Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry

Autores
Valdivieso, Ángel Gabriel; Marín, María C.; Clauzure, Mariángeles; Santa Coloma, Tomás A.
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K Cl-) for chloride in water solution was 115.0 ± 2.8 M-1, whereas the intracellular KCl- was 17.8 ± 0.8 M-1, for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.
Fil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Fil: Marín, María C.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Fil: Clauzure, Mariángeles. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Fil: Santa Coloma, Tomás A.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Materia
CFTR
CHLORIDE CHANNEL
CHLORIDE FLUORESCENCE SPECTROPHOTOMETRY
CYSTIC FIBROSIS
SPQ
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:11336/192913
Acceder
id CONICETDig_293b2fdca45bb8a42e402c9feec206c5
oai_identifier_str oai:ri.conicet.gov.ar:11336/192913
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometryValdivieso, Ángel GabrielMarín, María C.Clauzure, MariángelesSanta Coloma, Tomás A.CFTRCHLORIDE CHANNELCHLORIDE FLUORESCENCE SPECTROPHOTOMETRYCYSTIC FIBROSISSPQhttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K Cl-) for chloride in water solution was 115.0 ± 2.8 M-1, whereas the intracellular KCl- was 17.8 ± 0.8 M-1, for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.Fil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Marín, María C.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Clauzure, Mariángeles. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaFil: Santa Coloma, Tomás A.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; ArgentinaAcademic Press Inc Elsevier Science2011-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/192913Valdivieso, Ángel Gabriel; Marín, María C.; Clauzure, Mariángeles; Santa Coloma, Tomás A.; Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry; Academic Press Inc Elsevier Science; Analytical Biochemistry; 418; 2; 11-2011; 231-2370003-2697CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0003269711004994info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ab.2011.07.029info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:02:29Zoai:ri.conicet.gov.ar:11336/192913instacron: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 10:02:30.177CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
spellingShingle Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
Valdivieso, Ángel Gabriel
CFTR
CHLORIDE CHANNEL
CHLORIDE FLUORESCENCE SPECTROPHOTOMETRY
CYSTIC FIBROSIS
SPQ
title_short Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_full Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_fullStr Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_full_unstemmed Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
title_sort Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry
dc.creator.none.fl_str_mv Valdivieso, Ángel Gabriel
Marín, María C.
Clauzure, Mariángeles
Santa Coloma, Tomás A.
author Valdivieso, Ángel Gabriel
author_facet Valdivieso, Ángel Gabriel
Marín, María C.
Clauzure, Mariángeles
Santa Coloma, Tomás A.
author_role author
author2 Marín, María C.
Clauzure, Mariángeles
Santa Coloma, Tomás A.
author2_role author
author
author
dc.subject.none.fl_str_mv CFTR
CHLORIDE CHANNEL
CHLORIDE FLUORESCENCE SPECTROPHOTOMETRY
CYSTIC FIBROSIS
SPQ
topic CFTR
CHLORIDE CHANNEL
CHLORIDE FLUORESCENCE SPECTROPHOTOMETRY
CYSTIC FIBROSIS
SPQ
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K Cl-) for chloride in water solution was 115.0 ± 2.8 M-1, whereas the intracellular KCl- was 17.8 ± 0.8 M-1, for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.
Fil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Fil: Marín, María C.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Fil: Clauzure, Mariángeles. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
Fil: Santa Coloma, Tomás A.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentina
description Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K Cl-) for chloride in water solution was 115.0 ± 2.8 M-1, whereas the intracellular KCl- was 17.8 ± 0.8 M-1, for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 μM) and glibenclamide (100 μM) showed a significant reduction (P < 0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.
publishDate 2011
dc.date.none.fl_str_mv 2011-11
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/11336/192913
Valdivieso, Ángel Gabriel; Marín, María C.; Clauzure, Mariángeles; Santa Coloma, Tomás A.; Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry; Academic Press Inc Elsevier Science; Analytical Biochemistry; 418; 2; 11-2011; 231-237
0003-2697
CONICET Digital
CONICET
url http://hdl.handle.net/11336/192913
identifier_str_mv Valdivieso, Ángel Gabriel; Marín, María C.; Clauzure, Mariángeles; Santa Coloma, Tomás A.; Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry; Academic Press Inc Elsevier Science; Analytical Biochemistry; 418; 2; 11-2011; 231-237
0003-2697
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/S0003269711004994
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ab.2011.07.029
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
dc.publisher.none.fl_str_mv Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
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
_version_ 1842269758706155520
score 13.13397

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