Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells

Autores
Raychowdhury, Malay K.; Ibarra, Cristina Adriana; Damiano, Alicia Ermelinda; Jackson Jr., George R.; Smith, Peter R.; McLaughlin, Margaret; Prat, Adriana G.; Ausiello, Dennis A.; Lader, Alan S.; Cantiello, Horacio Fabio
Año de publicación
2004
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In this study, the presence of Na+-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na+-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an ∼3:1 Na+/K+ permeability-selectivity ratio. The Na+-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na+ channel activity. Spontaneous and vasopressin-induced Na+ channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the αENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although αENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on αENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na+-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na+ transport mechanisms in the mammalian proximal nephron.
Fil: Raychowdhury, Malay K.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados Unidos
Fil: Ibarra, Cristina Adriana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Damiano, Alicia Ermelinda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Jackson Jr., George R.. Massachusetts General Hospital East; Estados Unidos
Fil: Smith, Peter R.. University of Alabama at Birmingahm; Estados Unidos
Fil: McLaughlin, Margaret. Massachusetts General Hospital East; Estados Unidos
Fil: Prat, Adriana G.. Massachusetts General Hospital East; Estados Unidos. Universidad de Buenos Aires. Facultad de Medicina; Argentina
Fil: Ausiello, Dennis A.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados Unidos
Fil: Lader, Alan S.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados Unidos
Fil: Cantiello, Horacio Fabio. Massachusetts General Hospital East; Estados Unidos. Harvard Medical School; Estados Unidos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Materia
Na
LLC-PK1
Renal Epithelial Cells
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:11336/242485
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/242485
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cellsRaychowdhury, Malay K.Ibarra, Cristina AdrianaDamiano, Alicia ErmelindaJackson Jr., George R.Smith, Peter R.McLaughlin, MargaretPrat, Adriana G.Ausiello, Dennis A.Lader, Alan S.Cantiello, Horacio FabioNaLLC-PK1Renal Epithelial Cellshttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1In this study, the presence of Na+-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na+-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an ∼3:1 Na+/K+ permeability-selectivity ratio. The Na+-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na+ channel activity. Spontaneous and vasopressin-induced Na+ channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the αENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although αENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on αENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na+-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na+ transport mechanisms in the mammalian proximal nephron.Fil: Raychowdhury, Malay K.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados UnidosFil: Ibarra, Cristina Adriana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Damiano, Alicia Ermelinda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jackson Jr., George R.. Massachusetts General Hospital East; Estados UnidosFil: Smith, Peter R.. University of Alabama at Birmingahm; Estados UnidosFil: McLaughlin, Margaret. Massachusetts General Hospital East; Estados UnidosFil: Prat, Adriana G.. Massachusetts General Hospital East; Estados Unidos. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Ausiello, Dennis A.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados UnidosFil: Lader, Alan S.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados UnidosFil: Cantiello, Horacio Fabio. Massachusetts General Hospital East; Estados Unidos. Harvard Medical School; Estados Unidos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaAmerican Society for Biochemistry and Molecular Biology2004-12info: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/11336/242485Raychowdhury, Malay K.; Ibarra, Cristina Adriana; Damiano, Alicia Ermelinda; Jackson Jr., George R.; Smith, Peter R.; et al.; Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 279; 19; 12-2004; 20137-201460021-9258CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M311946200info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0021925820671290info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:11:00Zoai:ri.conicet.gov.ar:11336/242485instacron: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 10:11:01.252CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
title Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
spellingShingle Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
Raychowdhury, Malay K.
Na
LLC-PK1
Renal Epithelial Cells
title_short Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
title_full Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
title_fullStr Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
title_full_unstemmed Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
title_sort Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells
dc.creator.none.fl_str_mv Raychowdhury, Malay K.
Ibarra, Cristina Adriana
Damiano, Alicia Ermelinda
Jackson Jr., George R.
Smith, Peter R.
McLaughlin, Margaret
Prat, Adriana G.
Ausiello, Dennis A.
Lader, Alan S.
Cantiello, Horacio Fabio
author Raychowdhury, Malay K.
author_facet Raychowdhury, Malay K.
Ibarra, Cristina Adriana
Damiano, Alicia Ermelinda
Jackson Jr., George R.
Smith, Peter R.
McLaughlin, Margaret
Prat, Adriana G.
Ausiello, Dennis A.
Lader, Alan S.
Cantiello, Horacio Fabio
author_role author
author2 Ibarra, Cristina Adriana
Damiano, Alicia Ermelinda
Jackson Jr., George R.
Smith, Peter R.
McLaughlin, Margaret
Prat, Adriana G.
Ausiello, Dennis A.
Lader, Alan S.
Cantiello, Horacio Fabio
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Na
LLC-PK1
Renal Epithelial Cells
topic Na
LLC-PK1
Renal Epithelial Cells
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv In this study, the presence of Na+-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na+-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an ∼3:1 Na+/K+ permeability-selectivity ratio. The Na+-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na+ channel activity. Spontaneous and vasopressin-induced Na+ channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the αENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although αENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on αENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na+-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na+ transport mechanisms in the mammalian proximal nephron.
Fil: Raychowdhury, Malay K.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados Unidos
Fil: Ibarra, Cristina Adriana. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Damiano, Alicia Ermelinda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Jackson Jr., George R.. Massachusetts General Hospital East; Estados Unidos
Fil: Smith, Peter R.. University of Alabama at Birmingahm; Estados Unidos
Fil: McLaughlin, Margaret. Massachusetts General Hospital East; Estados Unidos
Fil: Prat, Adriana G.. Massachusetts General Hospital East; Estados Unidos. Universidad de Buenos Aires. Facultad de Medicina; Argentina
Fil: Ausiello, Dennis A.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados Unidos
Fil: Lader, Alan S.. Harvard Medical School; Estados Unidos. Massachusetts General Hospital East; Estados Unidos
Fil: Cantiello, Horacio Fabio. Massachusetts General Hospital East; Estados Unidos. Harvard Medical School; Estados Unidos. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica. Cátedra de Química General e Inorgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description In this study, the presence of Na+-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na+-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an ∼3:1 Na+/K+ permeability-selectivity ratio. The Na+-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na+ channel activity. Spontaneous and vasopressin-induced Na+ channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the αENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although αENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on αENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na+-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na+ transport mechanisms in the mammalian proximal nephron.
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/11336/242485
Raychowdhury, Malay K.; Ibarra, Cristina Adriana; Damiano, Alicia Ermelinda; Jackson Jr., George R.; Smith, Peter R.; et al.; Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 279; 19; 12-2004; 20137-20146
0021-9258
CONICET Digital
CONICET
url http://hdl.handle.net/11336/242485
identifier_str_mv Raychowdhury, Malay K.; Ibarra, Cristina Adriana; Damiano, Alicia Ermelinda; Jackson Jr., George R.; Smith, Peter R.; et al.; Characterization of Na+-permeable cation channels in LLC-PK1 renal epithelial cells; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 279; 19; 12-2004; 20137-20146
0021-9258
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M311946200
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0021925820671290
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 Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
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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