Dual Roles of the C₂B Domain of Synaptotagmin I in Synchronizing Ca²⁺-Dependent Neurotransmitter Release

Although the vesicular protein synaptotagmin I contains two Ca 2+ -binding domains (C 2 A and C 2 B), Ca 2+ binding to the C 2 B domain is more important for triggering synchronous neurotransmitter release. We have used point mutagenesis to determine the functional contributions of the five negatively charged aspartate (Asp) residues that constitute the Ca 2+ -binding sites in the C 2 B domain of synaptotagmin I. Transfecting wild-type synaptotagmin I DNA into cultured hippocampal neurons from synaptotagmin I knock-out mice rescued Ca 2+ -dependent synchronous transmitter release and reduced a slower, asynchronous component of release, indicating that synaptotagmin I suppresses asynchronous release. Mutating either the second or third Asp residues of the C 2 B domain potently inhibited the ability of synaptotagmin I to rescue synchronous release but did not change its ability to suppress asynchronous release. Synaptotagmin I with mutations in the first or fourth Asp residues of the C 2 B domain partially rescued synchronous release and partially suppressed asynchronous release, whereas neutralizing the fifth Asp residue had no effect on the ability of synaptotagmin I to rescue transmitter release. Thus, we conclude that the C 2 B domain of synaptotagmin I regulates neurotransmitter release in at least two ways. Synchronous release absolutely requires binding of Ca 2+ to the second and third Asp residues in this domain. For the suppression of asynchronous release, Ca 2+ binding to the C 2 B domain of synaptotagmin I apparently is not necessary because mutation of the second Asp residue inhibits Ca 2+ binding, yet still allows this protein to suppress asynchronous release.