OBJECTIVES: Mutations in connexin (Cx) 26 are the most common cause of nonsyndromic hereditary hearing impairments. Our goal is to investigate molecular mechanisms responsible for hearing impairment caused by various types of Cx26 mutations. STUDY DESIGN: Mutant Cxs linked to deafness were expressed in HEK293 cells. The permeability of reconstituted gap junctions (GJs) and hemichannels were studies. METHODS: HEK293 cells were used to reconstitute GJs and hemichannels in vitro. Ionic as well as biochemical permeabilities of reconstituted GJs were evaluated. RESULTS: We found two-point substitution mutations located in the first extracellular loop of Cx26, E47K, and G45E affected the GJ functions in dramatically different manners. E47K mutant Cx26 formed nonfunctional GJs that lacked GJ- and hemichannel-mediated biochemical and ionic coupling. In contrast, G45E mutation resulted in apoptosis and cell death within 24 hours of transfection. Increasing concentration of extracellular calcium ([Ca2+]0) rescued the cells in a dose-dependent manner. The rescued cells formed functional G45E GJs permeable to both ions and fluorescent tracer molecules. CONCLUSIONS: The primary effect of G45E Cx26 mutation is to cause leaky GJ hemichannels when cells are bathed in normal [Ca2+]0. Our data showed that abnormally open hemichannels with resultant cell death, in addition to GJ and hemichannel uncoupling, is a novel molecular mechanism by which Cx26 mutations may result in hearing impairment. One plausible therapeutic strategy for this type of Cx mutation, therefore, is to manipulate [Ca2+]0 and/or the Ca-binding affinity of GJ hemichannels.
OBJECTIVES: Mutations in connexin (Cx) 26 are the most common cause of nonsyndromic hereditary hearing impairments. Our goal is to investigate molecular mechanisms responsible for hearing impairment caused by various types of Cx26 mutations. STUDY DESIGN: Mutant Cxs linked to deafness were expressed in HEK293 cells. The permeability of reconstituted gap junctions (GJs) and hemichannels were studies. METHODS: HEK293 cells were used to reconstitute GJs and hemichannels in vitro. Ionic as well as biochemical permeabilities of reconstituted GJs were evaluated. RESULTS: We found two-point substitution mutations located in the first extracellular loop of Cx26, E47K, and G45E affected the GJ functions in dramatically different manners. E47K mutant Cx26 formed nonfunctional GJs that lacked GJ- and hemichannel-mediated biochemical and ionic coupling. In contrast, G45E mutation resulted in apoptosis and cell death within 24 hours of transfection. Increasing concentration of extracellular calcium ([Ca2+]0) rescued the cells in a dose-dependent manner. The rescued cells formed functional G45E GJs permeable to both ions and fluorescent tracer molecules. CONCLUSIONS: The primary effect of G45ECx26 mutation is to cause leaky GJ hemichannels when cells are bathed in normal [Ca2+]0. Our data showed that abnormally open hemichannels with resultant cell death, in addition to GJ and hemichannel uncoupling, is a novel molecular mechanism by which Cx26 mutations may result in hearing impairment. One plausible therapeutic strategy for this type of Cx mutation, therefore, is to manipulate [Ca2+]0 and/or the Ca-binding affinity of GJ hemichannels.