title: Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells.
abstract: Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.
authors.affiliation: Instituto de Neurociencias, Centro Mixto CSIC-Universidad Miguel Hernández, Sant Joan d'Alacant, 03550 Alicante, and Instituto Teófilo Hernando, Servicio de Farmacología Clínica, Hospital de la Princesa, Madrid, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Lopez
authors.forename: Daniel
authors.initials: D
authors.lastname: Giner
authors.forename: Ana
authors.initials: A
authors.lastname: Ruiz-Nuño
authors.forename: Jorge
authors.initials: J
authors.lastname: Fuentealba
authors.forename: Salvador
authors.initials: S
authors.lastname: Viniegra
authors.forename: Antonio G
authors.initials: AG
authors.lastname: Garcia
authors.forename: Bazbek
authors.initials: B
authors.lastname: Davletov
authors.forename: Luis M
authors.initials: LM
authors.lastname: Gutiérrez
mesh_terms: D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D005089:Exocytosis; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D022142:Secretory Vesicles; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050827:Syntaxin 1 / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D004299:Dopamine beta-Hydroxylase