{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T11:45:11Z","timestamp":1792755911969,"version":"4.5.0"},"reference-count":55,"publisher":"Rockefeller University Press","issue":"1","content-domain":{"domain":["rupress.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2013,7,1]]},"abstract":"<jats:p>Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N\/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N\/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)\u2212. D50C mutant hemichannels display properties similar to those of D50N\/Y mutants. Recovery of the negative charge with chemical modification by MTSES\u2212 restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.<\/jats:p>","DOI":"10.1085\/jgp.201210893","type":"journal-article","created":{"date-parts":[[2013,6,24]],"date-time":"2013-06-24T15:27:25Z","timestamp":1372087645000},"page":"23-35","update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":57,"title":["Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N\/Y)"],"prefix":"10.1085","volume":"142","author":[{"given":"William","family":"Lopez","sequence":"first","affiliation":[{"name":"Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jorge","family":"Gonzalez","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew L.","family":"Harris","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jorge E.","family":"Contreras","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2013,6,24]]},"reference":[{"key":"2023072622475049000_bib1","doi-asserted-by":"publisher","first-page":"3980","DOI":"10.1073\/pnas.261713499","article-title":"Voltage opens unopposed gap junction hemichannels formed by a connexin 32 mutant associated with X-linked Charcot-Marie-Tooth disease","volume":"99","author":"Abrams","year":"2002","journal-title":"Proc. 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