Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells

Autores
Weissmann, Carina; Di Guilmi, Mariano Nicolás; Urbano Suarez, Francisco Jose; Uchitel, Osvaldo Daniel
Año de publicación
2012
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by ‘whole-cell voltage-clamp’ recordings, showing a maximum current peak in the I–V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through CaV2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the CaV2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channels’ currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action.
Fil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Urbano Suarez, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Materia
Pregabalin
Calcium Channels
Hek293t Cells
Endocytosis
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/20141
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/20141
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cellsWeissmann, CarinaDi Guilmi, Mariano NicolásUrbano Suarez, Francisco JoseUchitel, Osvaldo DanielPregabalinCalcium ChannelsHek293t CellsEndocytosishttps://purl.org/becyt/ford/3.1https://purl.org/becyt/ford/3We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by ‘whole-cell voltage-clamp’ recordings, showing a maximum current peak in the I–V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through CaV2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the CaV2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channels’ currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action.Fil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Urbano Suarez, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaElsevier Inc2012-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/20141Weissmann, Carina; Di Guilmi, Mariano Nicolás; Urbano Suarez, Francisco Jose; Uchitel, Osvaldo Daniel; Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells; Elsevier Inc; Brain Research Bulletin; 90; 10-2012; 107-1130361-9230CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.brainresbull.2012.10.001info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0361923012002183info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:00:15Zoai:ri.conicet.gov.ar:11336/20141instacron: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 13:00:15.376CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
title Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
spellingShingle Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
Weissmann, Carina
Pregabalin
Calcium Channels
Hek293t Cells
Endocytosis
title_short Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
title_full Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
title_fullStr Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
title_full_unstemmed Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
title_sort Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
dc.creator.none.fl_str_mv Weissmann, Carina
Di Guilmi, Mariano Nicolás
Urbano Suarez, Francisco Jose
Uchitel, Osvaldo Daniel
author Weissmann, Carina
author_facet Weissmann, Carina
Di Guilmi, Mariano Nicolás
Urbano Suarez, Francisco Jose
Uchitel, Osvaldo Daniel
author_role author
author2 Di Guilmi, Mariano Nicolás
Urbano Suarez, Francisco Jose
Uchitel, Osvaldo Daniel
author2_role author
author
author
dc.subject.none.fl_str_mv Pregabalin
Calcium Channels
Hek293t Cells
Endocytosis
topic Pregabalin
Calcium Channels
Hek293t Cells
Endocytosis
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.1
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by ‘whole-cell voltage-clamp’ recordings, showing a maximum current peak in the I–V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through CaV2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the CaV2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channels’ currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action.
Fil: Weissmann, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Urbano Suarez, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
description We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by ‘whole-cell voltage-clamp’ recordings, showing a maximum current peak in the I–V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through CaV2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the CaV2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channels’ currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action.
publishDate 2012
dc.date.none.fl_str_mv 2012-10
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/11336/20141
Weissmann, Carina; Di Guilmi, Mariano Nicolás; Urbano Suarez, Francisco Jose; Uchitel, Osvaldo Daniel; Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells; Elsevier Inc; Brain Research Bulletin; 90; 10-2012; 107-113
0361-9230
CONICET Digital
CONICET
url http://hdl.handle.net/11336/20141
identifier_str_mv Weissmann, Carina; Di Guilmi, Mariano Nicolás; Urbano Suarez, Francisco Jose; Uchitel, Osvaldo Daniel; Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells; Elsevier Inc; Brain Research Bulletin; 90; 10-2012; 107-113
0361-9230
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.brainresbull.2012.10.001
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0361923012002183
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Inc
publisher.none.fl_str_mv Elsevier Inc
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 12.48226

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