Elucidating the Specificity of Membrane Interactions of Synaptotagmin-1 in Synchronous Neurotransmitter Release

Neurotransmission involves Ca2+ dependent fusion of a neurotransmitter containing vesicle to the presynaptic plasma membrane to release the vesicle contents into the synaptic cleft. Soluble N-ethylmaleimide sensitive factor attachment protein receptors drive membrane fusion. The SNARE complex is not sensitive to Ca2+, however, and thus the Ca2+ sensor Synaptotagmin-1 couples the depolarization and fusion events during synchronous neurotransmitter release. Synaptotagmin-1 is a vesicular-tethered membrane protein, with two homologous C2A and C2B domains attached through a flexible linker which extend outward on the cytoplasmic face of the synaptic vesicle. Upon Ca2+ influx, the hydrophobic loops of the two domains bind Ca2+ and insert into the membrane. The C2B domain contains several other regions capable of membrane interaction including a lysine rich polybasic face and the arginine apex. Some combination of these elements initiates the bridging of the Synaptotagmin-1 bound vesicle to the presynaptic membrane. Once this occurs, the SNARE complex drives the fusion and pore opening processes that lead to neurotransmitter release. This membrane interaction and insertion is lipid specific with certain head groups required to drive membrane bridging. This body of work focuses on further understanding the lipid specific interactions between Synaptotagmin-1 and the vesicle and plasma membranes. First, through mapping of the soluble C2AB domains insertion and orientation differences under different membrane compositions, highlighting differences in binding when PIP2 is present. Next, through moving to more physiologically similar work on the full-length version of Synaptotagmin-1. Initial purification optimization, characterization, and comparison to the soluble domains will be shown to validate purification choice. This is followed by work on the driving forces for membrane insertion for full-length Synaptotagmin-1, determining coordination and electrostatic forces drive membrane interactions. Finally, an examination of the mechanism for Synaptotagmin-1 will be explored through a series of measurements to determine if the calcium sensor may act as a distance regulator for SNARE-dependent fusion. Overall, the goal is to further understanding of Synaptotagmin-1 interactions with different lipids and its contribution to fusion by use of structural and functional studies using CW-EPR, pulsed-EPR, NMR, fluorescent techniques, TIRF and FLIC.