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

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.Intracellular Ca²⁺ signaling regulates a multitude of cellular processes. In sensory hair cells, Ca²⁺ is crucial for electrical frequency tuning, afferent synaptic transmission, and efferent modulation (reviewed in ref. 1). To separate these signaling pathways and maintain high temporal fidelity of neurotransmission, Ca²⁺ signals must be temporally limited and spatially confined to the site of action. Cells typically achieve this by localizing Ca²⁺ entry and by rapidly removing free Ca²⁺ ions via binding to cytosolic “buffers” and finally Ca²⁺ extrusion (2–4). Of the various EF-hand Ca²⁺-binding proteins, some seem to function primarily as Ca²⁺-dependent signaling proteins (e.g., calmodulin and Ca²⁺-binding proteins 1–8, CaBP1–8), whereas others [parvalbumin-α (PVα), calbindin-D28k (CB), and calretinin (CR)] are thought to mainly serve as mobile Ca²⁺ buffers.Hair cells of various species strongly express the Ca²⁺-binding proteins PV, CB, and, in some cases, CR. This possibly reflects the need for buffers with different biophysical properties to functionally isolate different Ca²⁺ signaling mechanisms, which are spatially not well separated in these compact epithelial cells. Ca²⁺-binding proteins are particularly abundant in frog and chicken hair cells, which contain millimolar concentrations of parvalbumin-3 (5) as well as of CR (6, 7). An immune-EM study in rats indicated hundreds of micromolar of proteinaceous Ca²⁺-binding sites in inner hair cells (IHCs) (8). A patch-clamp study in gerbil IHCs reported endogenous buffers equivalent to approximately 0.4 mM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (9). Genetic deletion of the EF-hand Ca²⁺-binding proteins PVα, CB, and CR in mice has facilitated the analysis of their function (10–13; reviewed in ref. 14), but the combined deletion of these proteins remains to be studied. IHCs provide an experimentally well-accessible presynaptic preparation that uses all three. Here, we studied IHC function and hearing in mice lacking the three buffers [triple buffer KO (TKO); Pv^−/−Cb^−/−Cr^−/−]. By using perforated and ruptured-patch recordings, we analyzed voltage-gated Ca²⁺ currents and exocytosis of Pv^−/−Cb^−/−Cr^−/− IHCs, in which we also substituted the deleted endogenous buffers with the synthetic Ca²⁺ chelators EGTA or BAPTA. Auditory systems function was probed by measuring otoacoustic emissions and auditory brainstem responses (ABRs) as well as by recordings from single spiral ganglion neurons (SGNs). We performed mathematical modeling to estimate concentrations of the endogenous mobile Ca²⁺ buffers and to better understand how these proteins control exocytosis at IHC synapses. We conclude that the endogenous buffer capacity of IHCs is well approximated by 1 mM synthetic Ca²⁺-binding sites with different kinetics. A tight spatial coupling between Ca²⁺ channels and sensors of exocytosis (Ca²⁺ channel-exocytosis coupling) precludes interference of PVα, CB, and CR with fusion of the readily releasable pool of vesicles (RRP). Instead, we suggest that these buffers jointly regulate IHC presynaptic function by restricting neurotransmitter release to active zones (AZs).