Comparison of cysteine string protein (Csp) and mutant alpha-SNAP overexpression reveals a role for csp in late steps of membrane fusion in dense-core granule exocytosis in adrenal chromaffin cells.
Assembly of the SNARE complex and its disassembly caused by the action of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) and NSF is crucial for the maintenance of vesicular traffic, including fusion of regulated exocytotic vesicles. Various other proteins may also have important roles in the processes leading to membrane fusion via interaction with the SNARE proteins, including the secretory vesicle cysteine string protein (Csp). Here we have examined the effect of overexpression of a dominant negative alpha-SNAP mutant or Csp on exocytosis of dense-core granules in single chromaffin cells monitored using amperometry to detect released catecholamine. Exocytosis of trans-Golgi network (TGN)-derived dense-core granules was substantially inhibited by expression of alpha-SNAP(L294A). The amplitude and characteristics of the individual release events were unaffected by expression of alpha-SNAP(L294A), consistent with an essential role for alpha-SNAP in early steps of priming but not in the fusion process. In contrast, Csp overexpression, which also inhibited the extent of exocytosis, also modified the kinetics of the individual release events seen as an increase in the rise time and a broadening of the residual amperometric spikes in Csp-transfected cells. These results suggest that unlike alpha-SNAP, Csp plays a key role in the protein interactions close to the fusion process or fusion pore opening during Ca(2+)-regulated exocytosis.
Graham
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ME
The Physiological Laboratory, The University of Liverpool, Liverpool L69 3BX, United Kingdom.
Burgoyne
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RD
D000313:Adrenal Medulla / Q000166:cytology / Q000502:physiology*; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000502:physiology*; D002837:Chromaffin Granules / Q000502:physiology*; D004594:Electrophysiology; D005089:Exocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins; D050956:HSP40 Heat-Shock Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D014162:Transfection; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D050956:HSP40 Heat-Shock Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C088460:cysteine string protein; D049452:Green Fluorescent Proteins