Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor
- Autores
- Fernández Nievas, Gaspar Antonio; Barrantes, Francisco Jose; Antollini, Silvia Susana
- Año de publicación
- 2007
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.
Fil: Fernández Nievas, Gaspar Antonio. 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
Fil: Antollini, Silvia Susana. 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
-
Achr
Fret - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/43914
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Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine ReceptorFernández Nievas, Gaspar AntonioBarrantes, Francisco JoseAntollini, Silvia SusanaAchrFrethttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.Fil: Fernández Nievas, Gaspar Antonio. 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; ArgentinaFil: Antollini, Silvia Susana. 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; ArgentinaAmerican Chemical Society2007-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/43914Fernández Nievas, Gaspar Antonio; Barrantes, Francisco Jose; Antollini, Silvia Susana; Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor; American Chemical Society; Biochemistry; 46; 11; 3-2007; 3503-35120006-2960CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/abs/10.1021/bi061388zinfo:eu-repo/semantics/altIdentifier/doi/10.1021/bi061388zinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:44:52Zoai:ri.conicet.gov.ar:11336/43914instacron: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:34982025-09-29 10:44:53.233CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
title |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
spellingShingle |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor Fernández Nievas, Gaspar Antonio Achr Fret |
title_short |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
title_full |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
title_fullStr |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
title_full_unstemmed |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
title_sort |
Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor |
dc.creator.none.fl_str_mv |
Fernández Nievas, Gaspar Antonio Barrantes, Francisco Jose Antollini, Silvia Susana |
author |
Fernández Nievas, Gaspar Antonio |
author_facet |
Fernández Nievas, Gaspar Antonio Barrantes, Francisco Jose Antollini, Silvia Susana |
author_role |
author |
author2 |
Barrantes, Francisco Jose Antollini, Silvia Susana |
author2_role |
author author |
dc.subject.none.fl_str_mv |
Achr Fret |
topic |
Achr Fret |
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 mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR. Fil: Fernández Nievas, Gaspar Antonio. 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 Fil: Antollini, Silvia Susana. 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 mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR. |
publishDate |
2007 |
dc.date.none.fl_str_mv |
2007-03 |
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/11336/43914 Fernández Nievas, Gaspar Antonio; Barrantes, Francisco Jose; Antollini, Silvia Susana; Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor; American Chemical Society; Biochemistry; 46; 11; 3-2007; 3503-3512 0006-2960 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/43914 |
identifier_str_mv |
Fernández Nievas, Gaspar Antonio; Barrantes, Francisco Jose; Antollini, Silvia Susana; Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor; American Chemical Society; Biochemistry; 46; 11; 3-2007; 3503-3512 0006-2960 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://pubs.acs.org/doi/abs/10.1021/bi061388z info:eu-repo/semantics/altIdentifier/doi/10.1021/bi061388z |
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 application/pdf |
dc.publisher.none.fl_str_mv |
American Chemical Society |
publisher.none.fl_str_mv |
American Chemical Society |
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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1844614487365124096 |
score |
13.070432 |