Connexin gap junctions play an important role in hearing function, but the mechanism by which this contribution occurs is unknown. Connexins in the cochlea are expressed only in supporting cells; no connexin expression occurs in auditory sensory hair cells. A gap junctional channel is formed by two hemichannels. Here, we show that connexin hemichannels in the cochlea can release ATP at levels that account for the submicromolar concentrations measured in the cochlear fluids in vivo. The release could be increased 3-to 5-fold by a reduction of extracellular Ca 2؉ or an increase in membrane stress, and blocked by gap junctional blockers. We also demonstrated that extracellular ATP at submicromolar levels apparently affected outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining cochlear sensitivity to sound stimulation in mammals. ATP reduced OHC electromotility and the slope factor of the voltage dependence and shifted the operating point to reduce the active amplifier gain. ATP also reduced the generation of distortion products. Immunofluorescent staining showed that purinergic receptors P2x2 and P2x7 were distributed on the OHC surface. Blockage of P2 receptors eliminated the effect of ATP on the OHC electromotility. The data revealed that there is a hemichannel-mediated, purinergic intercellular signaling pathway between supporting cells and hair cells in the cochlea to control hearing sensitivity. The data also demonstrated a potential source of ATP in the cochlea.active cochlear mechanics ͉ cochlear supporting cells ͉ connexin ͉ outer hair cell electromotility ͉ P2 receptor

Mammalian hearing relies upon active cochlear mechanics, which arises from outer hair cell (OHC) electromotility and hair bundle movement, to amplify acoustic stimulations increasing hearing sensitivity and frequency selectivity. Here we describe the novel finding that gap junctions between cochlear supporting cells also have a critical role in active cochlear amplification in vivo. We find that targeted-deletion of connexin26 (Cx26) in Deiters cells (DCs) and outer pillar cells (OPCs), which constrain OHCs standing on the basilar membrane, causes a leftward shift in OHC electromotility towards hyperpolarization, and reduces active cochlear amplification with hearing loss. Coincident with large reduction in distortion product otoacoustic emission (DPOAE) and severe hearing loss at high frequencies, the shift is larger in shorter OHCs. Our study demonstrates that active cochlear amplification in vivo is dependent on supporting cell gap junctions. These new findings also show that Cx26 deficiency can reduce active cochlear amplification to induce hearing loss.

Connexin26 (Cx26) and Cx30 are predominant isoforms of gap junction channels in the cochlea and play a critical role in hearing. In this study, the cellular distributions of Cx26 and Cx30 in the cochlear sensory epithelium of guinea pigs were examined by immunofluorescent staining and confocal microscopy in whole mounts of the cochlear sensory epithelium and dissociated cell preparations. The expression of Cx26 and Cx30 demonstrated a longitudinal gradient distribution in the epithelium and was reduced threefold from the cochlear apex to base. The reduction was more pronounced in the Deiters cells and pillar cells than in the Hensen cells. Cx26 was expressed in all types of supporting cells, but little Cx30 labeling was seen in the Hensen cells. Cx26 expression in the Hensen cells was concentrated mainly in the second and third rows, forming a distinct band along the sensory epithelium at its outer region. In the dissociated Deiters cells and pillar cells, Cx30 showed dense labeling at the cell bodies and processes in the reticular lamina. Cx26 labeling largely overlapped that of Cx30 in these regions. Cx26 and Cx30 were also coexpressed in the gap junctional plaques between Claudius cells. Neither Cx26 nor Cx30 labeling was seen in the hair cells and spiral ganglion neurons. These observations demonstrate that Cx26 and Cx30 have a longitudinal gradient distribution and distinct cellular expression in the auditory sensory epithelium. This further supports our previous reports that Cx26 and Cx30 can solely and concertedly perform different functions in the cochlea. Indexing termsgap junction; cochlear supporting cells; reticular lamina; spiral ganglion; inner ear; nonsyndromic hearing lossThe connexin gene family encodes gap junction channels in mammals. So far, more than 20 connexin genes and their corresponding isoforms have been identified . Each connexin shows tissue-or cell-specific expression, and most organs and tissues express more than one connexin (for reviews see Harris, 2001; Evans and Martin, 2002;Willecke et al., 2002). Connexin gap junctions perform electronic and metabolic communications between cells and play important roles in many aspects of cellular and physiological functions. In particular, gap junctions are known to play a critical role in hearing function. Connexin mutations can cause hearing loss and account for 70 -80% of nonsyndromic hearing loss in children (Kelsell et al., 1997;Denoyelle et al., 1997;Grifa et al., 1999 The mammalian cochlea is the auditory organ and contains sensory hair cells and nonsensory supporting cells. Gap junctional coupling is extensive in the cochlea (for reviews see Kikuchi et al., 2000;Zhao et al., 2006). In early histological studies, electron microscopy and dye injection demonstrated that intercellular communications existed in the organ of Corti (Jahnke, 1975;Gulley and Reese, 1976;Iurato et al., 1976Iurato et al., , 1977Hama and Saito, 1977;Santos-Sacchi and Dallos, 1983;Santos-Sacchi, 1987;Zwislocki et al., 1992). Kikuchi et al. (1995), using ...

Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations.

Hearing loss due to mutations in the connexin gene family, which encodes gap junctional proteins, is a common form of hereditary deafness. In particular, connexin 26 (Cx26, GJB2) mutations are responsible for ~50% of non-syndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate that congenital deafness mainly results from cochlear developmental disorders rather than hair cell degeneration and endocochlear potential reduction, while late-onset hearing loss results from reduction of active cochlear amplification, even though cochlear hair cells have no connexin expression. However, there is no apparent, demonstrable relationship between specific changes in connexin (channel) functions and the phenotypes of mutation-induced hearing loss. Moreover, new experiments further demonstrate that the hypothesized K+-recycling disruption is not a principal deafness mechanism for connexin deficiency induced hearing loss. Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), and Cx43 (GJA1) mutations can also cause hearing loss with distinct pathological changes in the cochlea. These new studies provide invaluable information about deafness mechanisms underlying connexin mutation-induced hearing loss and also provide important information for developing new protective and therapeutic strategies for this common deafness. However, the detailed cellular mechanisms underlying these pathological changes remain unclear. Also, little is known about specific mutation-induced pathological changes in vivo and little information is available for humans. Such further studies are urgently required.