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

Dysfunction of gap junctions (GJs) caused by mutations in connexin26 (Cx26) and Cx30 accounts for nearly half of all cases of hereditary nonsyndromic deafness cases. Although it is widely held that GJs connecting supporting cells in the organ of Corti mainly provide ionic pathways for rapid removal of K+ around the base of hair cells, the function of GJs in the cochlea remains unknown. Here we show that GJs were not assembled in the supporting cells of the organ of Corti until 3 days after birth in mice and then gradually matured to connect supporting cells before the onset of hearing. In organotypic cochlear cultures that were confirmed to express GJs, GJs mediated the propagation of intracellular Ca2+ concentration waves in supporting cells by allowing intercellular diffusion of inositol 1,4,5-trisphosphate. We found that a subset of structurally mild Cx26 mutations located at the second transmembrane region (V84L, V95M, and A88S) and a Cx30 mutation located at the first cytoplasmic segment (T5M) specifically affect the intercellular exchange of larger molecules but leave the ionic permeability intact. Our results indicated that Cx26 and Cx30 mutations that are linked to sensorineural deafness retained ionic coupling but were deficient in biochemical permeability. Therefore, GJ-mediated intercellular exchange of biochemically important molecules is required for normal cochlear functions.

[1]  D. Paul,et al.  Genetic diseases and gene knockouts reveal diverse connexin functions. , 1999, Annual review of physiology.

[2]  R. Pujol,et al.  Development of Sensory and Neural Structures in the Mammalian Cochlea , 1998 .

[3]  S. Finkbeiner,et al.  Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. , 1990, Science.

[4]  Wen‐Teng Chang,et al.  Functional analysis of connexin‐26 mutants associated with hereditary recessive deafness , 2003, Journal of neurochemistry.

[5]  T. W. White,et al.  Connexin disorders of the ear, skin, and lens. , 2004, Biochimica et biophysica acta.

[6]  D. Choo,et al.  Genotypic and phenotypic correlations of DFNB1-related hearing impairment in the Midwestern United States. , 2003, Archives of otolaryngology--head & neck surgery.

[7]  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.

[8]  Xi Lin,et al.  Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice. , 2003, Biochemical and biophysical research communications.

[9]  Yanping Zhang,et al.  Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts. , 2005, American journal of physiology. Cell physiology.

[10]  Arnold R. Kriegstein,et al.  Calcium Waves Propagate through Radial Glial Cells and Modulate Proliferation in the Developing Neocortex , 2004, Neuron.

[11]  C. Brosnan,et al.  Reciprocal Regulation of the Junctional Proteins Claudin-1 and Connexin43 by Interleukin-1β in Primary Human Fetal Astrocytes , 2000, The Journal of Neuroscience.

[12]  A. Raap,et al.  Connexin 26 mutations in cases of sensorineural deafness in eastern Austria , 2002, European Journal of Human Genetics.

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

[14]  T. W. White.  Unique and Redundant Connexin Contributions to Lens Development , 2002, Science.

[15]  Joe C. Adams,et al.  Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness , 2000, Medical Electron Microscopy.

[16]  S. Marlin,et al.  Loss‐of‐function and residual channel activity of connexin26 mutations associated with non‐syndromic deafness , 2003, FEBS letters.

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

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

[19]  O. Andreassen,et al.  Mice Deficient in Cellular Glutathione Peroxidase Show Increased Vulnerability to Malonate, 3-Nitropropionic Acid, and 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine , 2000, The Journal of Neuroscience.

[20]  W. H. Evans,et al.  Gap junctions: structure and function (Review) , 2002, Molecular membrane biology.

[21]  A. Forge,et al.  Gap junctions in the inner ear: Comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals , 2003, The Journal of comparative neurology.

[22]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[23]  Andrew Forge,et al.  Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant negative effect on connexin 30. , 2003, Human molecular genetics.

[24]  Eduardo López Granados,et al.  Molecular analysis of Bruton’s tyrosine kinase gene in Spain , 2001, Human Mutation.

[25]  D. Spray,et al.  Closure of gap junction channels by arylaminobenzoates. , 2003, Molecular pharmacology.

[26]  A. Taylor,et al.  trans-dominant inhibition of connexin-43 by mutant connexin-26: implications for dominant connexin disorders affecting epidermal differentiation. , 2001, Journal of cell science.

[27]  Xavier Estivill,et al.  Human connexin26 (GJB2) deafness mutations affect the function of gap junction channels at different levels of protein expression. , 2002, Human Genetics.

[28]  R D Horstmann,et al.  Pattern of connexin 26 (GJB2) mutations causing sensorineural hearing impairment in Ghana , 2001, Human mutation.

[29]  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.

[30]  K. Willecke,et al.  Connexin30 (Gjb6)-deficiency causes severe hearing impairment and lack of endocochlear potential. , 2003, Human molecular genetics.

[31]  Richard Smith,et al.  The Role of Connexins in Human Disease , 2003, Ear and hearing.

[32]  G. Dahl,et al.  Function of the voltage gate of gap junction channels: Selective exclusion of molecules , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Tullio Pozzan,et al.  Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness , 2005, Nature Cell Biology.

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

[35]  D. Jagger,et al.  The Inner Ear Contains Heteromeric Channels Composed of Cx26 and Cx30 and Deafness-Related Mutations in Cx26 Have a Dominant Negative Effect on Cx30 , 2003, Cell communication & adhesion.

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

[37]  R. Fay,et al.  Development of the Auditory System , 1998, Springer Handbook of Auditory Research.

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

[39]  F. Mammano,et al.  A Mechanism for Sensing Noise Damage in the Inner Ear , 2004, Current Biology.

[40]  Kurt Haas,et al.  Single-Cell Electroporationfor Gene Transfer In Vivo , 2001, Neuron.

[41]  H. Frank,et al.  Developmental expression patterns of connexin26 and -30 in the rat cochlea. , 1999, Developmental genetics.

[42]  Eric Londin,et al.  Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss , 2004, Human Genetics.

[43]  G. Utermann,et al.  Sensorineural hearing loss and the incidence of Cx26 mutations in Austria , 2001, European Journal of Human Genetics.