Differential regulation of exocytosis by α- and β-SNAPs

被引:26
作者
Xu, JH
Xu, YM
Ellis-Davies, GCR
Augustine, GJ
Tse, FW
机构
[1] Univ Alberta, Dept Pharmacol, Edmonton, AB T6G 2H7, Canada
[2] Univ Alberta, Ctr Neurosci, Edmonton, AB T6G 2H7, Canada
[3] Duke Univ, Med Ctr, Dept Neurobiol, Durham, NC 27710 USA
[4] Med Coll Penn & Hahnemann Univ, Dept Physiol & Pharmacol, Philadelphia, PA 19102 USA
关键词
SNAREs; catecholamine release; calcium dependence; whole-cell dialysis; flash photolysis; caged-calcium; amperometry; chromaffin cell;
D O I
10.1523/JNEUROSCI.22-01-00053.2002
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
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 similar to 170 nM produced a mean rate of exocytosis of approximately one amperometric event per minute. Including alpha -SNAP (60 or 500 nM) in the intracellular solution dramatically increased the mean rate of exocytosis. The stimulatory action of alpha -SNAP requires ATP hydrolysis mediated via NSF, because this action was blocked by intracellular dialysis of ATP-gamma -S (2 mM) and could not be mimicked by a mutant alpha -SNAP that does not stimulate the ATPase activity of NSF. This action of alpha -SNAP was significant only at [Ca2+](i) between 100 and 300 nM and was not shared by beta -SNAP (500 nM), suggesting that alpha -SNAP enhanced a component of exocytosis that is regulated by a high-affinity Ca2+ sensor. In cells dialyzed with both alpha- and beta -SNAP, the rate of exocytosis was smaller than that produced by alpha -SNAP alone, suggesting that alpha- and beta -SNAP interact competitively. Although only alpha -SNAP stimulated exocytosis at [Ca2+](i) between 100 and 300 nM, both alpha- and beta -SNAP isoforms equally slowed the time-dependent rundown of the exocytic response. Our results indicate that alpha- and beta -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.
引用
收藏
页码:53 / 61
页数:9
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