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Despite being a highly enriched synaptic vesicle (SV) protein and a candidate gene for autism, the physiological function of SCAMP5 remains mostly enigmatic. Here, using optical imaging and electrophysiological experiments, we demonstrate that SCAMP5 plays a critical role in release site clearance at the active zone. Truncation analysis revealed that the 2/3 loop domain of SCAMP5 directly interacts with adaptor protein 2, and this interaction is critical for its role in release site clearance. Knockdown (KD) of SCAMP5 exhibited pronounced synaptic depression accompanied by a slower recovery of the SV pool. Moreover, it induced a strong frequency-dependent short-term depression of synaptic release, even under the condition of sufficient release-ready SVs. Super-resolution microscopy further proved the defects in SV protein clearance induced by KD. Thus, reduced expression of SCAMP5 may impair the efficiency of SV clearance at the active zone, and this might relate to the synaptic dysfunction observed in autism.

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca 2+ dependent. However, the underlying mechanism of how small changes in basal Ca 2+ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca 2+ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca 2+ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca 2+ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca 2+ channels. A high concentration of intracellular Ca 2+ buffer or blockade of calmodulin abolished depolarization-induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization-induced increases in readily releasable pool size, and this increase was abolished by the blockade of calmodulin. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca 2+ channels in coupling subthreshold depolarization to the activation of Ca 2+ -dependent signaling molecules that regulate transmitter release.

Neurotransmitter release occurs either synchronously to action potentials or spontaneously, yet whether molecular machineries underlying evoked and spontaneous release are identical, especially whether voltage-gated Ca2+ channels (VGCCs) can trigger spontaneous events has been in debate. To elucidate this issue, we characterized Ca2+ dependency of miniature excitatory postsynaptic currents (mEPSCs) in autaptic cultured hippocampal neurons. We found that 58 % mEPSC frequency was dependent on extracellular Ca2+([Ca2+]o), and Ca2+cooperativity of spontaneous release was comparable to that of evoked release. Moreover, most (> 90 %) of [Ca2+]o-dependent mEPSCs was attributable to VGCCs. Coupling distance between VGCCs and Ca2+ sensors was estimated as tight for both spontaneous and evoked release (~22 nm). In hippocampal slices, VGCC-dependence on spontaneous release was also observed, but to a different extent, at different areas and ages. At the calyx of Held synapses, mEPSCs showed VGCC-dependence in type 1 mature synapses where VGCCs and Ca2+ sensors are tightly coupled, but not in immature synapses. These data strongly suggest that the distance between VGCCs and Ca2+ sensors is the key factor to determine VGCC dependence of spontaneous release.

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca2+-dependent. However, the underlying mechanism of how small changes in basal Ca2+caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca2+is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca2+that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca2+levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca2+channels. A high concentration of intracellular Ca2+buffer or blockade of calmodulin and phospholipase C abolished depolarization induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization induced increases in readily releasable pool size, and this increase was abolished by blockade of calmodulin or phospholipase C. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca2+channels in coupling subthreshold depolarization to the activation of Ca2+-dependent signaling molecules that regulate transmitter release.SIGNIFICANCENeuronal activities are encoded by action potentials, but subthreshold changes in resting membrane potentials also play important roles in regulating neuronal functions including synaptic transmission. It is, however, poorly understood how small changes in basal Ca2+induced by subthreshold depolarization regulate transmitter release triggered by a large increase in local Ca2+in presynaptic terminals. We demonstrate that L-type Ca2+channels are the major source of presynaptic Ca2+influx at basal state and during subthreshold depolarization, resulting in the activation of signaling molecules such as calmodulin and phospholipase C, which facilitate transmitter release by increasing both release probability and the readily releasable pool size. Our results provide mechanistic insight into how subthreshold potential changes contribute to regulating transmitter release.

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