Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca²⁺-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca²⁺–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca²⁺-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP₂)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca²⁺ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca²⁺-regulated interactions with t-SNAREs and PIP₂, target molecules known to play critical roles in exocytosis.