Differential but convergent functions of Ca²⁺ binding to synaptotagmin-1 C₂ domains mediate neurotransmitter release

Neurotransmitter release is triggered by cooperative Ca²⁺- binding to the Ca²⁺-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca²⁺ with similar properties and affinities. However, Ca²⁺ binding to the C2A domain is not required for release, whereas Ca²⁺ binding to the C2B domain is essential for release. We now demonstrate that despite its expendability, Ca²⁺-binding to the C2A domain significantly contributes to the overall triggering of neurotransmitter release, and determines its Ca²⁺ cooperativity. Biochemically, Ca ²⁺ induces more tight binding of the isolated C2A domain than of the isolated C2B domain to standard liposomes composed of phosphatidylcholine and phosphatidylserine. However, here we show that surprisingly, the opposite holds true when the double C2A/B-domain fragment of synaptotagmin-1 is used instead of isolated C2 domains, and when liposomes containing a physiological lipid composition are used. Under these conditions, Ca²⁺ binding to the C2B domain but not the C2A domain becomes the primary determinant of phospholipid binding. Thus, the unique requirement for Ca²⁺ binding to the C2B domain for synaptotagmin-1 in Ca²⁺-triggered neurotransmitter release may be accounted for, at least in part, by the unusual phospholipid-binding properties of its double C2A/B-domain fragment.