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. 1994). This occurs because the pores of Ca2+ channels contain glutamate residues that can bind H+ ions at a site close the channel mouth and this extremely fast binding reduces Ca2+ channel conductance (Prod’hom et al., 1987, 1989; Chen et al., 1996). To date, evidence for a block of Ca2+ currents by vesicular protons has come primarily from retinal ribbon-type synapses (DeVries, 2001; Palmer et al., 2003; Jarsky et al., 2010). At these synapses, presynaptic L-type Ca2+ channels are located a few nanometers from docked synaptic vesicles within specialized active zones that contain a synaptic ribbon organelle (Zenisek et al., 2003; Vaithianathan and Matthews, 2014). However, auditory and vestibular hair cells also contain synaptic ribbons (Liberman et al., 1990; Nouvian et al., 2006). Here, graded membrane potential changes, sometimes fluctuating in the submillisecond time scale and as small as 0.2 mV, are able Fn1 to gate the entry of Ca2+ ions through L-type Ca2+ channels (Russell and Sellick, 1983; Spassova et al., 2001). The resulting rapid changes in free Ca2+ ion concentration trigger glutamate release at the basal pole of hair cells in response to sound-evoked signals. This rapid secretion of neurotransmitter occurs via a highly synchronous form of multivesicular release (Glowatzki and Fuchs, 2002; Grant et al., 2010; Schnee et al., 2013), although fusion pore flickering of uniquantal release may also play a role (Chapochnikov et al., 2014). Recent studies suggest that protons are also released from vestibular hair cells and may act as nonquantal neurotransmitters that directly SB 252218 affect postsynaptic nerve terminals (Highstein et al., 2014). However, evidence for an effect of released protons on Ca2+ channels has not been described for hair cell synapses. Here, we show that a copious amount of protons are released from auditory hair cells in the bullfrog amphibian papilla. We reveal the features that can mask this effect, such as underlying T-type Ca2+currents, hyperpolarized resting membrane potentials, and specific drugs that change vesicle or extracellular pH buffering strength. We also show that multivesicular release promotes synaptic cleft acidification. Finally, we suggest that this proton-mediated SB 252218 Ca2+ channel block may function to reduce unnecessary Ca2+ influx, increase exocytosis efficiency, and modulate short-term plasticity. Materials and Methods Hair cell preparation. After an Oregon Health & Science University Institutional Animal Care and Use CommitteeCapproved animal care protocol, amphibian papillae were carefully dissected from adult female or male bullfrogs (= 20) and 23.7 1.7 M for afferent fibers (= 18). Whole-cell recordings were electronically compensated by 0% to 30% depending on the uncompensated series resistance to maintain a constant series resistance throughout the recordings. Capacitance measurements. The measurements of the whole-cell membrane capacitance (= 20). The increase of tests. Data are expressed as mean SEM. Results Ca2+ currents and synaptic transmission at different hair cell holding potentials We performed paired whole-cell recordings of hair cells and their afferent fibers. The depolarization of hair cells activates Ca2+ currents and the subsequent increase in Ca2+ ion concentration triggers glutamate release. However, the profile of the SB 252218 Ca2+ currents was different depending on the hair cell holding potential (Fig. 1= 19; Fig. 1= 19). Importantly, the average EPSC peak amplitudes were significantly larger when hair cells were.