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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Mutations in the GJB2 gene, which encodes the gap junction protein connexin26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of the allelic variant M34T in causing hereditary deafness remains controversial. By combining genetic, clinical, biochemical, electrophysiological and structural modeling studies, we have re-assessed the pathogenetic role of the M34T mutation. Genetic and audiological data indicate that the majority of heterozygous carriers and all five compound heterozygotes exhibited an impaired auditory function. Functional expression in transiently transfected HeLa cells showed that, although M34T was correctly synthesized and targeted to the plasma membrane, it inefficiently formed intercellular channels that displayed an abnormal electrical behavior and retained only 11% of the unitary conductance of the wild-type protein (HCx26wt). Moreover, M34T channels failed to support the intercellular diffusion of Lucifer Yellow and the spreading of mechanically induced intercellular Ca2+ waves. When co-expressed together with HCx26wt, M34T exerted dominant-negative effects on cell-cell coupling. Our findings are consistent with a structural model, predicting that the mutation leads to a constriction of the channel pore. These data support the view that M34T is a pathological variant of Cx26 associated with hearing impairment.

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