Cochlear inner hair cells (IHCs) develop from pre-sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) Ca V 1.3 channels formed stripe-like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic Ca V 1.3-channels were selectively reduced, (iv) the intrinsic Ca 2+ dependence of fast exocytosis probed by Ca 2+ uncaging remained unchanged but (v) the apparent Ca 2+ dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca 2+ influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca 2+ influx through an individual channel dominates the [Ca 2+ ] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca 2+ influx and exocytosis.

EF-hand Ca2+-binding proteins are thought to shape the spatiotemporal properties of cellular Ca2+ signaling and are prominently expressed in sensory hair cells in the ear. Here, we combined genetic disruption of parvalbumin-α, calbindin-D28k, and calretinin in mice with patch-clamp recording, in vivo physiology, and mathematical modeling to study their role in Ca2+ signaling, exocytosis, and sound encoding at the synapses of inner hair cells (IHCs). IHCs lacking all three proteins showed excessive exocytosis during prolonged depolarizations, despite enhanced Ca2+-dependent inactivation of their Ca2+ current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca2+ channels and release sites (effective “coupling distance” of 17 nm). Substitution experiments with synthetic Ca2+ chelators indicated the presence of endogenous Ca2+ buffers equivalent to 1 mM synthetic Ca2+-binding sites, approximately half of them with kinetics as fast as 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Synaptic sound encoding was largely unaltered, suggesting that excess exocytosis occurs extrasynaptically. We conclude that EF-hand Ca2+ buffers regulate presynaptic IHC function for metabolically efficient sound coding.