Synaptotagmins form a hierarchy of exocytotic Ca²⁺ sensors with distinct Ca²⁺ affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca²⁺-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca²⁺ affinities of synaptotagmin C₂-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca²⁺ affinities, with plasma membrane synaptotagmins binding Ca²⁺ with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca²⁺ affinities are functionally important, we examined the effects of synaptotagmin C₂-domains on Ca₂₊-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca²⁺ affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr²⁺, which was also selectively affected by high-affinity C₂-domains of synaptotagmins. Together, our results suggest that Ca²⁺ triggering of exocytosis involves tandem Ca²⁺ sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca²⁺ sensors involved in slow Ca²⁺dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca²⁺ sensors specialized for fast Ca²⁺-dependent exocytosis.