Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

Autores
Spitzmaul, Guillermo Federico; Corradi, Jeremias; Bouzat, Cecilia Beatriz
Año de publicación
2004
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.
Fil: Spitzmaul, Guillermo Federico. 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
Fil: Corradi, Jeremias. 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
Fil: Bouzat, Cecilia 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
Materia
Acetylcholine
Acetylcholine Receptor
Ion Channel
Patch Clamp
Site-Directed Mutagenesis
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:11336/53089
Acceder
id CONICETDig_bb4e411daf5ea94b30f8035f06a35655
oai_identifier_str oai:ri.conicet.gov.ar:11336/53089
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gatingSpitzmaul, Guillermo FedericoCorradi, JeremiasBouzat, Cecilia BeatrizAcetylcholineAcetylcholine ReceptorIon ChannelPatch ClampSite-Directed Mutagenesishttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.Fil: Spitzmaul, Guillermo Federico. 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; ArgentinaFil: Corradi, Jeremias. 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; ArgentinaFil: Bouzat, Cecilia 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; ArgentinaTaylor & Francis Ltd2004-01info: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/11336/53089Spitzmaul, Guillermo Federico; Corradi, Jeremias; Bouzat, Cecilia Beatriz; Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating; Taylor & Francis Ltd; Molecular Membrane Biology; 21; 1; 1-2004; 39-500968-7688CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.tandfonline.com/doi/abs/10.1080/09687680310001607341info:eu-repo/semantics/altIdentifier/doi/10.1080/09687680310001607341info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:45:09Zoai:ri.conicet.gov.ar:11336/53089instacron: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:34982025-09-03 09:45:09.666CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
title Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
spellingShingle Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
Spitzmaul, Guillermo Federico
Acetylcholine
Acetylcholine Receptor
Ion Channel
Patch Clamp
Site-Directed Mutagenesis
title_short Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
title_full Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
title_fullStr Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
title_full_unstemmed Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
title_sort Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating
dc.creator.none.fl_str_mv Spitzmaul, Guillermo Federico
Corradi, Jeremias
Bouzat, Cecilia Beatriz
author Spitzmaul, Guillermo Federico
author_facet Spitzmaul, Guillermo Federico
Corradi, Jeremias
Bouzat, Cecilia Beatriz
author_role author
author2 Corradi, Jeremias
Bouzat, Cecilia Beatriz
author2_role author
author
dc.subject.none.fl_str_mv Acetylcholine
Acetylcholine Receptor
Ion Channel
Patch Clamp
Site-Directed Mutagenesis
topic Acetylcholine
Acetylcholine Receptor
Ion Channel
Patch Clamp
Site-Directed Mutagenesis
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.
Fil: Spitzmaul, Guillermo Federico. 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
Fil: Corradi, Jeremias. 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
Fil: Bouzat, Cecilia 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
description The nicotinic receptor (AChR) is a pentamer of homologous subunits with an α2βεδ composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15′ of the M1 domain is phenylalanine in α subunits while it is isoleucine in non-α subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant α subunit (αF15′I) as well as those containing the reverse mutations in the non-α subunits (βI15′F, δI15′F, and εI15′F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the β subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of βI15′F AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant of the diliganded receptor, and a significant increased opening in the absence of agonist. Mutations at βI15′ showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an ∼70% closed-state-like environment for the β15′ position at the transition state of gating. The overall results identify position 15′ as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.
publishDate 2004
dc.date.none.fl_str_mv 2004-01
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/11336/53089
Spitzmaul, Guillermo Federico; Corradi, Jeremias; Bouzat, Cecilia Beatriz; Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating; Taylor & Francis Ltd; Molecular Membrane Biology; 21; 1; 1-2004; 39-50
0968-7688
CONICET Digital
CONICET
url http://hdl.handle.net/11336/53089
identifier_str_mv Spitzmaul, Guillermo Federico; Corradi, Jeremias; Bouzat, Cecilia Beatriz; Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating; Taylor & Francis Ltd; Molecular Membrane Biology; 21; 1; 1-2004; 39-50
0968-7688
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.tandfonline.com/doi/abs/10.1080/09687680310001607341
info:eu-repo/semantics/altIdentifier/doi/10.1080/09687680310001607341
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 Taylor & Francis Ltd
publisher.none.fl_str_mv Taylor & Francis 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 13.13397

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