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

Synaptotagmin-1 is a vesicular protein and Ca²⁺ sensor for Ca²⁺-dependent exocytosis. Ca²⁺ 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 Ca²⁺-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 [Ca²⁺] of 10-100 μM. High PIP₂ concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP₂ concentrations control the Ca²⁺-cooperativity of synaptotagmin-1. Our data provide evidence that Ca²⁺ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.