The large conductance voltage-and Ca 2؉ -activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming ␣-subunit coexpressed with the auxiliary ␤1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel ␣-(BK␣ ؊/؊ ) and ␤1-subunit (BK␤1 ؊/؊ ) knockout mice, we demonstrate normal hearing function and cochlear structure of BK␤1 ؊/؊ mice. During the first 4 postnatal weeks also, BK␣ ؊/؊ mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BK␣ ؊/؊ mice only from Ϸ8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.cochlea ͉ KCNQ4 C a 2ϩ -activated potassium (BK) channels are heterooctamers of four ␣-and four ␤-subunits. The pore-forming ␣-subunit (KCNMA1) is a member of the slo family of potassium channels (1), originally identified in Drosophila (2). Studies of BK channels from smooth muscle have identified an auxiliary ␤1-subunit (KCNMB1) whose presence in the channel complex confers an increased voltage and calcium sensitivity toward the poreforming ␣-subunit (3).In turtle and chick, there is evidence that differential splicing of the BK channel ␣-subunit in conjunction with a graded expression of the auxiliary ␤-subunit along the tonotopic axis provides the functional heterogeneity of BK channels that underlies electrical tuning (for review, see ref. 4).In inner hair cells (IHCs) of the mammalian organ of Corti, the predominant K ϩ conductance is a voltage-and Ca 2ϩ -activated K ϩ channel termed I K,f (5, 6). BK channel mRNA (7,8) and protein expression (8) were shown in IHCs, indicating that I K,f flows through BK channels. The presumed physiological roles of BK channels are (i) a decrease of the membrane time constant even at the resting potential and (ii) fast repolarization of the receptor potential. Both contribute to phase-locked receptor potentials up to high sound frequencies (6). In addition to IHCs, BK type Ca 2ϩ -activated K ϩ conductances have been measured in OHCs (9) and in efferent fibers onto outer hair cells (OHCs) (10). The role of BKs in either OHCs or efferents is still controversially discussed (9).Studying the expression of BK channel ␣-splice variants and ␤-isoforms in rat cochlea using in situ hybridization and PCR techniques revealed the strict coexpressio...

Mutations of the human otoferlin gene lead to an autosomal recessive nonsyndromic form of prelingual, sensorineural deafness (deafness autosomal recessive 9, DFNB9). Several studies have demonstrated expression of otoferlin in the inner ear and brain, and suggested a role of otoferlin in Ca(2+)-triggered exocytosis. So far, otoferlin expression profiles were solely based on the detection of mRNA. Here, we analysed the expression of otoferlin protein and mRNA using immunohistochemistry, in situ hybridization and RT-PCR in neonatal and mature Wistar rat tissue. In agreement with previous studies, otoferlin expression was found in the brain and in inner and vestibular hair cells. Otoferlin mRNA and protein was, however, also detected in mature outer hair cells of low-frequency processing cochlear turns and in auditory nerve fibres. In outer, inner and vestibular hair cells, otoferlin was subcellularly localized at a considerable distance from the presumed active release sites. Double-staining with the synaptic ribbon marker, C-terminal binding protein 2 (CtBP2), or the presynaptic Ca(2+)-channel, Ca(v)1.3, both assumed to mark the sites of vesicle fusion and transmitter release, did not colocalize with otoferlin expression and thus do not necessarily support a selected role of otoferlin in Ca(2+)-triggered exocytosis. The widespread distribution of otoferlin in neurons, nerve fibres and hair cells, and its subcellular distribution extending beyond the regions of synaptic vesicle fusion, i.e. coenrichment with the cytosolic Golgi matrix protein 130 (GM130) in inner hair cells or the early endosomal autoantigen 1 (EEA1) in outer hair cells support instead the idea of a more ubiquitous role of otoferlin in early/recycling endosome trans-Golgi network dynamics.

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Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.

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Ciliary neurotrophic factor (CNTF) signals via a tripartite receptor complex consisting of the glycosyl-phosphatidylinositol (GPI)-anchored CNTF receptor (CNTF-R), the leukaemia inhibitory factor receptor (LIF-R) and the interleukin-6 (IL-6) signal transducer gp130. We have recently reported that gp130 is endogenously expressed in the polarised epithelial model cell line Madin-Darby canine kidney (MDCK) and we have demonstrated a preferential basolateral localisation of this protein. In the present study we show that MDCK cells also express the LIF-R and respond to stimulation with human LIF by activation of tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3), both however in an unpolarised fashion. This suggests that MDCK cells may be target cells for LIF. We have furthermore stably expressed the human CNTF-R in MDCK cells and by two different assays we found an apical localisation. Consistent with these findings, stimulation of CNTF-R-positive cells resulted only in an activation of STAT3 when CNTF was added apically. These data demonstrate that each subunit of the CNTF receptor complex has a distinct distribution in polarised cells which may reflect the different roles the respective cytokines play in vivo.Since it is currently believed that lipid rafts are involved in signal transduction as well as protein sorting we studied the association of the three receptor complex components with membrane rafts using different protocols. Whereas the CNTF-R cofractionated quantitatively with lipid rafts independently of the method used, gp130 and the LIF-R were found to associate with lipid rafts only partially when detergents were used for isolation. These findings could indicate that either the three receptor complex subunits are localised to the same kind of raft but with different affinities to the liquid-ordered environment, or that they are localised to different types of rafts. CNTF-, LIF-, and IL-6-dependent STAT3 activation was sensitive to the cholesterol-depleting drug methyl-β-cyclodextrin (MCD) suggesting that the integrity of lipid rafts is important for IL-6-type cytokine-induced STAT activation.

Cochlear outer hair cells (OHCs) terminally differentiate prior to the onset of hearing. During this time period, thyroid hormone (TH) dramatically influences inner ear development. It has been shown recently that TH enhances the expression of the motor protein prestin via liganded TH receptor beta (TRbeta) while in contrast the expression of the potassium channel KCNQ4 is repressed by unliganded TRalpha1. These different mechanisms of TH regulation by TRalpha1 or TRbeta prompted us to analyse other ion channels that are required for the final differentiation of OHCs. We analysed the onset of expression of the Ca(2+) channel Ca(V)1.3, and the K(+) channels SK2 and BK and correlated the results with the regulation via TRalpha1 or TRbeta. The data support the hypothesis that proteins expressed in rodents prior to or briefly after birth like Ca(V)1.3 and prestin are either independent of TH (e.g. Ca(V)1.3) or enhanced through TRbeta (e.g. prestin). In contrast, proteins expressed in rodents later than P6 like KCNQ4 ( approximately P6), SK2 ( approximately P9) and BK ( approximately P11) are repressed through TRalpha1. We hypothesise that the precise regulation of expression of the latter genes requires a critical local TH level to overcome the TRalpha1 repression.