The Role of alpha- and beta-SNAP in Synaptic Vesicle Exocytosis

Abstract

Neurotransmitter release is mediated by the neuronal SNARE proteins, which are transmembrane proteins whose assembly into ternary complexes is thought to drive membrane fusion of synaptic vesicles at axonal terminals. After fusion, spent SNARE complexes need to be disassembled in order to regenerate fusion-competent active SNAREs. This recycling step is performed by a conserved protein machinery consisting of the chaperone NSF and the co-chaperones SNAPs (alpha- and beta-SNAP, but probably not gamma-SNAP). The aim of the present study was to elucidate the functional role of alpha- and beta-SNAP in synaptic vesicle exocytosis at central synapses. The experimental approach consisted of analysing synaptic transmission in glutamatergic hippocampal neurons under conditions of decreased total alpha/beta-SNAP expression levels. I generated SNAP double mutant mice by crossing beta-SNAP deletion mutant with alpha-SNAP hypomorphic HYH mutant mice. Crossing of the two mutations resulted in a ~70% reduction of total combined alpha/beta-SNAP levels. Electrophysiological analysis of synaptic transmission showed that, while the readily releasable pool of synaptic vesicles (RRP) is only slightly smaller in SNAP double mutant neurons as compared to controls (~25% reduction), total release induced either by strong increases in intracellular Ca2+ levels or by 100 Hz stimulation trains is drastically reduced by ~60%. Detailed analysis of neurotransmitter release during stimulus trains showed that this strong reduction mainly arises from changes in the tonic release component, while the phasic release component, much like the RRP, is only slightly decreased. I propose a two-pool model to describe neurotransmitter release during high-frequency stimulation. According to this model, one pool of primed synaptic vesicles supports slow, tonic release of transmitter, while a second, the RRP, supports fast, phasic release. The two pools operate in a successive fashion with the RRP drawing vesicles from the tonically releasable pool. My study indicates that alpha and beta-SNAP are key regulators of the efficacy of central synapses. The levels of alpha- and beta-SNAP are critical in determining the size of the primed synaptic vesicle pool that supports tonic neurotransmitter release during activity trains and feeds the RRP necessary for fast phasic transmitter release.