Abstract
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.
Original language | English (US) |
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Pages (from-to) | 53-61 |
Number of pages | 9 |
Journal | Journal of Neuroscience |
Volume | 22 |
Issue number | 1 |
DOIs | |
State | Published - Jan 1 2002 |
Externally published | Yes |
Keywords
- Amperometry
- Caged-calcium
- Calcium dependence
- Catecholamine release
- Chromaffin cell
- Flash photolysis
- SNAREs
- Whole-cell dialysis
ASJC Scopus subject areas
- General Neuroscience