Ca2+-induced changes in SNAREs and synaptotagmin I correlate with triggered exocytosis from chromaffin cells: insights gleaned into the signal transduction using trypsin and botulinum toxins.
Ca2+-triggered catecholamine exocytosis from chromaffin cells involves SNAP-25, synaptobrevin and syntaxin (known as SNAREs). Synaptotagmin I has been implicated as the Ca2+-sensor because it binds Ca2+, and this enhances its binding to syntaxin, SNAP-25 and phospholipids in vitro. However, most of these interactions are only mediated by [Ca2+]i two orders of magnitude higher than that needed to elicit secretion. Thus, the Ca2+ sensitivities of synaptotagmin I and the other SNAREs were quantified in situ. Secretion elicited from permeabilised cells by microM Ca2+ was accompanied, with almost identical Ca2+ dependencies, by changes in synaptotagmin I, SNAP-25, syntaxin and synaptobrevin that rendered them less susceptible to trypsin. The majority of the trypsin-resistant SNAREs were not associated with SDS-resistant complexes. None of these proteins acquired trypsin resistance in cells rendered incompetent for exocytosis by run-down. Removal of nine C-terminal residues from SNAP-25 by botulinum toxin A reduced both exocytosis and the SNAREs' acquisition of trypsin resistance but did not alter the Ca2+ sensitivity, except for synaptotagmin I. Even after synaptobrevin had been cleaved by botulinum toxin B, all the other proteins still responded to Ca2+. These data support a model whereby Ca2+ is sensed, probably by synaptotagmin I, and the signal passed to syntaxin and SNAP-25 before they interact with synaptobrevin.
Lawrence
Gary W
GW
Centre for Neurobiochemistry, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AY, UK.
Dolly
J Oliver
JO
D000255:Adenosine Triphosphate; D000313:Adrenal Medulla; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002135:Calcium-Binding Proteins; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins; D014357:Trypsin; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D002395:Catecholamines; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D033921:Vesicular Transport Proteins; C096323:rimabotulinumtoxinB; D050857:Synaptotagmins; D000255:Adenosine Triphosphate; D014357:Trypsin; D019286:Endopeptidase K; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D002118:Calcium