Effects of cholesterol on membrane binding by Synaptotagmin-7 C2 domains.

Abstract

Synaptotagmin-7 (Syt-7) is a calcium-dependent membrane binding protein that triggers membrane fusion and content release of large dense core vesicles in neuroendocrine cells, including insulin granules in pancreatic β-cells. Syt-7 is also important in synaptic facilitation for neurotransmitter release. Defining factors involved in Syt-7 membrane binding is important for understanding basic endocrine and neuronal biology as well as disease pathology. Syt-7 contains two C-terminal C2 domains that bind anionic lipid membranes with strong calcium sensitivity. In particular, the C2A domain binds physiological membranes in the presence of low micromolar calcium via a combination of electrostatic and hydrophobic interactions, while C2B interacts with both phosphatidylserine and phosphatidyl-4,5-bisphosphate via conserved lysine and arginine residues in the calcium-binding loops and polybasic regions. However, it remains unknown how other lipids fine-tune Syt-7 membrane binding in cells. Here, we present molecular dynamics simulation data which suggest that cholesterol may preferentially accumulate at the sites of C2 domain membrane interaction. Additionally, we used stopped flow fluorescence to measure kinetics of binding and release from synthetic liposomes, and our preliminary results indicate an increased dissociation rate when cholesterol is removed. Results will be presented for ongoing experiments with Syt-7 C2A, C2B, and C2AB domains. Overall, the study will improve our understanding of the factors that control the membrane affinity and calcium sensitivity of Syt-7.