Mechanisms of Fast Neurotransmitter Secretion

University Of Arizona, Tucson AZ

Investigators

Abstract

Regulated neurotransmitter exocytosis is a fundamental process for the intercellular communication among neurons. It is widely accepted that neurotransmitter secretion is a tightly regulated form of constitutive vesicular exocytosis shared by all eukaryotic cells. At the nerve terminal, a depolarization-induced Ca2+ influx through ion specific channels triggers the fusion of synaptic vesicles, which expel their neurotransmitter cargo onto the postsynaptic cell. A combination of biochemical and genetic approaches by many laboratories has lead to molecular models describing exocytotic and endocytotic mechanisms. In particular, recent advances uncovered the SNARE- or core complex driving constitutive membrane fusion. However, little is known about mechanism that mediate vesicle docking, arrest fusion-competent vesicles in the readily releasable pool, accomplish Ca2+ signaling from the sensor to the fusion machinery, adjust the probability of vesicle fusion, or regulate the size of readily releasable pool. We have initiated a genetic screen to identify further components mediating Ca2+-triggered exocytosis at nerve terminals and identified several novel mutations, which potentially affect neurotransmission. Of these, the mutation B682 is especially interesting because it inhibits a unique activity-dependent loss of evoked release, which coincides with a simultaneous increase in spontaneous neurotransmitter release, suggestive of an impaired releasable vesicle pool. A unifying hypothesis suggests that B682 protein mediates a late step in synaptic vesicle maturation that accumulates fusion-competent vesicles in the "readily releasable vesicle pool". Specifically, B682 may "clamp" these fusogenic vesicles such that they await the fusion-triggering Ca2+ signal. To test this hypothesis (and explore others if warranted), loss- and gain-of-function B682 mutations will be examined for their effects on synaptic function at NMJs. These effects will be examined by a multi-disciplinary approach including electrophysiology, Ca2+ imaging, FM1-43 imaging, confocal and electron microscopy, and biochemistry. The first two Objectives will lay basic and essential groundwork for evaluating B682's synaptic role. Specifically, we will verify that B682 is indeed a presynaptic protein that affects a physiological step of synaptic transmission but not neuromuscular synaptogenesis. Once these points are fully established the third Objective will test the essence of the B682 protein hypothesis. Specifically, it will test the prediction that B682 mutations do not impair Ca2+ entry and/or extrusion. In addition, this objective will test whether the abnormal vesicle distribution in B682 mutants is caused by a defect in vesicle recycling, steps of vesicle trafficking to active zones and/or refilling the readily releasable vesicle pool. Together, these studies will help to better understand the molecular mechanisms that mediate the regulation of fast, synchronous neurotransmitter exocytosis at nerve terminals.