Two forms of the membrane-bound state of the first C2 domain (C2A) of synaptotagmin I and calcium-triggered membrane insertion

The synaptic vesicle protein synaptotagmin I (syt I) is a vesicle transmembrane protein present in synaptic vesicles, which has been proposed as the Ca2+ sensor that regulates secretion. The C2A domain is the membrane proximal part of its cytoplasmic domain. The interaction between C2A and lipid bilayer has been considered to be essential for triggering neurotransmitter release. In the present work, the measurements of membrane surface tension and surface concentration showed that the C2A domain of syt I exhibited two membrane-bound states: the surface adsorption state and the membrane insertion state. The surface absorption state formed in a Ca2+-independent manner with lower affinity, while the membrane insertion state formed with high affinity was only found in the presence of Ca2+. Both the Ca2+-independent and Ca2+-dependent syt I membrane interactions required anionic phospholipids, such as phosphatidylserine (PS). When expressed into rat pheochromocytoma (PC12) cells and human embryonic kidney (HEK-293) cells, as demonstrated by immunofluorescence staining and subcellular fractionation, most of the C2A was found at the plasma membrane, even when the cells were depleted of Ca2+ by incubation with EGTA. These results suggested a new molecular mechanism of syt I as a Ca2+ sensor in membrane fusion. Ca2+-independent surface adsorption might attach syt I to the release site during the docking or priming step. When intracellular Ca2+ increased, syt I triggered the neurotransmitter release following the Ca2+-dependent penetration into the target membrane.