Gap junctions in the inner ear: Comparison of distribution patterns in different vertebrates and assessement of connexin composition in mammals

Abstract

The distribution and size of gap junctions (GJ) in the sensory epithelia of the inner ear have been examined in a reptile (gecko), birds (chicken and owl), and mammals (mouse, guinea pig, gerbil, and bat), and the connexin composition of GJs in the mammalian inner ear has been assessed. Freeze fracture revealed a common pattern of GJ distribution in auditory and vestibular sensory epithelia in the different vertebrate classes. In all these tissues, GJs are numerous, often occupying more than 25% of the plasma membrane area of supporting cells and sometimes composed of more than 100,000 channels. Screening for 12 members of the connexin family in the mammalian inner ear by RT-PCR, Western blotting, and immunohistochemistry revealed four connexin isotypes, cx26, cx30, cx31, and cx43, in the cochlea and three, cx26, cx30, and cx43, in the vestibular organs. With antibodies characterised for their specificity, cx26 and cx30 colocalised in supporting cells of the organ of Corti, in the basal cell region of the stria vascularis, and in type 1 fibrocytes of the spiral ligament. No other connexin was detected in these regions. Cx31 was localised among type 2 fibrocytes below the spiral prominence, a region where cx30 was not expressed and cx26 expression appeared to be low. Cx43 was detected only in the region of "tension fibrocytes" lining the inner aspect of the otic capsule. This suggests separate functional compartments in the cochlea. In addition to cx26 and cx30, cx43 was detected in supporting cells of the vestibular sensory epithelia. Where cx26 and cx30 were colocalised, double immunogold labelling of thin sections showed both cx26 and cx30 evenly distributed in individual GJ plaques, a pattern consistent with the presence of heteromeric connexons. Coimmunoprecipitation of cochlear membrane proteins solubilised with a procedure that preserves the oligomeric structure of connexons confirmed the presence of heteromeric cx26/cx30 connexons. Heteromeric cx26/cx30 connexons may be unique to the inner ear, which could be one factor underlying the non-syndromic character of the deafness caused by mutations in cx26.

Contractility in Type III Cochlear Fibrocytes Is Dependent on Non-muscle Myosin II and Intercellular Gap Junctional Coupling

“…cx31 immunofluorescence was restricted to apparently cytoplasmic sites, with few if any gap junction plaques being detected. cx31 has been described previously in type II fibrocytes (Xia et al 2000;Forge et al 2003), an observation we confirmed in guinea pig sections (data not shown). We also observed intracellular cx31 immunofluorescence in type III fibrocytes but not within intercellular plaque-like arrangements.…”

Immunocytochemical Traits of Type IV Fibrocytes and Their Possible Relations to Cochlear Function and Pathology

“…Similar findings have been reported for both connexin 26 and connexin 30 (Ahmad et al 2003;Forge et al 2003). Sparse staining for connexin 26 for cells in this region was also noted in the initial report on its localization in rat (Kikuchi et al 1995).…”

“…These observations are not compelling, however, because tissue preservation was not optimal due to the tissue processing protocol having been optimized for immunostaining sensitivity. Nevertheless, the assumption that all three noise bands used in the present study may have damaged the lower basal turn organ of Corti seems reasonable, based upon considerations of basilar membrane mechanics discussed above and upon previous reports of hair cell loss near the hook in a number of species following noise exposures with a variety of noise types (Fried et al 1976;Johnsson and Hawkins 1976;Liberman and Kiang 1978;Moody et al 1978;Salvi et al 1982;Wang et al 2002;Harding et al 2005).…”

The Membrane Properties of Cochlear Root Cells are Consistent with Roles in Potassium Recirculation and Spatial Buffering

“…This maintenance ensures the longevity of sensory hair cells and the generation of the endocochlear potential. The ion transporting mechanisms that regulate these concentrations have been characterized in a number of cochlear tissues, leading to widely held hypotheses of K + "recirculation" and "spatial buffering" (Forge et al 2003a;Hibino and Kurachi 2006;Kikuchi et al 2000;Kikuchi et al 1995;Nickel and Forge 2008;Nin et al 2008;Spicer and Schulte 1996;Takeuchi et al 2000;Wangemann 2006;Weber et al 2001;. In common with these mechanisms studied in the CNS and retina (Kofuji and Newman 2004), cochlear K + recirculation and spatial buffering both rely on gap junctional coupling throughout the epithelial cell network, and the ability of cells at the edges of the network to secrete K + .…”

“…The supporting cells in the organ of Corti, together with the adjacent epithelial cells spanning the radial distance to the lateral wall, collectively form a syncytium known as the "epithelial gap junction network" (Kikuchi et al 2000). Gap junctions in the syncytium are formed from connexin26 (Cx26) and/or Cx30 subunits (Forge et al 2003a;Forge et al 2003b;Zhao and Yu 2006). Mutations in the Cx26 and Cx30 genes are a common cause of autosomal recessive non-syndromic deafness in humans (Nickel and Forge 2008).…”