Electrostatic regulation of the cis- and trans-membrane interactions of synaptotagmin-1

Synaptotagmin-1 is a vesicular protein and Ca 2+ sensor for Ca 2+ -dependent exocytosis. Ca 2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis- interaction, thus preventing the trans -interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis - and trans -membrane interaction of synaptotagmin-1 was poorly understood in different Ca 2+ -buffering conditions. Here we provide an assay to monitor the cis - and trans -membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis -membrane interaction of synaptotagmin-1 in free [Ca 2+ ] of 10–100 μM. High PIP 2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP 2 concentrations control the Ca 2+ -cooperativity of synaptotagmin-1. Our data provide evidence that Ca 2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis -interaction of synaptotagmin-1.