A role for soluble NSF attachment proteins (SNAPs) in regulated exocytosis in adrenal chromaffin cells

EMBO J. 1995 Jan 16;14(2):232-9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenal Medulla / cytology
  • Adrenal Medulla / metabolism*
  • Animals
  • Carrier Proteins / physiology*
  • Cattle
  • Cells, Cultured
  • Chromaffin Granules / metabolism
  • Exocytosis*
  • Membrane Proteins / physiology*
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Vesicular Transport Proteins*

Substances

  • Carrier Proteins
  • Membrane Proteins
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Vesicular Transport Proteins