SNAP25 disease mutations change the energy landscape for synaptic exocytosis due to aberrant SNARE interactions

Abstract

AbstractSNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the Synaptotagmin-1 (Syt1) binding interface, and I67N, which destabilizes the SNARE-complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusionin vitroand in neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced Readily Releasable Pool (RRP) size, due to increased spontaneous (mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found inSyt1knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y>V48F) than in theSyt1KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the apparent height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.