Functional analysis of connexin‐26 mutants associated with hereditary recessive deafness

The physiological importance of connexin‐26 (Cx26) gap junctions in regulating auditory function is indicated by the finding that autosomal recessive DFNB1 deafness is associated with mutations of the Cx26 gene. To investigate the pathogenic role of Cx26 mutation in recessive hearing loss, four putative DFNB1 Cx26 mutants (V84L, V95M, R127H, and R143W) were stably expressed in N2A cells, a communication‐deficient cell line. In N2A cells expressing (R127H) Cx26 gap junctions, macroscopic junctional conductance and ability of transferring neurobiotin between transfected cells were greatly reduced. Despite the formation of defective junctional channels, immunoreactivity of (R127H) Cx26 was mainly localized in the cell membrane and prominent in the region of cell–cell contact. Mutant (V84L), (V95M), or (R143W) Cx26 protein formed gap junctions with a junctional conductance similar to that of wild‐type Cx26 junctional channels. (V84L), (V95M), or (R143W) Cx26 gap junctions also permitted neurobiotin transfer between pairs of transfected N2A cells. The present study suggests that (R127H) mutation associated with hereditary sensorineural deafness results in the formation of defective Cx26 gap junctions, which may lead to the malfunction of cochlear gap junctions and hearing loss. Further studies are required to determine the exact mechanism by which mutant (V84L), (V95M), and (R143W) Cx26 proteins, which are capable of forming functional homotypic junctional channels in N2A cells, cause the cochlear dysfunction and sensorineural deafness.

[1]  P. Willems,et al.  Nonsyndromic hearing impairment: unparalleled heterogeneity. , 1997, American journal of human genetics.

[2]  A Chakravarti,et al.  Two different connexin 26 mutations in an inbred kindred segregating non-syndromic recessive deafness: implications for genetic studies in isolated populations. , 1997, Human molecular genetics.

[3]  Hong-Bo Zhao,et al.  Voltage Gating of Gap Junctions in Cochlear Supporting Cells: Evidence for Nonhomotypic Channels , 2000, The Journal of Membrane Biology.

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

[5]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[6]  N. Hakuba,et al.  Exacerbation of Noise-Induced Hearing Loss in Mice Lacking the Glutamate Transporter GLAST , 2000, The Journal of Neuroscience.

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

[8]  R. Bruzzone,et al.  Connexin channels in Schwann cells and the development of the X-linked form of Charcot-Marie-Tooth disease , 2000, Brain Research Reviews.

[9]  J. W. Askew,et al.  Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. , 1998, American journal of human genetics.

[10]  H L Wang,et al.  A Conserved Arginine in the Distal Third Intracellular Loop of the μ‐Opioid Receptor Is Required for G Protein Activation , 1999, Journal of neurochemistry.

[11]  Sam W. Lee,et al.  Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines , 1992, The Journal of cell biology.

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

[13]  Lee Mj,et al.  Heteromeric gap junction channels in rat hepatocytes in which the expression of connexin26 is induced. , 1998, Molecules and cells.

[14]  D. Spray,et al.  Changes in the Properties of Gap Junctions during Neuronal Differentiation of Hippocampal Progenitor Cells , 1998, The Journal of Neuroscience.

[15]  J. Weissenbach,et al.  A non–syndromic form of neurosensory, recessive deafness maps to the pericentromeric region of chromosome 13q , 1994, Nature Genetics.

[16]  Jian-ting,et al.  Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA , 1989, The Journal of cell biology.

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

[18]  W. Kimberling,et al.  Connexin 26: required for normal auditory function , 2000, Brain Research Reviews.

[19]  H. Hauri,et al.  cDNA Cloning and Molecular Characterization of Human Brain Metalloprotease MP100 , 1999 .

[20]  R. Bruzzone,et al.  Connections with connexins: the molecular basis of direct intercellular signaling. , 1996, European journal of biochemistry.

[21]  K. Stauffer.  The Gap Junction Proteins β1-Connexin (Connexin-32) and β2-Connexin (Connexin-26) Can Form Heteromeric Hemichannels , 1995 .

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

[23]  R. Bruzzone,et al.  Connexin 26 gene linked to a dominant deafness , 1998, Nature.

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

[25]  D. Gros,et al.  Assignment of connexin 26 (GJB2) and 46 (GJA3) genes to human chromosome 13q11-->q12 and mouse chromosome 14D1-E1 by in situ hybridization. , 1996, Cytogenetics and cell genetics.

[26]  B Müller-Myhsok,et al.  Connexin 26 R143W mutation associated with recessive nonsyndromic sensorineural deafness in Africa. , 1998, The New England journal of medicine.

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

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

[29]  P. Lefebvre,et al.  Connexins, hearing and deafness: clinical aspects of mutations in the connexin 26 gene. , 2000, Brain research. Brain research reviews.

[30]  H L Wang,et al.  Basic Amino Acids at the C‐Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin‐B Receptors , 1997, Journal of neurochemistry.

[31]  W. Fang,et al.  Point mutation associated with X-linked dominant Charcot-Marie-Tooth disease impairs the P2 promoter activity of human connexin-32 gene. , 2000, Brain research. Molecular brain research.

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

[33]  M. J. Lee,et al.  Heteromeric gap junction channels in rat hepatocytes in which the expression of connexin26 is induced. , 1998, Molecules and cells.

[34]  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.

[35]  K. Fischbeck,et al.  X‐linked Charcot‐Marie‐Tooth Disease and Connexin32 , 1999, Annals of the New York Academy of Sciences.

[36]  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.

[37]  M. Tekin,et al.  Advances in hereditary deafness , 2001, The Lancet.

[38]  K. Willecke,et al.  Molecular Cloning and Functional Expression of Mouse Connexin-30,a Gap Junction Gene Highly Expressed in Adult Brain and Skin* , 1996, The Journal of Biological Chemistry.

[39]  T. Bargiello,et al.  Mutations in connexin 32: the molecular and biophysical bases for the X-linked form of Charcot–Marie–Tooth disease , 2000, Brain Research Reviews.

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

[41]  X. Estivill,et al.  Connexin 31 (GJB3) is expressed in the peripheral and auditory nerves and causes neuropathy and hearing impairment. , 2001, Human molecular genetics.

[42]  K. Willecke,et al.  Targeted Ablation of Connexin26 in the Inner Ear Epithelial Gap Junction Network Causes Hearing Impairment and Cell Death , 2002, Current Biology.

[43]  C. Petit.  Genes responsible for human hereditary deafness: symphony of a thousand , 1996, Nature Genetics.

[44]  A J Hudspeth,et al.  How Hearing Happens , 1997, Neuron.

[45]  G. Green,et al.  Genetic testing for hereditary hearing loss: Connexin 26 (GJB2) allele variants and two novel deafness‐causing mutations (R32C and 645‐648delTAGA) , 2000, Human mutation.

[46]  M. Bennett,et al.  Changes in Permeability Caused by Connexin 32 Mutations Underlie X-Linked Charcot-Marie-Tooth Disease , 1997, Neuron.