The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (⌬C m ) in low-frequency (apical region, ϳ300 Hz) and high-frequency (basal, ϳ30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mM extracellular Ca 2ϩ and body temperature). With maturation, the Ca 2ϩ efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca 2ϩ cooperativity of exocytosis despite the smaller ⌬C m in apical cells. After maturation, ⌬C m in high-frequency IHCs increased linearly with the Ca 2ϩ current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca 2ϩ sensor otoferlin or the spatial coupling between Ca 2ϩ channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells. However, the nature of the Ca(2+) sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca(2+) dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca(2+) dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca(2+) sensitivity of vesicle fusion at hair cell synapses.

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...

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type CaV1.3 Ca2+ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca2+ channels in immature mouse IHCs under near-physiological recording conditions. CaV1.3 Ca2+ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca2+ action potential activity characteristic of these immature cells. The CaV1.3 Ca2+ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca2+ currents indicated that very few Ca2+ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca2+ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca2+ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.