The neuron has a remarkable ability to release quanta of neurotransmitters in less than amillisecond in response to the pulse of Ca2+. This fast synchronized release constitutes thefundamental basis of major brain activities. The release requires vesicle fusion, which isorchestrated by the fusion machine SNARE (soluble N-ethylmaleimide-sensitive factorattachment protein receptor) proteins, a priming agent Complexin (Cpx), and the Ca2+ switchSynatotagmin 1 (Syt1). This research project uses the innovative single-vesicle fusion assay to investigatethe mechanism by which SNARE-dependent vesicle fusion is regulated by Cpx, Syt1, andCa2+. The single-vesicle fusion assay makes it possible to dissect the sequential intermediatestages of vesicle fusion, overcoming major drawbacks of conventional bulk fusion assays.The technique also allows us to track the dynamic transitions in a single fusion event withmillisecond time resolution. With this powerful approach, we intend to reconstitutesynchronized vesicle fusion in a test tube. Specifically, we test a mechanistic modelproposing that Cpx arrests an intermediate stage called hemifusion and that Syt1 delivers thefinal blow to the fusion machinery at the Ca2+ spike. In parallel, this research program usessite-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR), a powerfultechnique for the investigation of the protein structure at the membrane interface. With EPRwe intend to comprehend the structural basis for the regulation of SNARE-dependent fusionnecessary for synchronization. The combined approach of the single fusion assay and SDSL EPR will provideimportant insights into the mechanism by which the synchronized fast release ischoreographed in the neuron.
Neurotransmitter release at synapses underlies major brain activities, such as cognition, memory, and emotion. Dysfunction of the release machinery is linked to many gruesome mental diseases, including schizophrenia, epilepsy, attention deficit syndrome (ADS), and autism. SNAREs are the newly emerged drug targets for neuromuscular diseases. A new line of SNARE inhibitors has been identified as a result of this project. The outcomes of proposed studies will help expand the repertoire on which the development of drugs for mental diseases may be based.
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