Phase-locking requires efficient ca21 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 Ca21 changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca21 imaging to examine Ca21 dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca21 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 Ca21 ATPase (PMCA), the mitochondrial Ca21 uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca21 ATPase (SERCA), but not the Na1/Ca21 exchanger (NCX). Furthermore, we found that both the basal Ca21 level and the Ca21 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 Ca21 buffer, multiple Ca21 extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.