Disruption of Otoferlin Alters the Mode of Exocytosis at the Mouse Inner Hair Cell Ribbon Synapse

Abstract

Sound encoding relies on Ca²⁺-mediated exocytosis at the ribbon synapse between cochlear inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs). Otoferlin, a multi-C₂ domain protein, is proposed to regulate Ca²⁺-triggered exocytosis at this synapse, but the precise mechanisms of otoferlin function remain to be elucidated. Here, performing whole-cell voltage-clamp recordings of excitatory postsynaptic currents (EPSCs) from SGNs in otoferlin mutant mice, we investigated the impact of <i>Otof</i> disruption at individual synapses with single release event resolution. <i>Otof</i> deletion decreased the spontaneous release rate and abolished the stimulus-secretion coupling. This was evident from failure of potassium-induced IHC depolarization to stimulate release and supports the proposed role of otoferlin in Ca²⁺ sensing for fusion. A missense mutation in the <i>Otof</i> gene (pachanga), in which otoferlin level at the IHC plasma membrane was lowered without changing its Ca²⁺ binding, also reduced the spontaneous release rate but spared the stimulus-secretion coupling. The slowed stimulated release rate supports the hypothesis that a sufficient abundance of otoferlin at the plasma membrane is crucial for the vesicle supply. Large-sized monophasic EPSCs remained present upon <i>Otof</i> deletion despite the drastic reduction of the rate of exocytosis. However, EPSC amplitude, on average, was modestly decreased. Moreover, a reduced contribution of multiphasic EPSC was observed in both <i>Otof</i> mutants. We argue that the presence of large monophasic EPSCs despite the exocytic defect upon <i>Otof</i> deletion supports the uniquantal hypothesis of transmitter release at the IHC ribbon synapse. Based upon the reduced contribution of multiphasic EPSC, we propose a role of otoferlin in regulating the mode of exocytosis in IHCs.