title: Calcium binding by synaptotagmin's C2A domain is an essential element of the electrostatic switch that triggers synchronous synaptic transmission.
abstract: Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C2A that inhibit Ca2+ binding support efficient synaptic transmission, suggesting that Ca2+ binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E in Drosophila) that inhibited Ca2+ binding but maintained the negative charge of the pocket. This C2A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+ affinity of release. Previous aspartate-to-asparagine mutations in C2A partially mimicked Ca2+ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+ binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+ binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+ binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+ influx, but Ca2+ binding by both C2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.
authors.affiliation: Department of Biomedical Sciences, Program in Molecular, Cellular, and Integrative Neuroscience, Colorado State University, Fort Collins, Colorado, 80523, USA.
authors.forename: Amelia R
authors.initials: AR
authors.lastname: Striegel
authors.forename: Laurie M
authors.initials: LM
authors.lastname: Biela
authors.forename: Chantell S
authors.initials: CS
authors.lastname: Evans
authors.forename: Zhao
authors.initials: Z
authors.lastname: Wang
authors.forename: Jillian B
authors.initials: JB
authors.lastname: Delehoy
authors.forename: R Bryan
authors.initials: RB
authors.lastname: Sutton
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
authors.forename: Noreen E
authors.initials: NE
authors.lastname: Reist
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology*; D004331:Drosophila melanogaster; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009433:Neural Inhibition / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D055672:Static Electricity; D009435:Synaptic Transmission / Q000502:physiology*; D050857:Synaptotagmins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D013816:Thermodynamics
chemical_list: D002135:Calcium-Binding Proteins; D050857:Synaptotagmins; D002118:Calcium