Role of complexin-lipid interactions in controlling snare-mediated membrane fusion.

Abstract

Fast and reliable neurotransmitter release is essential for signaling fidelity at neuronal synapses. Synaptic exocytosis is a tightly controlled process accomplished through Ca2+ triggered fusion of neurotransmitter-laden vesicles with the presynaptic plasma membrane. This process is mediated by SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins that zipper into a four-helix bundle to drive membrane fusion. The neuronal SNARE complex, consisting of Syntaxin-1a, SNAP-25, and Synaptobrevin-2, is intrinsically capable of catalyzing slow and Ca2+ independent fusion in reconstituted fusion assays in vitro. A single model of how interactions with SNARE regulatory proteins impart physiological properties seen in neurons, such as Ca2+ specificity, has yet to be established. Complexin-1 (Cpx) is a small, soluble regulatory protein that binds SNAREs and lipid membranes. Cpx is proposed to facilitate fusion in the presence of Ca2+ as well as suppress fusion in the absence of Ca2+ through interactions of its central domain with SNARE complexes in their prefusion and postfusion states. Additionally, the Cpx C-terminal domain interacts with membranes, an interaction that has been proposed to facilitate function. Recent work has shown that Cpx's lipid-binding domains can differentiate between membranes of different compositions, though the functional relevance of this finding has yet to be established. Our group has previously developed a hybrid fusion assay that combines synthetic, reconstituted, and native components to reproduce key features of synaptic exocytosis. This method mimics intracellular conditions more closely than a reconstituted system with only purified components while maintaining the ability to easily alter lipid, protein, and ion concentrations. We utilize this system with wild-type and mutant Cpx, along with other protein and lipid regulators of SNAREs, to investigate how interaction with and composition of the fusing membranes controls Cpx's regulatory function.