The C₂B domain of synaptotagmin is critical for synaptic efficacy in Drosphila

Abstract

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca2+ sensor for fast, evoked neurotransmitter release and also may function in vesicle docking or recycling. Genetic deletion of synaptotagmin strongly suppresses synaptic transmission in every species examined, demonstrating that synaptotagmin plays a central role in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin consists of two homologous C2 domains, C2A and C2B. Early studies ascribed all of synaptotagmin's binding activities to the C2A domain, neglecting any potential function by C2B. Here, I have tested the importance of two highly conserved motifs in C2B domain of synaptotagmin: a polylysine motif on β-strand four of C2B and a cluster of acidic residues that constitute a binding pocket for Ca2+ ions. Biochemical studies demonstrated that synaptotagmin bound the clathrin adapter protein AP-2, neuronal Ca2+ channels, inositol high polyphosphates, and other synaptotagmin molecules all via the polylysine motif. Electrophysiological analyses of Drosophila site directed mutants (K379,380,384Q) revealed that evoked transmitter release is decreased by ~36% and spontaneous release is increased two fold relative to sytnull flies expressing a wild type syt transgene, suggesting that this motif is important for full protein function. Alternatively, Ca2+-binding aspartate residues may mediate synaptotagmin binding to anionic phospholipids, SNARE (soluble Nethylmaleimide-sensitive factor attachment protein receptor) proteins, or self-oligomerization. Each of these processes may constitute a necessary step in vesicle fusion, however, there has been no direct evidence that any of the Ca2+-binding residues are required in vivo. Here, I show that mutating two of the Ca2+-binding aspartate residues in the C2B domain (D416.418N) decreased evoked transmitter release by > 95% and decreased the apparent Ca2+ affinity of evoked transmitter release. Taken together, these studies demonstrate that synaptotagmin's C2B Ca2+-binding motif is essential for synaptic transmission. Furthermore, these data support the idea that synaptotagmin functions as part of the Ca2+ sensing mechanism in the nerve terminal.