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Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca 2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca 2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca 2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca 2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X 3 receptors, the increase and synchronization of the Ca 2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca 2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca 2+ signals from sensory and non-sensory cells., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1(Bth/Bth)) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca(2+) permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1(Bth/Bth) OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca(2+) dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1(Bth/Bth) OHCs, whereas raising the intracellular concentration of the Ca(2+) chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca(2+) permeability of the mutated MET channel indirectly causing the Ca(2+) sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca(2+) influx as a compensatory mechanism., Significance Statement: In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca(2+) permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca(2+)-dependent adaptation appear stronger, in compensation for the reduced Ca(2+) entry., (Copyright © 2016 Corns et al.)

Mechanotransduction in the auditory and vestibular systems depends on mechanosensitive ion channels in the stereociliary bundles that project from the apical surface of the sensory hair cells. In lower vertebrates, when the mechanoelectrical transducer (MET) channels are opened by movement of the bundle in the excitatory direction, Ca(2+) entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca(2+)-dependent adaptation is also present in mammalian hair cells. Hair bundles of both outer and inner hair cells from mice were deflected by using sinewave or step mechanical stimuli applied using a piezo-driven fluid jet. We found that when cochlear hair cells were depolarized near the Ca(2+) reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca(2+) concentration (40 µM), all manifestations of adaptation, including the rapid decline of the MET current and the reduction of the available resting MET current, were abolished. MET channel adaptation was also reduced or removed when the intracellular Ca(2+) buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was increased from a concentration of 0.1 to 10 mM. The findings show that MET current adaptation in mouse auditory hair cells is modulated similarly by extracellular Ca(2+), intracellular Ca(2+) buffering, and membrane potential, by their common effect on intracellular free Ca(2+).

Developmental changes in the coupling between Ca2+ entry and exocytosis were studied in mouse inner hair cells (IHCs) which, together with the afferent endings, form the primary synapse of the mammalian auditory system. Ca2+ currents (ICa) and changes in membrane capacitance (DeltaCm) were recorded using whole-cell voltage clamp from cells maintained at body temperature, using physiological (1.3 mM) extracellular Ca2+. The magnitudes of both ICa and DeltaCm increased with maturation from embryonic stages until postnatal day 6 (P6). Subsequently, ICa gradually declined to a steady level of about -100 pA from P13 while the Ca2+-induced DeltaCm remained relatively constant, indicating a developmental increase in the Ca2+ efficiency of exocytosis. Although the size of ICa changed during development, its activation properties did not, suggesting the presence of a homogeneous population of Ca2+ channels in IHCs throughout development. The Ca2+ dependence of exocytosis changed with maturation from a fourth power relation in immature cells to an approximately linear relation in mature cells. This change applies to the release of both a readily releasable pool (RRP) and a slower secondary pool of vesicles, implying a common release mechanism for these two kinetically distinct pools that becomes modified during development. The increased Ca2+ efficiency and linear Ca2+ dependence of mature IHC exocytosis, especially over the physiological range of intracellular Ca2+, could improve the high-fidelity transmission of both brief and long-lasting stimulation. These properties make the mature cell ideally suited for fine intensity discrimination over a wide dynamic range.

Ca(2+)-activated K(+) currents were studied in inner hair cells (IHCs) of mature mice. I(K,f), the large-conductance Ca(2+)-activated K(+) current (BK) characteristic of mature IHCs, had a fast activation time constant (0.4 ms at -25 mV at room temperature) and did not inactivate during 170 ms. Its amplitude, measured at -25 mV, and activation time constant were similar between IHCs in the apical and basal regions of the cochlea. I(K,f) was selectively blocked by 30 nm IbTx but was unaffected by superfusion of Ca(2+)-free solution, nifedipine or Bay K 8644, excluding the direct involvement of voltage-gated Ca(2+) channels in I(K,f) activation. Increasing the intracellular concentration of the Ca(2+) chelator BAPTA from 0.1 mm to 30 mm reduced the amplitude of I(K,f) at -25 mV and shifted its activation by 37 mV towards more depolarized potentials. A reduction in the size of I(K,f) and a depolarizing shift of its activation were also seen when either thapsigargin and caffeine or ryanodine were added intracellularly, suggesting that I(K,f) is modulated by voltage-dependent release from intracellular Ca(2+) stores. Mature IHCs had a small additional Ca(2+)-activated K(+) current (I(K(Ca))), activated by Ca(2+) flowing through L-type Ca(2+) channels. This current was still present during superfusion of either IbTx (60 nm) or apamin (300 nm) but was abolished in Cs(+)-based intracellular solution or during superfusion of 5 mm TEA, suggesting the presence of an additional BK-channel type. Current clamp experiments at body temperature show that I(K,f), but not I(K(Ca)), is essential for fast voltage responses of mature IHCs.

From just after birth, mouse inner hair cells (IHCs) expressed a Ca²-activated K+ current that was reduced by intracellular BAPTA at concentrations ≥ 1 mM. The block of this current by nifedipine suggests the direct involvement of Cav 1.3 Ca2+ channels in its activation. On the basis of its high sensitivity to apamin (KD 360 pM) it was identified as a small-conductance Ca2+-activated K+ current (SK), probably SK2. A similar current was also found in outer hair cells (OHCs) from the beginning of the second postnatal week. In both cell types the appearance of the SK current coincided with their becoming responsive to acetylcholine (ACh), the main efferent neurotransmitter in the cochlea. The effect of ACh on IHCs was abolished when they were simultaneously superfused with strychnine, consistent with the presence of nicotinic ACh receptors (nAChRs). Extracellular Ca2+ either potentiated or blocked the nAChR current depending on its concentration, as previously reported for the recombinant α9α10 nAChR. Outward currents activated by ACh were reduced by blocking the SK current with apamin or by preventing SK current activation with intracellular BAPTA (≥ 10 mM). The endogenous mobile Ca2+ buffer concentration was estimated to be equivalent to about 1 mM BAPTA, suggesting that in physiological conditions the SK channel is significantly activated by Ca2+ influx through both Cav 1.3 Ca2+ channels and α9α10 nAChRs. Current clamp experiments showed that in IHCs the SK current is required for sustaining a train of action potentials and also modulates their frequency when activated by ACh. [ABSTRACT FROM AUTHOR]