EF-hand protein Ca ²⁺ buffers regulate Ca ²⁺ influx and exocytosis in sensory hair cells

Abstract

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.

Hair cells use active zones with different voltage dependence of Ca 2+ influx to decompose sounds into complementary neural codes

“…The present study corroborates the notion that the Ca 2+ -channel number is proportional to ribbon size in cochlear hair cells (11,30). We speculate that release rate for a given open probability scales linearly with the number of Ca 2+ channels, assuming nanodomain coupling (29,65,66), regardless of the AZ size. Future simultaneous measurements of AZ Ca 2+ influx and exocytosis or SGN firing will be required to analyze the consequences of presynaptic heterogeneity for transmitter release at IHC AZs.…”

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated Ca V 1.3 Ca 2+ channels at hair cell active zones

“…5C) and Ca 2+ efficiency (Fig. 5D) suggests that coupling of Ca 2+ influx to exocytosis (1,4,54) was largely unaltered in RIM2α-deficient IHCs, which contrasts findings at the in RIM1/2-deficient IHCs calyx of Held (18), which might reflect compensation by other molecular links between channels and release sites at the IHC AZ. Alternatively, a subtle alteration of Ca 2+ channel position relative to the vesicular Ca 2+ sensor on the scale of few nanometers might have gone undetected in our measurements of exocytic membrane capacitance changes.…”

Ca 2+ -binding protein 2 inhibits Ca 2+ -channel inactivation in mouse inner hair cells

“…This excess exocytosis was prevented after the coapplication of the synthetic Ca 2+ chelators ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) in a ruptured-patch configuration (Fig. S5C) at concentrations (0.5 mM each) that were previously approximately found to mimic the action of endogenous Ca 2+ buffers in IHCs (42). The excess of sustained IHC exocytosis in Cabp2-deficient mice might indicate a contribution of CaBP2 in Ca 2+ buffering at the active zone.…”

“…Lack of Ca 2+ buffering by CaBP2 might enable wider Ca 2+ spread, promoting extrasynaptic exocytosis. Such a scenario has been proposed as an explanation for excessive sustained exocytosis in triple EF-hand, Ca 2+ -buffer KO mice (42)…”

“…One way to probe synaptic release largely independent from potential extrasynaptic exocytosis is to record the sound-evoked spiking of single SGNs, each of which is thought to be driven by a single active zone (42). Therefore, and to analyze how Cabp2 deficiency affects synaptic sound encoding, we performed in vivo extracellular recordings from single SGNs where they enter the cochlear nucleus (43).…”