Human connexin26 (GJB2) deafness mutations affect the function of gap junction channels at different levels of protein expression.

Abstract. Mutations in the connexin26 (GJB2) gene account for about half of inherited non-syndromic deafness cases in Western countries. The connexin26 protein is a subunit of gap junctions that form a network of intercellular communication among supporting cells and fibrocytes in the mammalian inner ear. Here we describe functional implications of mutations in the coding region of connexin26 genes (M1V, M34T, L90P, R127H, F161S, P173R, and R184P), identified in patients and stably transfected in human HeLa cells. While all mutated connexin26 cDNAs were transcribed, only M34T, L90P, R127H, F161S, and R184P were translated in HeLa cells. Analysis of indirect immunofluorescence showed membranous localization, strong for M34T, L90P, R127H, and very weak for F161S, but no signal corresponding to M1V, P173R and R184P. Tracer coupling experiments revealed diffusion of microinjected neurobiotin into neighbouring cells in the case of M34T and R127H, whereas M1V, L90P, F161S, P173R and R184P mutants did not show intercellular coupling. The results of oligomerisation studies suggested a partly disturbed assembly of hemichannels in M34T and L90P mutants but complete absence of hemichannel formation in the R184P mutant. The R127H mutation did not affect channel formation and is likely to represent a polymorphism. Our results show that mutations in the connexin26 gene can affect gap junctional intercellular communication at the level of protein translation, trafficking or assembly of hemichannels.

[1]  K. Willecke,et al.  Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels , 2001, The Journal of Membrane Biology.

[2]  C. Petit,et al.  Clinical features of the prevalent form of childhood deafness, DFNB1, due to a connexin-26 gene defect: implications for genetic counselling , 1999, The Lancet.

[3]  C. Petit,et al.  Connexin 26 gene mutations in congenitally deaf children: pitfalls for genetic counseling. , 2001, Archives of otolaryngology--head & neck surgery.

[4]  X. Estivill,et al.  Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus , 1999, Nature Genetics.

[5]  S. Antonarakis,et al.  Nomenclature for the description of human sequence variations , 2001, Human Genetics.

[6]  X. Estivill,et al.  High carrier frequency of the 35delG deafness mutation in European populations , 2000, European Journal of Human Genetics.

[7]  K. Willecke,et al.  Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells , 1995, The Journal of cell biology.

[8]  T. W. White.  Functional analysis of human Cx26 mutations associated with deafness , 2000, Brain Research Reviews.

[9]  C. Berlin,et al.  Autosomal recessive nonsyndromic neurosensory deafness at DFNB1 not associated with the compound-heterozygous GJB2 (connexin 26) genotype M34T/167delT. , 2000, American journal of human genetics.

[10]  C. R. Fourtner,et al.  Identification of a proline residue as a transduction element involved in voltage gating of gap junctions , 1993, Nature.

[11]  G. Green,et al.  The M34T allele variant of connexin 26. , 2000, Genetic testing.

[12]  V. Sheffield,et al.  Connexin mutations and hearing loss , 1998, Nature.

[13]  N. Gilula,et al.  The Gap Junction Communication Channel , 1996, Cell.

[14]  Joe C. Adams,et al.  Gap junction systems in the mammalian cochlea , 2000, Brain Research Reviews.

[15]  Joe C. Adams,et al.  Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis. , 1994, Acta oto-laryngologica.

[16]  J. Degen,et al.  Structural and Functional Diversity of Connexin Genes in the Mouse and Human Genome , 2002, Biological chemistry.

[17]  K. Xia,et al.  Mutations in the gene encoding gap junction protein β-3 associated with autosomal dominant hearing impairment , 1998, Nature Genetics.

[18]  A. Pagnamenta,et al.  Genetic analysis of the connexin-26 M34T variant: identification of genotype M34T/M34T segregating with mild-moderate non-syndromic sensorineural hearing loss , 2001, Journal of medical genetics.

[19]  D. Kelsell,et al.  Connexin mutations in deafness , 1998, Nature.

[20]  D. Goodenough,et al.  Diverse functions of vertebrate gap junctions. , 1998, Trends in cell biology.

[21]  R. Errington,et al.  Multiple pathways in the trafficking and assembly of connexin 26, 32 and 43 into gap junction intercellular communication channels. , 2001, Journal of cell science.

[22]  X. Estivill,et al.  Molecular genetics of hearing impairment due to mutations in gap junction genes encoding beta connexins , 2000, Human mutation.

[23]  F. Zacchello,et al.  Cx26 deafness: mutation analysis and clinical variability , 1999, Journal of medical genetics.

[24]  A Forge,et al.  Properties of connexin26 gap junctional proteins derived from mutations associated with non-syndromal heriditary deafness. , 1999, Human molecular genetics.

[25]  C. Petit,et al.  Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene. , 1997, Human molecular genetics.

[26]  K. Steel,et al.  A genetic approach to understanding auditory function , 2001, Nature Genetics.

[27]  X. Estivill,et al.  Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. , 1997, Human molecular genetics.

[28]  Joe C. Adams,et al.  Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis , 1995, Anatomy and Embryology.

[29]  Mathew W. Wright,et al.  The HUGO Gene Nomenclature Committee (HGNC) , 2001, Human Genetics.

[30]  G. Goldberg,et al.  The molecular basis of selective permeability of connexins is complex and includes both size and charge. , 2000, Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas.

[31]  X. Estivill,et al.  Connexin-26 mutations in sporadic and inherited sensorineural deafness , 1998, The Lancet.

[32]  D. Kelsell,et al.  Connexin 26 mutations in hereditary non-syndromic sensorineural deafness , 1997, nature.

[33]  H. Frank,et al.  Expression of the gap-junction connexins 26 and 30 in the rat cochlea , 1998, Cell and Tissue Research.

[34]  K. Steel,et al.  Mutations in connexin31 underlie recessive as well as dominant non-syndromic hearing loss. , 2000, Human molecular genetics.