The Interplay Between SNAREs and Membranes: How Complexin's Binding Interactions Regulate Membrane Fusion

Synaptic vesicle fusion with the neuronal plasma membrane is a highly regulated process that is essential for the release of neurotransmitters. Energetic barriers inhibit the spontaneous fusion of membranes and formation of the SNARE complex provides the energy needed to overcome these barriers. The SNARE complex, composed of Syntaxin, Synaptobrevin and SNAP25, can fuse membranes alone, but additional regulatory mechanisms are required to ensure rapid, calcium-triggered membrane fusion. One of these regulatory proteins involved is Complexin, which functions to both clamp spontaneous fusion and facilitate calcium-triggered release. Complexin interacts with both the SNARE complex and membrane bilayers and these interactions are critical for its functions. However, the precise mechanisms by which these interactions support its functions are not completely understood. This dissertation explores Complexin’s interactions with SNAREs and membrane bilayers using fluorescence anisotropy and continuous wave electron paramagnetic resonance. Our studies revealed that Complexin’s membrane binding is sensitive to membrane properties. Increase acyl chain unsaturation or PIP2 concentration resulted in an increase membrane binding of complexin. Complexin can also inhibit its own membrane binding at low protein:lipid concentrations. Although individual SNARE proteins in solution did not alter Complexin’s membrane binding, membrane-anchored SNAP25 significantly enhanced this binding while membrane anchored Syntaxin and Synaptobrevin inhibited it. The ability of SNAP25 to increase Complexin’s membrane binding was due to the action of its N-terminal SN1 domain. SNARE complexes formed with either SNAP25 and Syntaxin or SNAP25, Syntaxin and Synaptobrevin increases Cpx’s membrane binding when anchored to lipid vesicles while the soluble complexes reduced Complexin membrane binding We also found that Complexin can bind multiple SNARE complexes in solution, including Syntaxin alone, Syntaxin and SNAP25 complex or Syntaxin, SNAP25 and Synaptobrevin complex. However, when these same complexes were attached to membranes, Complexin only interacted with the ternary SNARE complex. These findings highlight the importance of membrane environment in altering the conformation of SNAREs. Complexin’s affinity for unsaturated lipids suggests that packing defects are important for its membrane binding. SNAP25 alone or complexed with other SNARE proteins may function to increase these packing defects that alter Cpx-membrane binding. By binding to these packing defects that promote fusion, Complexin can help stabilize these defects and therefore clamp fusion before the arrival of the calcium signal.