Role of the Synaptobrevin C-terminus in Fusion Pore Formation

Abstract

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII) also called VAMP2, syntaxin and SNAP-25 generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to fusion pore formation remains elusive.To determine a possible role of the sybII TM domain in fusion pore formation, sybII constructs in which one or two polar or non-polar residues were added at the C-terminus of the protein were expressed in double knock-out (DKO) embryonic mouse chromaffin cells deficient in sybII and cellubrevin1. Exocytosis was stimulated by flash photolysis of caged-calcium (NP-EGTA), and the capability of the mutated constructs to support exocytosis was monitored by whole-cell patch clamp capacitance measurements while the associated transmitter release was monitored by carbon fiber amperometry. We found that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C-terminus depending on their energy of transfer from water to the membrane interface2, following a Boltzmann distribution. These results suggest that C-terminal zipping of the SNARE complex pulls the C-terminus of sybII deeper into the vesicle membrane, with this movement disrupting the vesicular membrane continuity and leading to fusion pore formation. In contrast to current models, fusion thus begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed endoplasmic leaflets.1 M. Borisovska, Y. Zhao, Y. Tsytsyura et al., Embo J 24 (12), 2114 (2005).2 W. C. Wimley and S. H. White, Nat Struct Biol 3 (10), 842 (1996).Supported by NIH grants R01NS38200, R01GM85808, T32GM8267, the Nanobiotechnology Center (NSF) and DFG grants SFB523-B16 and SFB530-C10.