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Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

ABSTRACT: The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca(2+) entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca(2+) entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone. DOI: http://dx.doi.org/10.7554/eLife.01180.001.

SUBMITTER: Zhou K

PROVIDER: S-EPMC3821175 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

Zhou Keming K Stawicki Tamara M TM Goncharov Alexandr A Jin Yishi Y

eLife 20131112

The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca(2+) entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulate ...[more]

PMID: 24220508

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Project description:Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

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Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

Publications

Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics.

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