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The Journal of Neuroscience, January 1, 2002, 22(1):53-61

Differential Regulation of Exocytosis by - and -SNAPs

Jianhua Xu¹, Yimei Xu², Graham C. R. Ellis-Davies³, George J. Augustine², and Frederick W. Tse¹

¹ Department of Pharmacology and Center for Neuroscience, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada, ² Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, and ³ Department of Pharmacology and Physiology, MCP Hahnemann University, Philadelphia, Pennsylvania 19102

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 -SNAP (60 or 500 nM) in the intracellular solution dramatically increased the mean rate of exocytosis. The stimulatory action of -SNAP requires ATP hydrolysis mediated via NSF, because this action was blocked by intracellular dialysis of ATP--S (2 mM) and could not be mimicked by a mutant -SNAP that does not stimulate the ATPase activity of NSF. This action of -SNAP was significant only at [Ca²⁺]_i between 100 and 300 nM and was not shared by -SNAP (500 nM), suggesting that -SNAP enhanced a component of exocytosis that is regulated by a high-affinity Ca²⁺ sensor. In cells dialyzed with both - and -SNAP, the rate of exocytosis was smaller than that produced by -SNAP alone, suggesting that - and -SNAP interact competitively. Although only -SNAP stimulated exocytosis at [Ca²⁺]_i between 100 and 300 nM, both - and -SNAP isoforms equally slowed the time-dependent rundown of the exocytic response. Our results indicate that - and -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.

Key words: SNAREs; catecholamine release; calcium dependence; whole-cell dialysis; flash photolysis; caged-calcium; amperometry; chromaffin cell

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Copyright © 2002 Society for Neuroscience  0270-6474/02/22153-09$05.00/0

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