Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells.
NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules.
Xu
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Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany.
Ashery
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Burgoyne
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Neher
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology*; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002837:Chromaffin Granules / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D003956:Dialysis; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004553:Electric Conductivity; D004705:Endocytosis / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D007700:Kinetics; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000494:pharmacology*; D009154:Mutation; D052066:N-Ethylmaleimide-Sensitive Proteins; D018408:Patch-Clamp Techniques; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D002395:Catecholamines; D008565:Membrane Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C085112:2-nitrophenyl-EGTA; C022571:adenosine 5'-O-(3-thiotriphosphate); D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D052066:N-Ethylmaleimide-Sensitive Proteins; D005033:Ethylmaleimide; D002118:Calcium