| Neurons communicate by releasing neurotransmitters into the synaptic cleft where they bind and activate post-synaptic receptors. Release is triggered by increases in intracellular Ca2+ concentration and is mediated by the fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Here, we provide insights to the molecular dynamics and function of the putative Ca2+ sensor, synaptotagmin I (syt), that regulates exocytosis.; Syt is anchored to the vesicle membrane via a single membrane-spanning domain near its N-terminus and harbors a large cytoplasmic domain that is composed of two Ca2+-binding modules, designated as C2A and C2B. Here, we demonstrate that both C2A and C2B rapidly penetrate into lipid bilayers in response to Ca2+. The C2B domain of syt forms pre-complexes with PIP2, a plasma membrane lipid. This precomplex steers the membrane penetration activity of syt towards the plasma membrane, and increases the speed-of-response of syt to sub-millisecond scales. In addition, syt binds directly to syntaxin and SNAP-25, the two t-SNARE components of the conserved membrane fusion complex. Ca2+ triggered syt•t-SNARE interactions also occur rapidly enough to couple Ca2+ to fusion. Expression of mutant syts with selective and graded reductions in t-SNARE binding activity results in the graded destabilization of fusion pores in PC12 cells. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and t-SNAREs.; This thesis presents a model in which syt binds to anionic lipids and t-SNAREs in the target membrane. These interactions would pull the vesicle and target membranes together to initiate hemi-fusion and pore opening. In this model, the initial open state of the pore can be envisioned as being lined with the membrane anchors of synaptobrevin and syntaxin. Syt would subsequently drive the lateral separation of SNARE complexes to mediate dilation of the pore. |
