Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30T5M/T5M mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca2+ signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

a b s t r a c tDistinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.

Mutations in the GJB2 gene coding for connexin26 (Cx26) can cause a variety of deafness and hereditary hyperproliferative skin disorders in humans. In this study, we investigated the Cx26S17F mutation in mice, which had been identified to cause the keratitis-ichthyosis-deafness (KID) syndrome in humans. The KID syndrome is characterized by keratitis and chronic progressive corneal neovascularization, skin hyperplasia, sensorineural hearing loss and increased carcinogenic potential. We have generated a conditional mouse mutant, in which the floxed wild-type Cx26-coding DNA can be deleted and the Cx26S17F mutation is expressed under control of the endogenous Cx26 promoter. Homozygous mutants are not viable, whereas the surviving heterozygous mice show hyperplasia of tail and foot epidermis, wounded tails and annular tail restrictions, and are smaller than their wild-type littermates. Analyses of auditory brainstem responses (ABRs) indicate an ∼35 dB increased hearing threshold in these mice, which is likely due to the reduction of the endocochlear potential by 20-40%. Our results indicate that the Cx26S17F protein, which does not form functional gap junction channels or hemichannels, alters epidermal proliferation and differentiation in the heterozygous state. In the inner ear, reduced intercellular coupling by heteromeric channels composed of Cx26S17F and Cx30 could contribute to hearing impairment in heterozygous mice, while remaining wild-type Cx26 may be sufficient to stabilize Cx30 and partially maintain cochlear homeostasis. The phenotype of heterozygous mice resembles many of the symptoms of the human KID syndrome. Thus, these mice represent an appropriate model to further investigate the disease mechanism.

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