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

Abstract

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.