Competition Between Synaptotagmin 1 and Complexin for SNARE Complex Binding, Controls Fast Synaptic Vesicle Exocytosis

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2007-05-23

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Tang, Jiong

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Abstract

Calcium binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that were primed for release by SNARE complex assembly. Besides synaptotagmin 1, fast Ca2+- triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. To clarify previous debates on calcium dependent and independent binding between synaptotagmin 1 and SNARE proteins, I systematically examined the interactions between synaptotagmin 1 and purified SNARE monomer, heterodimer and core complex separately. This would avoid the problem of doing binding assays in an undefined protein mixture. We found the calcium dependency of synaptotagmin 1 and SNARE interactions relied on the accurate binding conditions that include protein concentration and ionic strength. In addition, at physiological conditions, calcium dependent binding is favored. Based on this system, I discovered the competition between complexin and synaptotagmin 1 for SNARE complex binding. Although in hydrophilic environment, complexin shows much higher affinity for SNARE complex than synaptotagmin 1, synaptotagmin 1 can more efficiently replace complexin from membrane embedded SNARE complex in a strictly calcium dependent manner. Expression of synaptic vesicle targeted complexin (by fusion to synaptobrevin 2) in cultured cortical neurons severely blocks fast synchronous release, but not asynchronous release, which is very similar to that of synaptotagmin 1 knockout mice. Based on electrophysiological data and biochemical confirmation of competition, we suggest that the phenotype could result from the replacement of synaptotagmin 1 from SNARE complex by local high concentration of fused complexin. We propose our model as: complexin binding promotes the assembly of SNARE complex and further stabilizes it. As a result, vesicles are activated into a "superprimed" metastable state, and are clamped at the same time waiting for triggering signals. Synaptotagmin 1 replaces complexin and releases this clamp through SNARE complex binding upon calcium entry. The simultaneous binding of synaptotagmin 1 with SNARE complex and phospholipids finally triggers membrane fusion and vesicle release.

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