The inner ear contains heteromeric channels composed of Cx26 and Cx30 and deafness-related mutations in Cx26 have a dominant negative effect on Cx30

Cx26 and cx30 co-localize in tissues of the mammalian cochlea. Transfected HeLa cells were used to examine interactions between cx26 and cx30 and the effects on cx30 of four point mutations in cx26 that are associated with dominantly inherited hearing loss--W44S, G59A, D66H and R75W. When co-expressed, wtcx26 and wtcx30 trafficked to the same gap junction plaques. Cells transferred neurobiotin but not Lucifer Yellow, which passes freely through cx26 channels, suggesting cx30 affects the properties of cx26. G59A and D66H had a perinuclear localization when expressed alone but trafficked to the membrane when co-expressed with cx30. Co-expression of W44S, G59A or R75W with cx30, significantly reduced neurobiotin transfer in comparison with cells expressing cx30 only. These results indicate that cx26 and cx30 can oligomerize to form heteromeric connexons and demonstrate a dominant negative effect of some cx26 mutants on cx30. Immunogold labeling of thin sections of the cochlea showed both cx26 and cx30 distributed evenly on both sides of individual gap junction profiles. Immunoprecipitation of cochlear membrane proteins, isolated by procedures that preserve connexons, with either cx30 or cx26 antibodies precipitated both cx26 and cx30. Following co-injection of Lucifer Yellow and neurobiotin into individual supporting cells of the organ of Corti in cochlear slices, neurobiotin transferred to many cells, but Lucifer Yellow was retained in the injected cell. These observations are consistent with junctions composed of cx26/cx30 heteromeric connexons in the cochlea. The functional disruption caused by some cx26 mutations upon such heteromeric channels may underlie the non-syndromic nature of their effects on hearing.