Ca2+/phospholipid-binding and syntaxin-binding of native synaptotagmin I.

ABSTRACT

Synaptotagmin, a synaptic vesicle protein endowed with multiple properties, is the putative calcium sensor in neuroexocytosis. Ca2+/phospholipid binding and syntaxin binding activity of synaptotagmin were previously investigated using recombinant fusion proteins. In phospholipid binding the EC50 for calcium obtained was different when fusion proteins containing one (C2A) or both (C2A+C2B) binding domains were used. It was alternatively proposed that one or both synaptotagmin binding domains are important for calcium-sensing and triggering of transmitter release. In this study the binding activity of native full-length synaptotagmin, immobilized on beads, was investigated. We found the kinetic parameters of Ca2+/phospholipid binding to be compatible with the role of calcium sensor for synaptotagmin (EC50 for calcium = 72 +/- 7 microM), with the two C2 domains supporting separate and complementary calcium sensing properties. The binding of native syntaxin to synaptotagmin was measurable in the absence of calcium, but was markedly stimulated (2.2-fold) in the presence of mM calcium. It may be speculated that the two domains have a synergistic action in fast synchronous transmitter release, whereas C2B domain alone may support slow asynchronous release, working as a high affinity calcium sensor.