Didier Dulon

The relationship between intracellular free calcium and the motile responses of outer hair cells isolated from the guinea pig cochlea was examined. Calcium levels were modulated by the addition of the calcium ionophores ionomycin or A23187 to the incubation medium and monitored with the fluorescent calcium indicator fluo-3. In the presence of 1.25 mM external calcium, the application of either ionophore (10 microM) led to an increase in intracellular free calcium from 157 +/- 76 nM to 1200 +/- 500 nM within 30-60 sec. Concurrently, cells elongated by 1-2 microns, cell diameter decreased, and cell volume shrank by 269 +/- 220 microns 3 (5.0 +/- 4.1%). The reduction in diameter was most pronounced in the middle portion of the cell (4.4% +/- 4.2%), also evident in the apical region (3.1% +/- 4.8%) but not significant in the basal region near the nucleus. This response was observed in outer hair cells from basal and apical turns of the cochlea and was reversed when the cells were rinsed with calcium-free medium supplemented with 2 mM EGTA. Optical imaging of the cell membrane with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta] [2-(di-n-butylaminol)-6-naphthyl vinyl] pyridinium betaine during the elevation of intracellular calcium demonstrated features of contractility at the lateral cell membrane. A rise in intracellular calcium as well as the motile response was still observed after a 5-min exposure of the cells to a calcium-free solution (supplemented with 2 mM EGTA), indicating that the ionophore was also able to liberate calcium from intracellular sites. However, depletion of calcium stores through prolonged incubation of the cells in calcium-free medium (30-60 min) suppressed both the calcium signal and the cell response. The calmodulin inhibitors trifluoperazine and pimozide (30 microM) blocked the cell motility induced by ionomycin while they left the increase of intracellular calcium unaffected. These observations suggest that calcium-dependent circumferential contractions in outer hair cells are mediated by calmodulin. The application to the extracellular medium of putative neurotransmitters of the cochlear efferent system such as acetylcholine and GABA led to neither an increase in intracellular calcium nor a modification of cell shape. Therefore, these neurotransmitters may not be directly involved in calcium-induced contractions in outer hair cells. The circumferential contractions altered the stiffness of the plasma membrane and the turgor of the cell. Under normal conditions, changes in cell volume were inversely proportional to the osmotic pressure of the extracellular medium following van't Hoff's law.(ABSTRACT TRUNCATED AT 400 WORDS)

The cholinergic efferent inhibition of mammalian outer hair cells (OHCs) is mediated by a hyperpolarizing K+ current. We have made whole-cell tight-seal recordings from single OHCs isolated from the guinea pig cochlea to characterize the mechanism by which acetylcholine (ACh) activates K+ channels. After ACh application, OHCs exhibited a biphasic response: an early depolarizing current preceding the predominant hyperpolarizing K+ current. The current-voltage (I-V) relationship of the ACh-induced response displayed an N-shape, suggesting the involvement of Ca2+ influx. When whole-cell recording was combined with confocal calcium imaging, we simultaneously observed the ACh-induced K+ current (IK(ACh)) and a Ca2+ response restricted to the synaptic area of the cell. This IK(ACh) could be prevented by loading OHCs with 10 mM of the fast Ca2+ buffer bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (or BAPTA), therefore allowing the observation of the ACh-induced early current in isolation. This early current revealed nicotinic features because it activated with an intrinsic delay in the millisecond range, reversed nearly in between potassium and sodium equilibrium potentials, and was blocked by curare. However, it was strongly reduced in the absence of external Ca2+, and its I-V relationship displayed an unusual outward rectification at positive membrane potentials and an inward rectification below -60 mV. The results indicate that the cholinergic response of mammalian OHCs involves a "nicotinic-like" nonspecific cation channel through which Ca2+ enters and triggers activation of nearby Ca2+-dependent K+ channels.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof Ala515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Otoferlin, a C2-domain-containing Ca(2+) binding protein, is required for synaptic exocytosis in auditory hair cells. However, its exact role remains essentially unknown. Intriguingly enough, no balance defect has been observed in otoferlin-deficient (Otof(-/-)) mice. Here, we show that the vestibular nerve compound action potentials evoked during transient linear acceleration ramps in Otof(-/-) mice display higher threshold, lower amplitude, and increased latency compared with wild-type mice. Using patch-clamp capacitance measurement in intact utricles, we show that type I and type II hair cells display a remarkable linear transfer function between Ca(2+) entry, flowing through voltage-activated Ca(2+) channels, and exocytosis. This linear Ca(2+) dependence was observed when changing the Ca(2+) channel open probability or the Ca(2+) flux per channel during various test potentials. In Otof(-/-) hair cells, exocytosis displays slower kinetics, reduced Ca(2+) sensitivity, and nonlinear Ca(2+) dependence, despite morphologically normal synapses and normal Ca(2+) currents. We conclude that otoferlin is essential for a high-affinity Ca(2+) sensor function that allows efficient and linear encoding of low-intensity stimuli at the vestibular hair cell synapse.