Differential regulation of exocytosis by α- and β-SNAPs

We examined the role of SNAPs, soluble proteins that attach N-ethylmaleimide-sensitive factor (NSF), in regulating exocytosis in single rat adrenal chromaffin cells. Whole-cell dialysis of Ca²⁺-buffered solution or photolysis of caged-Ca²⁺ was used to manipulate cytosolic Ca²⁺ concentration ([Ca²⁺]_i), whereas exocytosis was measured via carbon fiber amperometry or membrane capacitance. Buffering [Ca²⁺]_i to ∼170 nM produced a mean rate of exocytosis of approximately one amperometric event per minute. Including α-SNAP (60 or 500 nM) in the intracellular solution dramatically increased the mean rate of exocytosis. The stimulatory action of α-SNAP requires ATP hydrolysis mediated via NSF, because this action was blocked by intracellular dialysis of ATP-γ-S (2 mM) and could not be mimicked by a mutant α-SNAP that does not stimulate the ATPase activity of NSF. This action of α-SNAP was significant only at [Ca²⁺]_i between 100 and 300 nM and was not shared by β-SNAP (500 nM), suggesting that α-SNAP enhanced a component of exocytosis that is regulated by a high-affinity Ca²⁺ sensor. In cells dialyzed with both α- and β-SNAR the rate of exocytosis was smaller than that produced by α-SNAP alone, suggesting that α- and β-SNAP interact competitively. Although only α-SNAP stimulated exocytosis at [Ca²⁺]_i between 100 and 300 nM, both α- and β-SNAP isoforms equally slowed the time-dependent rundown of the exocytic response. Our results indicate that α- andβ-SNAP have different actions in exocytosis. Thus, the ratio of different isoforms of SNAPs can determine release probability at the levels of [Ca²⁺]_i that are involved in regulation of exocytosis.