Abstract
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 (OtofAla515,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.
Highlights
The extremely precise encoding of sound temporal features by the first synapse of the mammalian auditory system, that is, between the sensory inner hair cell (IHC) and the primary auditory neuron, is crucial for many perceptive tasks
We investigated the roles of otoferlin in the IHC synaptic vesicle cycle through a mutagenesis strategy similar to that previously used to demonstrate that Syt1 and Syt2 function as Ca2+ sensors for fast exocytosis, and that Syt7 functions as the Ca2+ sensor for synaptic facilitation, at central nervous system synapses (Fernandez-Chacon et al, 2001; Schneggenburger et al, 2012; Jackman et al, 2016)
We investigated the role of otoferlin in the kinetics of readily releasable pool (RRP) synaptic vesicle fusion further, by analyzing the DCm elicited by brief depolarizations, of 2 to 50 ms duration, to À10 mV (Figure 5B1), first in low intracellular Ca2+-buffering conditions with an intracellular solution containing 0.5 mM EGTA
Summary
The extremely precise encoding of sound temporal features by the first synapse of the mammalian auditory system, that is, between the sensory inner hair cell (IHC) and the primary auditory neuron, is crucial for many perceptive tasks. Sound-evoked mechanical stimulation of the IHC sensory antenna, the hair bundle, induces changes in membrane potential, modulating synaptic exocytosis with submillisecond precision (Glowatzki and Fuchs, 2002; Goutman, 2012; Li et al, 2014) This temporal precision exceeds that for most conventional synapses, and allows sound-evoked action potentials of the primary auditory neurons to be phase-locked to the sinusoidal acoustic signal up to frequencies of ~4 kHz (Fuchs, 2005; Moser et al, 2006; Safieddine et al, 2012). Most of the vesicles in each synapse are tethered to a ribbon-shaped osmiophilic structure ( the name ‘ribbon synapse’), presumably forming a pool of primed vesicles for the immediate and sustained replenishment of the pool of fusion-competent vesicles located between the base of the ribbon and the presynaptic plasma membrane (von Gersdorff and Matthews, 1997; Lenzi et al, 1999; Moser and Beutner, 2000)
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