Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells.
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.
Lopez
Inmaculada
I
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.
Giner
Daniel
D
Ruiz-Nuño
Ana
A
Fuentealba
Jorge
J
Viniegra
Salvador
S
Garcia
Antonio G
AG
Davletov
Bazbek
B
Gutiérrez
Luis M
LM
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*
D015220:Calcium Channels; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D004299:Dopamine beta-Hydroxylase