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

PEDICONI, M.F.; GALLEGOS, C.E.; DE LOS SANTOS, E.B.; BARRANTES, F.J.

NEUROSCIENCE

PERGAMON-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2004 vol. 128 p. 239 - 249

0306-4522

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 a2bde 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 grwon for 48 hs in lipid-deificient medium supplement with  0.5 mM Mevinolin, total cholesterol was significantly reduced (40%). Concomitantly, the maxim number of binding sites (Bmax) of the cell-surface AChR for the competitive antagonist a-bungarotoxin was reduced from 647 ± 30 to 352±34 fmol/mg protein, i.e. by 46%. The apparent dissociation constant (Kdapp) for a-bungarotoxin of the AChR 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 a-bungarotoxin labeled cell-surface AChRs was reduced by approximately 70% in Mevinolin-treated cells. The distribution of intracellular AChR also changed: Alexa594-a-bungarotoxin-labelled 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 fluorescnet  trans-Golgi/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.