Proton-mediated block of Ca²⁺ channels during multivesicular release regulates short-term plasticity at an auditory hair cell synapse

Synaptic vesicles release both neurotransmitter and protons during exocytosis, which may result in a transient acidification of the synaptic cleft that can block Ca²⁺ channels located close to the sites of exocytosis. Evidence for this effect has been reported for retinal ribbon-type synapses, but not for hair cell ribbon synapses. Here, we report evidence for proton release from bullfrog auditory hair cells when they are held at more physiological, in vivo-like holding potentials (V_h = —60 mV) that facilitate multivesicular release. During paired recordings of hair cells and afferent fibers, L-type voltage-gated Ca²⁺ currents showed a transient block, which was highly correlated with the EPSC amplitude (or the amount of glutamate release). This effect was masked at V_h = — 90 mV due to the presence of a T-type Ca²⁺ current and blocked by strong pH buffering with HEPES or TABS. Increasing vesicular pH with internal methylamine in hair cells also abolished the transient block. High concentrations of intracellular Ca²⁺ buffer (10 mM BAPTA) greatly reduced exocytosis and abolished the transient block of the Ca²⁺ current. We estimate that this transient block is due to the rapid multivesicular release of ~ 600 -1300 H⁺ ions per synaptic ribbon. Finally, during a train of depolarizing pulses, paired pulse plasticity was significantly changed by using 40 mM HEPES in addition to bicarbonate buffer. We propose that this transient block of Ca²⁺ current leads to more efficient exocytosis per Ca²⁺ ion influx and it may contribute to spike adaptation at the auditory nerve.