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

Autores
Pediconi, Maria Filomena; Gallegos, Cristina Eugenia; de Los Santos, Elisa Beatriz; Barrantes, Francisco Jose
Año de publicación
2004
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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.
Fil: Pediconi, Maria Filomena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Fil: Gallegos, Cristina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Fil: de Los Santos, Elisa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Materia
Cholinergic Receptor
Golgi Apparatus
Lipid Domains
Receptor-Lipid Interactions
Sterol
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/53014
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/53014
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptorPediconi, Maria FilomenaGallegos, Cristina Eugeniade Los Santos, Elisa BeatrizBarrantes, Francisco JoseCholinergic ReceptorGolgi ApparatusLipid DomainsReceptor-Lipid InteractionsSterolhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The 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.Fil: Pediconi, Maria Filomena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Gallegos, Cristina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: de Los Santos, Elisa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; ArgentinaPergamon-Elsevier Science Ltd2004-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/53014Pediconi, 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; 239-2490306-4522CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0306452204004750info:eu-repo/semantics/altIdentifier/doi/10.1016/j.neuroscience.2004.06.007info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:08:25Zoai:ri.conicet.gov.ar:11336/53014instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-10 13:08:26.143CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
title Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
spellingShingle Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
Pediconi, Maria Filomena
Cholinergic Receptor
Golgi Apparatus
Lipid Domains
Receptor-Lipid Interactions
Sterol
title_short Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
title_full Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
title_fullStr Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
title_full_unstemmed Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
title_sort Metabolic cholesterol depletion hinders cell-surface trafficking of the nicotinic acetylcholine receptor
dc.creator.none.fl_str_mv Pediconi, Maria Filomena
Gallegos, Cristina Eugenia
de Los Santos, Elisa Beatriz
Barrantes, Francisco Jose
author Pediconi, Maria Filomena
author_facet Pediconi, Maria Filomena
Gallegos, Cristina Eugenia
de Los Santos, Elisa Beatriz
Barrantes, Francisco Jose
author_role author
author2 Gallegos, Cristina Eugenia
de Los Santos, Elisa Beatriz
Barrantes, Francisco Jose
author2_role author
author
author
dc.subject.none.fl_str_mv Cholinergic Receptor
Golgi Apparatus
Lipid Domains
Receptor-Lipid Interactions
Sterol
topic Cholinergic Receptor
Golgi Apparatus
Lipid Domains
Receptor-Lipid Interactions
Sterol
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv 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.
Fil: Pediconi, Maria Filomena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Fil: Gallegos, Cristina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Fil: de Los Santos, Elisa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Unesco; Argentina
description 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.
publishDate 2004
dc.date.none.fl_str_mv 2004-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/53014
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
0306-4522
CONICET Digital
CONICET
url http://hdl.handle.net/11336/53014
identifier_str_mv 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
0306-4522
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0306452204004750
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.neuroscience.2004.06.007
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Pergamon-Elsevier Science Ltd
publisher.none.fl_str_mv Pergamon-Elsevier Science Ltd
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.993085

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