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Artículo

Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor

Pediconi, Maria FilomenaIcon ; Gallegos, Cristina EugeniaIcon ; de Los Santos, Elisa BeatrizIcon ; Barrantes, Francisco JoseIcon
Fecha de publicación: 12/2004
Editorial: Pergamon-Elsevier Science Ltd
Revista: Neuroscience
ISSN: 0306-4522
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Otras Ciencias Biológicas

Resumen

The effects of metabolic inhibition of cholesterol biosynthesis on the trafficking of the nicotinic acetylcholine receptor (AChR) to the cell membrane were studied in living CHO-K1/A5, a Chinese hamster ovary clonal line that heterologously expresses adult α 2βδε mouse AChR. To this end, we submitted CHO-K1/A5 cells to long-term cholesterol deprivation, elicited by Mevinolin, a potent inhibitor of 3-hydroxy-3-methyl- glutaryl-CoA reductase and applied a combination of biochemical, pharmacological and fluorescence microscopy techniques to follow the fate of the AChR. When CHO-K1/A5 cells were grown for 48 h in lipid-deficient medium supplemented with 0.5 μM Mevinolin, total cholesterol was significantly reduced (40%). Concomitantly, the maximum number of binding sites (B max) of the cell-surface AChR for the competitive antagonist α-bungarotoxin was reduced from 647±30 to 352±34 fmol/mg protein, i.e. by 46%. The apparent dissociation constant (Kd app) for α-bungarotoxin of the AChRs remaining at the cell surface was not modified by cholesterol depletion. Similarly, the half-concentration inhibiting the specific binding of the radioligand (IC 50) for another competitive antagonist, d-tubocurarine, did not differ from that in control cells. The decrease in cell-surface AChR was paralleled by an increase in intracellular AChR levels, which rose from 44±2.1% in control cells to 74±3.3% in Mevinolin-treated cells. When analyzed by wide-field fluorescence microscopy, the fluorescence signal arising from α-bungarotoxin labeled cell-surface AChRs was reduced by approximately 70% in Mevinolin-treated cells. The distribution of intracellular AChR also changed: Alexa 594-α- bungarotoxin-labeled AChR exhibited a highly compartmentalized pattern, concentrating at the perinuclear and Golgi-like regions. Temperature-arrest of protein trafficking magnified this effect, emphasizing the Golgi localization of the AChR. Colocalization studies using the transiently expressed fluorescent trans-Golgi/trans-Golgi network marker pEYFP/human β1,4- galactosyltransferase and the trans-Golgi network marker syntaxin 6 provided additional support for the Golgi localization of intracellular AChRs. The low AChR cell-surface expression and the increase in intracellular AChR pools in cholesterol-depleted cells raise the possibility that cholesterol participates in the trafficking of the receptor protein to the plasmalemma and its stability at this surface location.
Palabras clave: Cholinergic Receptor , Golgi Apparatus , Lipid Domains , Receptor-Lipid Interactions , Sterol
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
Identificadores
URI: http://hdl.handle.net/11336/53014
URL: https://www.sciencedirect.com/science/article/pii/S0306452204004750
DOI: http://dx.doi.org/10.1016/j.neuroscience.2004.06.007
Colecciones
Articulos(CCT - BAHIA BLANCA)
Articulos de CTRO.CIENTIFICO TECNOL.CONICET - BAHIA BLANCA
Articulos(INIBIBB)
Articulos de INST.DE INVEST.BIOQUIMICAS BAHIA BLANCA (I)
Articulos(SEDE CENTRAL)
Articulos de SEDE CENTRAL
Citación
Pediconi, Maria Filomena; Gallegos, Cristina Eugenia; de Los Santos, Elisa Beatriz; Barrantes, Francisco Jose; Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor; Pergamon-Elsevier Science Ltd; Neuroscience; 128; 2; 12-2004; 239-249
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