Loss‐of‐function and residual channel activity of connexin26 mutations associated with non‐syndromic deafness

Abstract

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.

Clinical features of patients with GJB2 (connexin 26) mutations: severity of hearing loss is correlated with genotypes and protein expression patterns

“…A series of in vitro studies, including protein expression study, cell-to-cell communication properties, or physiological conductance experiments, sometimes provided discrepant results when compared to the phenotypic results, and limitations have been suggested (see discussion in Cryns et al 2004). In the case of V37I, a complete loss of junctional properties has been reported (Bruzzone et al 2003) in spite of a rather mild phenotype shown in a series of studies. The protein expression experiments in the current study, however, were in line with the phenotype associated with this mutation.…”

Pediatric cholesteatoma and variants in the gene encoding connexin 26

“…This polymorphism has been shown to confer loss of function on connexin 26 in vitro. 19 Phenotypic variation in expression of GJB2 mutations does suggest the possibility of modification of expression by other genes and environmental influences, 20 such as the digenic inheritance seen with GJB6 deletion. 11 However there are no reports of autosomal dominant effects from V37I, and the small sample size in our study makes it inappropriate to assume that V37I might contribute to recidivism.…”

Gap junction-mediated intercellular biochemical coupling in cochlear supporting cells is required for normal cochlear functions

“…Mutations that result in no formation of GJs in the cell membrane include R143W, V153I and L214P, W77R (10,32). Many more Cx26 point mutants, e.g., V37I, L90P, S113R, delE120, M163V, R184P, 235delC, W44S, G59A, D66H, R75W, and R127H (8,9,33), are incorporated into GJs in the membrane. They interfere with either alignment of connexons or GJ permeability to abolish normal intercellular communications.…”

“…Consistent with the K ϩ recycling theory, some Cx26 mutations are found to nullify the GJ conductance. However, some deafness-linked Cx mutants (T8M, V84L, V95M, and N206S) do not significantly affect GJ-mediated intercellular ionic coupling (8)(9)(10). A recent report further shows that the V84L mutant impairs GJ permeability to inositol 1,4,5-trisphosphate (IP 3 ) (11).…”