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Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin.

ABSTRACT: Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca²⁺ sensor for SV release that binds Ca²⁺ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca²⁺-triggered fusion.

SUBMITTER: Courtney NA

PROVIDER: S-EPMC6733930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A conformational switch in complexin is required for synaptotagmin to trigger synaptic fusion.

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Complexin has opposite effects on two modes of synaptic vesicle fusion.

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Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1.

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Synaptotagmin-mediated vesicle fusion regulates cell migration.

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Evidence for synaptotagmin as an inhibitory clamp on synaptic vesicle release in Aplysia neurons.

Project description:While previous studies have demonstrated that synaptotagmin plays an essential role in evoked neurotransmitter release, it has been difficult to determine whether it acts to facilitate or inhibit release. To address this question, we used acute genetic manipulations to alter the expression of synaptotagmin in Aplysia neurons. Transient overexpression of synaptotagmin in acutely dissected cholinergic neurons and in cultured glutaminergic neurons decreased the amplitude of the excitatory postsynaptic potential (EPSP) by 32% and 26%, respectively. In contrast, treatment of cultured presynaptic neurons with synaptotagmin antisense oligonucleotides increased the amplitude of the EPSP by 50-75%. These results are consistent with a role of synaptotagmin as an inhibitor of release.

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Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons.

Project description:The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

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C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo.

Project description:Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.

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