Phase-Locking Requires Efficient Ca²⁺ Extrusion at the Auditory Hair Cell Ribbon Synapse

Abstract

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca²⁺ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca²⁺ imaging to examine Ca²⁺ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca²⁺ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca²⁺ ATPase (PMCA), the mitochondrial Ca²⁺ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca²⁺ ATPase (SERCA), but not the Na⁺/Ca²⁺ exchanger (NCX). Furthermore, we found that both the basal Ca²⁺ level and the Ca²⁺ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca²⁺ buffer, multiple Ca²⁺ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.

SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca²⁺ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca²⁺ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca²⁺ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca²⁺ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca²⁺ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.