The C2BCa2+-binding motif of synaptotagmin is required for synaptic transmission in vivo

Synaptotagmin is a synaptic vesicle protein that is postulated to be the Ca2+ sensor for fast, evoked neurotransmitter release(1). Deleting the gene for synaptotagmin (syt(null)) strongly suppresses synaptic transmission in every species examined(2), showing that synaptotagmin is central in the synaptic vesicle cycle. The cytoplasmic region of synaptotagmin contains two C-2 domains, C(2)A and C2B. Five, highly conserved, acidic residues in both the C(2)A and C2B domains of synaptotagmin coordinate the binding of Ca2+ ions(3-5), and biochemical studies have characterized several in vitro Ca2+-dependent interactions between synaptotagmin and other nerve terminal molecules(6). But there has been no direct evidence that any of the Ca2+-binding sites within synaptotagmin are required in vivo. Here we show that mutating two of the Ca2+-binding aspartate residues in the C2B domain (D-416,N-418 in Drosophila) decreased evoked transmitter release by >95%, and decreased the apparent Ca2+ affinity of evoked transmitter release. These studies show that the Ca2+-binding motif of the C2B domain of synaptotagmin is essential for synaptic transmission.