Functional mapping of synaptotagmin 1 Ca2+ ligands that mediate excitation-secretion coupling in neurons

Synaptotagmin (SYT) 1 is a Ca2+ sensor that triggers rapid synaptic vesicle exocytosis, but how it is activated by Ca2+ remains enigmatic. SYT1 contains two Ca2+-sensing motifs, C2A and C2B. Structural studies have proposed that five acidic residues in each C2 domain mediate Ca2+-binding, but their functional relevance remains unclear. Here, we mutated each of the ten putative ligands and conducted side-by-side comparisons of Ca2+-binding activity and evoked glutamate release. Remarkably, all ten putative ligands are crucial for evoked synchronous release, overturning the view that binding of Ca2+ to C2A is dispensable. Importantly, Ca2+ binding to C2B is essential for synchronization. Furthermore, we report that two new human mutations, corresponding to Ca2+ ligands in C2B, are dominant-negative. Finally, four new crystal structures reveal how the choice of neutralizing substitution differentially alters SYT1 structure and, consequently, function.