FUNCTIONAL-ANALYSIS OF THE C2A DOMAIN OF SYNAPTOTAGMIN-1 - IMPLICATIONS FOR CALCIUM-REGULATED SECRETION

Synaptotagmin 1 is proposed to function as a low affinity calcium sens or for calcium-triggered exocytosis from neural and neuroendocrine cel ls. Because of the calcium-binding properties of the C2A domain of syn aptotagmin 1, calcium-dependent interactions through this domain may m odulate neurotransmitter release. We addressed this question by using alanine-scanning mutagenesis to generate a series of mutations within the C2A domain of synaptotagmin 1. The effects of these mutations on s ynaptotagmin 1 C2A function were analyzed for (1) calcium-dependent ph ospholipid binding, (2) calcium-dependent binding to syntaxin 1A, a pl asma membrane protein critical for vesicle docking or fusion, and (3) calcium-regulated secretion after microinjection into neuroendocrine p heochromocytoma (PC12) cells. Our analyses reveal that a polylysine mo tif at residues 189-192 confers an inhibitory effect on secretion by r ecombinant synaptotagmin C2A fragments. The synaptotagmin 1 C2A polyly sine motif functions independently of calcium-mediated interactions wi th phospholipids and syntaxin 1A. Furthermore, alpha-latrotoxin revers es the inhibitory effect of injected recombinant C2A fragments, sugges ting that they perturb the cellular calcium-sensing machinery by inter fering with synaptotagmin 1 activity in vivo. Our results indicate tha t novel calcium-independent interactions mediated through the C2A poly lysine motif of synaptotagmin 1 function to modulate neurotransmitter release.