Differential regulation of exocytosis by alpha- and beta-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 approximately 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.
Xu
Jianhua
J
Department of Pharmacology and Center for Neuroscience, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Xu
Yimei
Y
Ellis-Davies
Graham C R
GC
Augustine
George J
GJ
Tse
Frederick W
FW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000379:methods; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D017551:Microdialysis; D052066:N-Ethylmaleimide-Sensitive Proteins; D018408:Patch-Clamp Techniques; D010782:Photolysis; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012117:Resins, Synthetic; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D012117:Resins, Synthetic; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C016979:Ambersorb XE-344 resin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D002118:Calcium