EF-hand protein Ca2+ buffers regulate Ca2+ influx and exocytosis in sensory hair cells

EF-hand Ca(2+)-binding proteins are thought to shape the spatiotemporal properties of cellular Ca(2+) 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 Ca(2+) 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 Ca(2+)-dependent inactivation of their Ca(2+) current. Exocytosis of readily releasable vesicles remained unchanged, in accordance with the estimated tight spatial coupling of Ca(2+) channels and release sites (effective "coupling distance" of 17 nm). Substitution experiments with synthetic Ca(2+) chelators indicated the presence of endogenous Ca(2+) buffers equivalent to 1 mM synthetic Ca(2+)-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 Ca(2+) buffers regulate presynaptic IHC function for metabolically efficient sound coding.