Document Detail


Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor.
MedLine Citation:
PMID:  15350637     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
M F Pediconi; C E Gallegos; E B De Los Santos; F J Barrantes
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Neuroscience     Volume:  128     ISSN:  0306-4522     ISO Abbreviation:  Neuroscience     Publication Date:  2004  
Date Detail:
Created Date:  2004-09-07     Completed Date:  2004-12-09     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  7605074     Medline TA:  Neuroscience     Country:  United States    
Other Details:
Languages:  eng     Pagination:  239-49     Citation Subset:  IM    
Affiliation:
Instituto de Investigaciones Bioquímicas de Bahía Blanca, CC 857, B8000FWB, Argentina.
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MeSH Terms
Descriptor/Qualifier:
Animals
Binding Sites
Binding, Competitive
Bungarotoxins / metabolism
CHO Cells
Cell Membrane / metabolism*
Cholesterol / deficiency,  physiology*
Cricetinae
Cricetulus
Hydroxymethylglutaryl-CoA Reductase Inhibitors / pharmacology
Intracellular Membranes / metabolism
Kinetics
Ligands
Lipid Metabolism
Lovastatin / pharmacology
Mice
Microscopy, Fluorescence
Protein Transport / physiology
Receptors, Nicotinic / metabolism*
Succinimides
Tissue Distribution
Grant Support
ID/Acronym/Agency:
1-RO3-TW01225-01/TW/FIC NIH HHS
Chemical
Reg. No./Substance:
0/Alexa Fluor 488 carboxylic acid succinimidyl ester; 0/Bungarotoxins; 0/Hydroxymethylglutaryl-CoA Reductase Inhibitors; 0/Ligands; 0/Receptors, Nicotinic; 0/Succinimides; 57-88-5/Cholesterol; 75330-75-5/Lovastatin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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