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Altered CO2 sensitivity of connexin26 mutant hemichannels in vitro.

ABSTRACT: Connexin26 (Cx26) mutations underlie human pathologies ranging from hearing loss to keratitis ichthyosis deafness (KID) syndrome. Cx26 hemichannels are directly gated by CO2 and contribute to the chemosensory regulation of breathing. The KID syndrome mutation A88V is insensitive to CO2, and has a dominant negative action on the CO2 sensitivity of Cx26WT hemichannels, and reduces respiratory drive in humans. We have now examined the effect of further human mutations of Cx26 on its sensitivity to CO2 : Mutated Cx26 subunits, carrying one of A88S, N14K, N14Y, M34T, or V84L, were transiently expressed in HeLa cells. The CO2-dependence of hemichannel activity, and their ability to exert dominant negative actions on cells stably expressing Cx26WT, was quantified by a dye-loading assay. The KID syndrome mutation, N14K, abolished the sensitivity of Cx26 to CO2 Both N14Y and N14K exerted a powerful dominant negative action on the CO2 sensitivity of Cx26WT None of the other mutations (all recessive) had a dominant negative action. A88S shifted the affinity of Cx26 to slightly higher levels without reducing its ability to fully open to CO2 M34T did not change the affinity of Cx26 for CO2 but reduced its ability to open in response to CO2 V84L had no effect on the CO2-sensitivity of Cx26. Some pathological mutations of Cx26 can therefore alter the CO2 sensitivity of Cx26 hemichannels. The loss of CO2 sensitivity could contribute to pathology and consequent reduced respiratory drive could be an unrecognized comorbidity of these pathologies.

SUBMITTER: de Wolf E

PROVIDER: S-EPMC5357999 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Altered CO2 sensitivity of connexin26 mutant hemichannels in vitro.

de Wolf Elizabeth E van de Wiel Joseph J Cook Jonathan J Dale Nicholas N

Physiological reports 20161101 22

Connexin26 (Cx26) mutations underlie human pathologies ranging from hearing loss to keratitis ichthyosis deafness (KID) syndrome. Cx26 hemichannels are directly gated by CO2 and contribute to the chemosensory regulation of breathing. The KID syndrome mutation A88V is insensitive to CO2, and has a dominant negative action on the CO2 sensitivity of Cx26WT hemichannels, and reduces respiratory drive in humans. We have now examined the effect of further hu ...[more]

PMID: 27884957

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Project description: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 Ca(2+) concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca(2+) 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 Ca(2+) concentration, indicating that Ca(2+) facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca(2+)-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca(2+) insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca(2+), and (b) facilitating closing and stabilization of the closed state in the presence of Ca(2+). To explore the role of a negatively charged residue at position 50 in regulation by Ca(2+), this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)(-). D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES(-) restores the wild-type Ca(2+) regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca(2+) 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 Ca(2+). Mutant cycle analysis supports a Ca(2+)-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca(2+) 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 Ca(2+).

Project description:BACKGROUND:Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS:We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS:Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION:Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

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Altered CO2 sensitivity of connexin26 mutant hemichannels in vitro.

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Altered CO2 sensitivity of connexin26 mutant hemichannels in vitro.

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