Phospholipid composition dependence of Ca²⁺-dependent phospholipid binding to the C2A domain of synaptotagmin IV

Synaptotagmins I and II are Ca²⁺- and phospholipid-binding proteins of synaptic vesicles that may function as Ca²⁺ receptors for neurotransmitter release via their first C2 domains. Herein, we describe the phospholipid binding properties of C2A domains of multiple synaptotagmins (II-VI). We demonstrate that all synaptotagmins can bind negatively charged phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI)) in a Ca²⁺-dependent manner, although it was previously reported that synaptotagmins IV and VI do not bind phospholipids. The Ca²⁺-dependent interaction of the C2A domain of synaptotagmin IV with PS was found to have two components with EC₅₀ values of approximately 5 and 120 μM free Ca²⁺ and exhibited positive cooperativity (Hill coefficient of approximately 2 for both components). This value is lower than that of the C2A domain of synaptotagmin II (Hill coefficient of approximately 3). All other isoforms bound PS with high affinity (EC₅₀ of 0.3-1 μM free Ca²⁺; Hill coefficient of 3-3.5). In addition, the C2A domain of synaptotagmin IV cannot bind liposomes consisting of PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine, PE) (1:1, w/w), indicating that the binding to negatively charged phospholipids is inhibited by the presence of PC or PE. In contrast, other isoforms bound all of the liposomes, which include either PS or PI, in a Ca²⁺-dependent manner. Mutational analysis indicated that this phospholipid composition-dependent Ca²⁺ binding of synaptotagmin IV results in the substitution of Asp for Ser at position 244. The cytoplasmic domain of synaptotagmin IV also shows this unique phospholipid binding. However, it binds PS with a positive cooperativity and an affinity similar to those of the C2A domains of other isoforms. Our results suggest that synaptotagmin IV is also a potential Ca²⁺ sensor for neurotransmitter release.