Exocytosis at mammalian vestibular ribbon synapses shows a high-order Ca2+ dependence and does not require synaptotagmin-4

Inner ear sensory synapses faithfully transduce information over a wide range of stimulus intensities for prolonged periods of time. The efficiency of such demanding and stringent exocytotic activity depends on the presence of specialised presynaptic ribbons in the sensory hair cells. Ribbons are electron dense structures able to tether a large number of releasable vesicles at the synaptic active zone and can maintain high rates of vesicle release. Calcium entry through CaV1.3 (L-type) Ca2+ channels in response to cell depolarization causes local increases in Ca2+ at the ribbon synapses, which is detected by the exocytotic Ca2+ sensors. At ribbon synapses of mature vestibular hair cells (VHCs), the coupling between Ca2+ channels and the exocytotic Ca2+ sensor remains unclear. We studied the Ca2+ dependence of exocytosis and the release kinetics of different vesicle pool populations in mature synaptotagmin-4 (Syt-4) mouse VHCs using patch-clamp capacitance measurements under physiological recording conditions. Exocytosis in VHCs showed a high order dependence on Ca2+ entry, which contrasts with the linear Ca2+ dependence observed in adult mammalian auditory inner hair cells (IHCs). The synaptic properties of mature VHCs, including the characteristics of the Ca2+ current and dynamics of vesicle release, were not affected by an absence of Syt-4. Our findings show that the coupling between Ca2+ influx and neurotransmitter release at VHC ribbon synapses is described by a non-linear relation that is likely to be more appropriate for the faithful encoding of low frequency vestibular information, consistent with that observed in very low frequency mammalian IHCs.