Synaptotagmins form a hierarchy of exocytotic Ca 2+ sensors with distinct Ca 2+ affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca 2+ ‐triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca 2+ affinities of synaptotagmin C 2 ‐domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca 2+ affinities, with plasma membrane synaptotagmins binding Ca 2+ with a 5‐ to 10‐fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca 2+ affinities are functionally important, we examined the effects of synaptotagmin C 2 ‐domains on Ca 2+ ‐triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca 2+ affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr 2+ , which was also selectively affected by high‐affinity C 2 ‐domains of synaptotagmins. Together, our results suggest that Ca 2+ triggering of exocytosis involves tandem Ca 2+ sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high‐affinity Ca 2+ sensors involved in slow Ca 2+ ‐dependent exocytosis, whereas vesicular synaptotagmins function as low‐affinity Ca 2+ sensors specialized for fast Ca 2+ ‐dependent exocytosis.