Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.
We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.
Chamberlain
L H
LH
Physiological Laboratory, University of Liverpool, United Kingdom.
Roth
D
D
Morgan
A
A
Burgoyne
R D
RD
D048948:14-3-3 Proteins; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000502:physiology; D002147:Calmodulin / Q000494:pharmacology*; D002352:Carrier Proteins / Q000494:pharmacology*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000502:physiology; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D008565:Membrane Proteins / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis; D011506:Proteins / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D014446:Tyrosine 3-Monooxygenase; D033921:Vesicular Transport Proteins
D048948:14-3-3 Proteins; D002147:Calmodulin; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011506:Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D014446:Tyrosine 3-Monooxygenase; D017301:Ca(2+) Mg(2+)-ATPase; D004072:Digitonin