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The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca2+- and stimulus-dependent compensating CME in mature IHCs. [ABSTRACT FROM AUTHOR]

Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism. [ABSTRACT FROM AUTHOR]

The exquisite performance of the mammalian hearing organ results from a finely orchestrated array of cell types, and their highly specialized functions are determined by their gene expression profile. In rodents, this profile is established mainly during the first 2 weeks of postnatal maturation. In this paper, we used the differential display technique on the rat organ of Corti to uncover transcripts upregulated in expression between postnatal stages P0 and P14. A total of 176 different genes were identified, the mRNA amount of which increased during early postnatal development. The transcripts code for proteins serving a broad spectrum of cellular functions including intracellular signaling, control of growth/differentiation, regulation of protein synthesis/degradation/modification, metabolism and synaptic function. In addition, the set of upregulated transcripts contained several proteins of yet unknown function, as well as hypothetical proteins and so far unknown mRNA sequences. Thus, this study unravels the broad and specific transcription program that operates the maturation of the mammalian hearing organ. Further, as 49 of the genes found here map to at least one unspecified deafness locus, our study provides candidate genes for these and novel deafness loci. [ABSTRACT FROM AUTHOR]

Large-conductance voltage- and Ca2+-activated K+ channels (BKCa) are involved in shaping spiking patterns in many neurons. Less is known about their role in mammalian inner hair cells (IHCs), mechanosensory cells with unusually large BKCa currents. These currents may be involved in shaping the receptor potential, implying crucial importance for the properties of afferent auditory signals. We addressed the function of BKCa by recording sound-induced responses of afferent auditory nerve (AN) fibers from mice with a targeted deletion of the pore-forming α-subunit of BKCa (BKα-/-) and comparing these with voltage responses of current-clamped IHCs. BKCa-mediated currents in IHCs were selectively abolished in BKα-/-, whereas cochlear physiology was essentially normal with respect to cochlear sensitivity and frequency tuning. BKα-/- AN fibers showed deteriorated precision of spike timing, measured as an increased variance of first spike latency in response to tone bursts. This impairment could be explained by a slowed voltage response in the presynaptic IHC resulting from the reduced K+ conductance in the absence of BKCa. Maximum spike rates of AN fibers were reduced nearly two-fold in BKα-/-, contrasting with increased voltage responses of IHCs. In addition to presynaptic changes, which may be secondary to a modest depolarization of BKα-/- IHCs, this reduction in AN rates suggests a role of BKCa in postsynaptic AN neurons, which was supported by increased refractory periods. In summary, our results indicate an essential role of IHC BKCa channels for precise timing of high-frequency cochlear signaling as well as a function of BKCa in the primary afferent neuron. [ABSTRACT FROM AUTHOR]

Presents a study that investigated the presence of KCNQ currents in cochlear inner hair cells (IHCs). Background on IHCs; Materials and methods; Results of the study.

Prestin, a transmembrane protein found in the outer hair cells of the cochlea, represents a new type of molecular motor, which is likely to be of great interest to molecular cell biologists. In contrast to enzymatic-activity-based motors, prestin is a direct voltage-to-force converter, which uses cytoplasmic anions as extrinsic voltage sensors and can operate at microsecond rates. As prestin mediates changes in outer hair cell length in response to membrane potential variations, it might be responsible for sound amplification in the mammalian hearing organ. [ABSTRACT FROM AUTHOR]

Investigates reciprocal electromechanical properties of the rat homologue of prestin, a protein with similarity to pendrin-related anion transporters. Unique property of outer hair cells of the mammalian cohclea; Mechanical force generated by prestin in response to electrical stimulation; Mechanoelectrical properties of prestin.

Studies the contribution of the membrane protein prestin to the electromotility of the outer hair cells (OHC) of the mammalian cochlea. Characterization of the prestin molecule; Possibility that the voltage sensor may be a charged particle extrinsic to the protein; Elimination of voltage-dependent motility of OHC after removal of chloride.