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The structural phenotype of neural connections in the auditory brainstem is sculpted by spontaneous and stimulus-induced neural activities during development. However, functional and molecular mechanisms of spontaneous action potentials (SAPs) in the developing cochlea are unknown. Additionally, it is unclear how regenerating hair cells establish their neural ranking in the constellation of neurons in the brainstem. We have demonstrated that a transient Ca(2+) current produced by the Ca(v)3.1 channel is expressed early in development to initiate spontaneous Ca(2+) spikes. Ca(v)1.3 currents, typical of mature hair cells, appeared later in development. Moreover, there is a surprising disappearance of the Ca(v)3.1 current that coincides with the attenuation of the transient Ca(2+) current as the electrical properties of hair cells transition to the mature phenotype. Remarkably, this process is recapitulated during hair-cell regeneration, suggesting that the transient expression of Ca(v)3.1 and the ensuing SAPs are signatures of hair cell development and regeneration.

Functional interactions between ligand-gated, voltage-, and Ca(2+)-activated ion channels are essential to the properties of excitable cells and thus to the working of the nervous system. The outer hair cells in the mammalian cochlea receive efferent inputs from the brain stem through cholinergic nerve fibers that form synapses at their base. The acetylcholine released from these efferent fibers activates fast inhibitory postsynaptic currents mediated, to some extent, by small-conductance Ca(2+)-activated K+ channels (SK) that had not been cloned. Here we report the cloning, characterization, and expression of a complete SK2 cDNA from the mouse cochlea. The cDNAs of the mouse cochlea alpha9 and alpha10 acetylcholine receptors were also obtained, sequenced, and coexpressed with the SK2 channels. Human cultured cell lines transfected with SK2 yielded Ca(2+)-sensitive K+ current that was blocked by dequalinium chloride and apamin, known blockers of SK channels. Xenopus oocytes injected with SK2 in vitro transcribed RNA, under conditions where only outward K+ currents could be recorded, expressed an outward current that was sensitive to EGTA, dequalinium chloride, and apamin. In HEK-293 cells cotransfected with cochlear SK2 plus alpha9/alpha10 receptors, acetylcholine induced an inward current followed by a robust outward current. The results indicate that SK2 and the alpha9/alpha10 acetylcholine receptors are sufficient to partly recapitulate the native hair cell efferent synaptic response.

KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild-type ( WT) channel function. Most importantly, overexpression of HSP90β, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved. [ABSTRACT FROM AUTHOR]

Multiple Ca2+ channels confer diverse functions to hair cells of the auditory and vestibular organs in the mammalian inner ear. We used gene-targeting technology to generate alpha1D Ca2+ channel-deficient mice to determine the physiological role of these Ca2+ channels in hearing and balance. Analyses of auditory-evoked brainstem recordings confirmed that alpha1D-/- mice were deaf and revealed that heterozygous (alpha1D+/-) mice have increased hearing thresholds. However, hearing deficits in alpha1D+/- mice were manifested mainly by the increase in threshold of low-frequency sounds. In contrast to impaired hearing, alpha1D-/- mice have balance performances equivalent to their wild-type littermates. Light and electron microscope analyses of the inner ear revealed outer hair cell loss at the apical cochlea, but no apparent abnormality at the basal cochlea and the vestibule. We determined the mechanisms underlying the auditory function defects and the normal vestibular functions by examining the Ba2+ currents in cochlear inner and outer hair cells versus utricular hair cells in alpha1D+/- mice. Whereas the whole-cell Ba2+ currents in inner hair cells consist mainly of the nimodipine-sensitive current (approximately 85%), the utricular hair cells express only approximately 50% of this channel subtype. Thus, differential expression of alpha1D channels in the cochlear and utricular hair cells confers the phenotype of the alpha1D null mutant mice. Because vestibular and cochlear hair cells share common features and null deletion of several genes have yielded both deafness and imbalance in mice, alpha1D null mutant mice may serve as a model to disentangle vestibular from auditory-specific functions. [ABSTRACT FROM AUTHOR]