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

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.