Differential regulation of synchronous versus asynchronous neurotransmitter release by the C2 domains of synaptotagmin 1

Abstract

Synaptic vesicle fusion at many synapses has been kinetically separated into two distinct Ca[superscript 2+]-dependent temporal components consisting of a rapid synchronous phase followed by a slower asynchronous component. Mutations in the synaptic vesicle Ca[superscript 2+] sensor Synaptotagmin 1 (Syt 1) reduce synchronous neurotransmission while enhancing the slower asynchronous phase of release. Syt 1 regulation of vesicle fusion requires interactions mediated by its tandem cytoplasmic C2 domains (C2A and C2B). Although Ca[superscript 2+] binding by Syt 1 is predicted to drive synchronous release, it is unknown if Ca[superscript 2+] interactions with either C2 domain is required for suppression of asynchronous release. To determine if Ca[superscript 2+] binding by Syt 1 regulates these two phases of release independently, we performed electrophysiological analysis of transgenically expressed Syt 1 mutated at Ca[superscript 2+] binding sites in C2A or C2B in the background of Drosophila Syt 1-null mutants. Transgenic animals expressing mutations that disrupt Ca[superscript 2+] binding to C2A fully restored the synchronous phase of neurotransmitter release, whereas the asynchronous component was not suppressed. In contrast, rescue with Ca[superscript 2+]-binding mutants in C2B displayed little rescue of the synchronous release component, but reduced asynchronous release. These results suggest that the tandem C2 domains of Syt 1 play independent roles in neurotransmission, as Ca[superscript 2+] binding to C2A suppresses asynchronous release, whereas Ca[superscript 2+] binding to C2B mediates synchronous fusion.