Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The speci¢c role of connexin channels in the homeostasis of di¡erent organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in una¡ected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identi¢ed in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identi¢ed as a polymorphism in heterozygous una¡ected individuals, was devoid of function and thus may be pathologically signi¢-cant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identi¢cation of functional di¡erences of disease causing mutations may lead to de¢ne which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.

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