title: Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor.
abstract: 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 alpha2betadeltaepsilon 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 microM Mevinolin, total cholesterol was significantly reduced (40%). Concomitantly, the maximum number of binding sites (Bmax) of the cell-surface AChR for the competitive antagonist alpha-bungarotoxin was reduced from 647+/-30 to 352+/-34 fmol/mg protein, i.e. by 46%. The apparent dissociation constant (Kdapp) for alpha-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 (IC50) 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 alpha-bungarotoxin labeled cell-surface AChRs was reduced by approximately 70% in Mevinolin-treated cells. The distribution of intracellular AChR also changed: Alexa594-alpha-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 beta1,4-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.
authors.affiliation: Instituto de Investigaciones Bioquímicas de Bahía Blanca, CC 857, B8000FWB, Argentina.
authors.forename: M F
authors.initials: MF
authors.lastname: Pediconi
authors.forename: C E
authors.initials: CE
authors.lastname: Gallegos
authors.forename: E B
authors.initials: EB
authors.lastname: De Los Santos
authors.forename: F J
authors.initials: FJ
authors.lastname: Barrantes
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000172:deficiency / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D050356:Lipid Metabolism; D008148:Lovastatin / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000502:physiology; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013388:Succinimides; D014018:Tissue Distribution
chemical_list: C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D002038:Bungarotoxins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008024:Ligands; D011978:Receptors, Nicotinic; D013388:Succinimides; D002784:Cholesterol; D008148:Lovastatin