Electrostatic regulation of thecis- andtrans-membrane interactions of synaptotagmin-1

Abstract

AbstractSynaptotagmin-1 is a vesicular protein and Ca2+sensor for Ca2+-dependent exocytosis. Ca2+induces synaptotagmin-1 binding to its own vesicle membrane, called thecis-interaction, thus preventing thetrans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of thecis- andtrans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor thecis- andtrans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse thecis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10 to 100 μM. High PIP2concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2concentrations 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 thecis-interaction of synaptotagmin-1.