RESUMO
Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18-1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho-proteomics abolished the stimulatory effect of Munc18-1 on SNARE complex formation ("SNARE-templating") and membrane fusion in vitro Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18-1-null neurons expressing Munc18-1Y473D Synaptic transmission was temporarily restored by high-frequency stimulation, as well as by a Munc18-1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non-phosphorylatable Munc18-1 supported normal synaptic transmission. We propose that SFK-dependent Munc18-1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post-docking SNARE-templating role of Munc18-1, resulting in a largely abolished pool of releasable synaptic vesicles.
Assuntos
Proteínas Munc18/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Quinases da Família src/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteínas Munc18/genética , Mutação , Fosforilação/fisiologia , Estrutura Secundária de Proteína , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/genética , Vesículas Sinápticas/genética , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Quinases da Família src/genéticaRESUMO
Cellular trafficking pathways end with fusion reactions at the target. These reactions have been studied extensively for many decades, but recent studies have been particularly productive in providing new solutions to old problems, especially in some of the most complex fusion reactions, like synaptic vesicle secretion in neurons. Here, we discuss new studies that begin to merge ideas on three central questions: (A) are all releasable vesicles equally likely to undergo fusion, (B) do different fusion modes contribute to synaptic transmission, and (C) which molecular events are 'upstream' and which ones 'downstream' of SNARE complex assembly.