesting; however, you have no direct evidence that there are carbamylation changes in the A88V mutant. Hence, speculation regarding specific mechanism should be removed from the manuscript, since we all agree that your observations may not be the result of changes in carbamylation. We had an active discussion about the value of asking you to include expanded information on the patient and particularly about familial history of the disease in order to link the mutation to the respiratory phenotype. In the end we think it would be informative if you provide information on the prevalence of KID patients with respiratory problems and on their longevity.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04249.007</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Your findings reported here are very interesting; however, you have no direct evidence that there are carbamylation changes in the A88V mutant. Hence, speculation regarding specific mechanism should be removed from the manuscript, since we all agree that your observations may not be the result of changes in carbamylation. We had an active discussion about the value of asking you to include expanded information on the patient and particularly about familial history of the disease in order to link the mutation to the respiratory phenotype. In the end we think it would be informative if you provide information on the prevalence of KID patients with respiratory problems and on their longevity</italic>.</p><p>We have removed any speculation that the A88V mutation alters carbamylation of Cx26.</p><p>We agree that more background information on KID syndrome would be helpful for the reader. This is a complex area as different mutations in Cx26 give forms of the syndrome that differ in their severity. The mutation A88V causes a highly severe form that is fatal in early childhood. In the first report linking A88V to KID syndrome, a requirement for mechanical ventilation was noted; a second report on A88V did not note any respiratory problems. A different mutation, G45E, also caused a severe form of KID syndrome, which was fatal in the first year of life. There are two reports of abnormal breathing in these patients. However no detailed cardiorespiratory recordings were presented in these prior descriptions of KID syndrome, and thus we cannot assess whether the abnormalities in breathing resulted from a loss of central respiratory drive.</p><p>For other mutations linked to KID syndrome such as D50N, which generally give a less severe form of the disease, there are no reports of breathing abnormalities in the literature. However we would be cautious in concluding from this that such abnormalities do not exist. It is possible that they were not sufficiently severe to be a clinical problem or were not actively investigated.</p><p>To address this issue in the paper, we have summarized the reported incidence of respiratory problems in A88V and G45E patients, and have simply stated that there are no reports of any such problems in KID syndrome patients with other Cx26 mutations.</p><p>An interesting feature of the missense mutations that underlie KID syndrome is that they are dominant. The Cx26<sup>A88V</sup> subunit should therefore act in a dominant negative fashion to remove CO<sub>2</sub> sensitivity from cells expressing wild type Cx26 hemichannels. We have now had an opportunity to test this idea, and now present this evidence in a new <xref ref-type="fig" rid="fig3">Figure 3</xref>. We believe that this additional evidence strengthens the potential link between the loss of CO<sub>2</sub> sensitivity in Cx26<sup>A88V</sup> and the pathologies evident in these patients.</p></body></sub-article></article> |