title: Conserved glycine at position 45 of major cochlear connexins constitutes a vital component of the Ca²⁺ sensor for gating of gap junction hemichannels.
abstract: Mutations in gap junction (GJ) family of proteins, especially in the connexin (Cx) 26, are responsible for causing severe congenital hearing loss in a significant portion of patients (30-50% in various ethnic groups). Substitution of glycine at the position 45 of Cx26 to glutamic acid (p.G45E mutation) causes the Keratitis-ichthyosis-deafness (KID) syndrome. Previous studies have suggested that this point mutation caused a gain-of-function defect. However, the molecular mechanism of KID syndrome remains unclear. Since glycine at this position is conserved in many Cxs expressed in the cochlea, we tested the hypothesis that glycine at position 45 is an important component of the sensor regulating the Ca(2+) gating of GJ hemichannels. Using reconstituted Cx30, 32 and 43 expressed in the HEK 293 cells, we compared the functions of wild type and p.G45E mutant Cxs. We found that G45E in Cx30 resulted in similar deleterious cellular effects as Cx26 did. Cell death occurred within 24h of transfection, which was rescued by increasing extracellular Ca(2+) concentration ([Ca(2+)]o). Dye loading assay showed that Cx30 G45E, similar to Cx26 G45E, had leaky hemichannels at physiological [Ca(2+)]o (1.2 mM). Higher [Ca(2+)]o reduced the dye loading in a dose-dependent manner. Whole cell membrane current recordings also indicated that G45E caused increased hemichannel activities. p.G45E mutations of Cx32 and 43 also resulted in leaky hemichannels compared to their respective wild types in lower [Ca(2+)]o. Our data in this study provided further support for the hypothesis that glycine at position 45 is a conserved Ca(2+) sensor for the gating of GJ hemichannels among multiple Cx subtypes expressed in the cochlea.
authors.affiliation: Department of Otolaryngology, 309th Hospital of Chinese PLA, Road Heishanhu, Haidian District, Beijing 100091, China. yanping309@yahoo.com
authors.forename: Yanping
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hongxia
authors.initials: H
authors.lastname: Hao
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D016923:Cell Death; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability; D003051:Cochlea / Q000378:metabolism / Q000473:pathology*; D000072259:Connexin 26; D000075302:Connexin 30; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D003638:Deafness / Q000235:genetics / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007057:Ichthyosis / Q000235:genetics / Q000473:pathology; D007634:Keratitis / Q000235:genetics / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
chemical_list: D002118:Calcium; D015220:Calcium Channels; D000072259:Connexin 26; D000075302:Connexin 30; D018031:Connexin 43; D017630:Connexins; D018698:Glutamic Acid; D005998:Glycine; D000097002:Gap Junction beta-1 Protein; C500216:GJA1 protein, human; C000607042:GJB2 protein, human; C491020:GJB6 protein, human
keywords: Calcium; Connexin 26; GJB6 protein human; Gap junctions; Ion channel gating