Otoferlin, a C2-domain-containing

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca2+ spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca2+ efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca2+ sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca2+-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1−/−,Syt2−/− and Syt7−/− mice). We only found a defect in RRP recovery in Syt1−/− mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2−/− and Syt7−/− mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca2+-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca2+-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.

A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.

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.