A role for soluble NSF attachment proteins (SNAPs) in regulated exocytosis in adrenal chromaffin cells.
Digitonin-permeabilized chromaffin cells secrete catecholamines by exocytosis in response to micromolar Ca2+ concentrations, but lose the ability to secrete in response to Ca2+ as the cells lose soluble proteins through the plasma membrane pores. Such secretory run-down can be retarded by cytosolic fractions, thus providing an assay for proteins potentially involved in the exocytotic process. We have used this assay to investigate the role of N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) in regulated exocytosis. Recombinant alpha- and gamma-SNAP stimulated Ca(2+)-dependent exocytosis, although recombinant NSF was ineffective, despite the fact that NSF and alpha-SNAP leak from the permeabilized cells with similar time courses. However, around one third of cellular NSF was found to be present in a non-cytosolic form and so it is possible that this is sufficient for exocytosis and that exogenous SNAPs stimulate the exocytotic mechanism by acting on the leakage-insensitive NSF. The stimulatory effect of alpha-SNAP displayed a biphasic dose-response curve and was maximal at 20 micrograms/ml. The effect of alpha-SNAP was Ca(2+)- and MgATP-dependent and was inhibited by N-ethylmaleimide and botulinum A neurotoxin, indicating a bona fide action on the exocytotic mechanism. Furthermore, Ca2+ concentrations which trigger catecholamine secretion acted to prevent the leakage of NSF and alpha-SNAP from permeabilized cells. These findings provide functional evidence for a role of SNAPs in regulated exocytosis in chromaffin cells.
Morgan
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Physiological Laboratory, University of Liverpool, UK.
Burgoyne
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RD
D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000378:metabolism; D005089:Exocytosis; D008565:Membrane Proteins / Q000502:physiology*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins