Gap Junction Mediated Intercellular Metabolite Transfer in the Cochlea Is Compromised in Connexin30 Null Mice

Abstract

Connexin26 (Cx26) and connexin30 (Cx30) are two major protein subunits that co-assemble to form gap junctions (GJs) in the cochlea. Mutations in either one of them are the major cause of non-syndromic prelingual deafness in humans. Because the mechanisms of cochlear pathogenesis caused by Cx mutations are unclear, we investigated effects of Cx30 null mutation on GJ-mediated ionic and metabolic coupling in the cochlea of mice. A novel flattened cochlear preparation was used to directly assess intercellular coupling in the sensory epithelium of the cochlea. Double-electrode patch clamp recordings revealed that the absence of Cx30 did not significantly change GJ conductance among the cochlear supporting cells. The preserved electrical coupling is consistent with immunolabeling data showing extensive Cx26 GJs in the cochlea of the mutant mice. In contrast, dye diffusion assays showed that the rate and extent of intercellular transfer of multiple fluorescent dyes (including a non-metabolizable D-glucose analogue, 2-NBDG) among cochlear supporting cells were severely reduced in Cx30 null mice. Since the sensory epithelium in the cochlea is an avascular organ, GJ-facilitated intercellular transfer of nutrient and signaling molecules may play essential roles in cellular homeostasis. To test this possibility, NBDG was used as a tracer to study the contribution of GJs in transporting glucose into the cochlear sensory epithelium when delivered systemically. NBDG uptake in cochlear supporting cells was significantly reduced in Cx30 null mice. The decrease was also observed with GJ blockers or glucose competition, supporting the specificity of our tests. These data indicate that GJs facilitate efficient uptake of glucose in the supporting cells. This study provides the first direct experimental evidence showing that the transfer of metabolically-important molecules in cochlear supporting cells is dependent on the normal function of GJs, thereby suggesting a novel pathogenesis process in the cochlea for Cx-mutation-linked deafness.

Timed conditional null of connexin26 in mice reveals temporary requirements of connexin26 in key cochlear developmental events before the onset of hearing

“…The absence of Cx26 in the supporting cells of the organ of Corti during the critical postnatal period may greatly reduce the intercellular diffusion of molecules (e.g., glucose (Chang et al, 2008)) essential for normal cellular activities. Significantly-reduced supply of growth factors and nutrient molecules may hinder the postnatal maturation of the organ of Corti in the cCx26 null mice.…”

“…Flattened cochlear preparations were made according to the method detailed in our published papers (Chang et al, 2008). Spontaneous depolarizing activities (SDAs) were recorded from outer sulcus cells under loose-patch configurations using the voltage clamp mode of the Axopatch 200B (Molecular Devices, Sunnyvale, CA).…”

“…The GJ network in the early postnatal cochlea may facilitate the intercellular transport of ions, nutrient (Chang et al, 2008;Zhang et al, 2005) and signaling (Beltramello et al, 2005) molecules. Early establishment of the cochlear GJ network may facilitate cochlear development by helping the transport of signal and nutrient molecules in the avascular sensory epithelium of the cochlea.…”

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions

“…In the inner ear, these large Cx30 gap junction plaques are found between supporting cells within the avascular organ of Corti (Sun et al, 2005;Jagger and Forge, 2006), which plays a key role in K + buffering and metabolite transfer (Jagger and Forge, 2014). Therefore, large gap junction plaques may be crucial for maintaining homeostasis by maximizing cell-cell transfer of metabolites and nutrients (Chang et al, 2008). In addition, longlived Cx30 gap junction plaques may play an important physiological role in the skin.…”

Mutations in Cx30 that are linked to skin disease and non-syndromic hearing loss exhibit several distinct cellular pathologies

“…Importantly, in the inner ear, this mutant might be affecting Cx26 levels, potentially reducing the frequency of heteromeric and heterotypic channel formation that is necessary for K + buffering. In addition, Cx30 has been reported to be involved in glucose uptake and metabolic coupling between mouse cochlear cells (Chang et al, 2008). Therefore, the T5M mutation, although able to pass ions and molecular dyes, might reduce, but not inhibit, the permeability of metabolites, such as glucose, between supporting cells in the avascular sensory epithelium.…”