SCAMP5 plays a critical role in axonal trafficking and synaptic localization of NHE6 to adjust quantal size at glutamatergic synapses.

Glutamate uptake into synaptic vesicles (SVs) depends on cation/H+ exchange activity, which converts the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane at the presynaptic terminals. Thus, the proper recruitment of cation/H+ exchanger to SVs is important in determining glutamate quantal size, yet little is known about its localization mechanism. Here, we found that secretory carrier membrane protein 5 (SCAMP5) interacted with the cation/H+ exchanger NHE6, and this interaction regulated NHE6 recruitment to glutamatergic presynaptic terminals. Protein-protein interaction analysis with truncated constructs revealed that the 2/3 loop domain of SCAMP5 is directly associated with the C-terminal region of NHE6. The use of optical imaging and electrophysiological recording showed that small hairpin RNA-mediated knockdown (KD) of SCAMP5 or perturbation of SCAMP5/NHE6 interaction markedly inhibited axonal trafficking and the presynaptic localization of NHE6, leading to hyperacidification of SVs and a reduction in the quantal size of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 KD without causing additional defects. Together, our results reveal that as a key regulator of axonal trafficking and synaptic localization of NHE6, SCAMP5 could adjust presynaptic strength by regulating quantal size at glutamatergic synapses. Since both proteins are autism candidate genes, the reduced quantal size by interrupting their interaction may underscore synaptic dysfunction observed in autism.

Release Mode Dynamically Regulates the RRP Refilling Mechanism at Individual Hippocampal Synapses.

Synaptic strength and reliability are determined by the number of vesicles released per action potential and the availability of release-competent vesicles in the readily releasable pool (RRP). Compared with release of a single vesicle (univesicular release), multivesicular release (MVR) would speed up RRP depletion, yet whether the RRP is refilled differently during the two different release modes has not been investigated. Here, we address this question by quantitative optical imaging with an axon-targeting glutamate sensor, iGluSnFRpre. We found that hippocampal synapses preferentially release multiple vesicles per action potential at high extracellular calcium or by paired-pulse stimulation. When MVR prevails, the RRP is recovered very rapidly with a time constant of 430 ms. This rapid recovery is mediated by dynamin-dependent endocytosis followed by direct reuse of retrieved vesicles. Furthermore, our simulation proved that the portion of retrieved vesicles that directly refill the RRP increases dramatically (>70%) in MVR compared with that in univesicular release (<10%). These results suggest that the contribution of rapid and direct recruitment of retrieved vesicle to the RRP changes dynamically with release mode at the level of individual synapses, which suggests a form of presynaptic homeostatic plasticity for reliable synaptic transmission during various synaptic activity.SIGNIFICANCE STATEMENT The number of vesicles released in response to an action potential and the number of release competent vesicles in the readily releasable pool (RRP) are the fundamental determinants of synaptic efficacy. Despite its functional advantages, releasing multiple vesicles, especially at small synapses, can deplete the RRP after a couple of action potentials. To prevent failure of synaptic transmission, the RRP should be refilled rapidly, yet whether the RRP replenishment process is regulated by the release mode has not been investigated. Here, using quantitative optical glutamate imaging and simulation, we demonstrate that the contribution of the fast refilling mechanism changes with release mode at the level of individual synapses, suggesting a rapid form of presynaptic homeostatic plasticity during various synaptic activity.