TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility

Autores
Farmer, Louise K.; Rollason, Ruth; Whitcomb, Daniel J.; Ni, Lan; Goodliff, Alexander; Lay, Abigail C.; Birnbaumer, Lutz; Heesom, Kate J.; Xu, Shang Zhong; Saleem, Moin A.; Welsh, Gavin I.
Año de publicación
2019
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified rolewithin the target cell for FSGS, the kidney podocyte. Methods We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. Results Compared with wild-type cells, TRPC62/2 podocytes are less motile and more adhesive, with an altered actin cytoskeleton.We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- A nd actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, andmotility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton,motility, and adhesion-characteristics of TRPC6 2/2 podocytes. Conclusions Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.
Fil: Farmer, Louise K.. Bristol Medical School; Reino Unido
Fil: Rollason, Ruth. Bristol Medical School; Reino Unido
Fil: Whitcomb, Daniel J.. Bristol Medical School; Reino Unido
Fil: Ni, Lan. Bristol Medical School; Reino Unido
Fil: Goodliff, Alexander. Bristol Medical School; Reino Unido
Fil: Lay, Abigail C.. Bristol Medical School; Reino Unido
Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina
Fil: Heesom, Kate J.. No especifíca;
Fil: Xu, Shang Zhong. University of Hull; Reino Unido
Fil: Saleem, Moin A.. Bristol Medical School; Reino Unido
Fil: Welsh, Gavin I.. Bristol Medical School; Reino Unido
Materia
focal segmental glomerulosclerosis
glomerulus
podocyte
renal cell biology
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:11336/120497
Acceder
id CONICETDig_72422382479a41aba4123c2be55d1522
oai_identifier_str oai:ri.conicet.gov.ar:11336/120497
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motilityFarmer, Louise K.Rollason, RuthWhitcomb, Daniel J.Ni, LanGoodliff, AlexanderLay, Abigail C.Birnbaumer, LutzHeesom, Kate J.Xu, Shang ZhongSaleem, Moin A.Welsh, Gavin I.focal segmental glomerulosclerosisglomeruluspodocyterenal cell biologyhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Background Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified rolewithin the target cell for FSGS, the kidney podocyte. Methods We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. Results Compared with wild-type cells, TRPC62/2 podocytes are less motile and more adhesive, with an altered actin cytoskeleton.We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- A nd actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, andmotility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton,motility, and adhesion-characteristics of TRPC6 2/2 podocytes. Conclusions Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.Fil: Farmer, Louise K.. Bristol Medical School; Reino UnidoFil: Rollason, Ruth. Bristol Medical School; Reino UnidoFil: Whitcomb, Daniel J.. Bristol Medical School; Reino UnidoFil: Ni, Lan. Bristol Medical School; Reino UnidoFil: Goodliff, Alexander. Bristol Medical School; Reino UnidoFil: Lay, Abigail C.. Bristol Medical School; Reino UnidoFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Heesom, Kate J.. No especifíca;Fil: Xu, Shang Zhong. University of Hull; Reino UnidoFil: Saleem, Moin A.. Bristol Medical School; Reino UnidoFil: Welsh, Gavin I.. Bristol Medical School; Reino UnidoSynthesis-Stuttgart2019-10info: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/11336/120497Farmer, Louise K.; Rollason, Ruth; Whitcomb, Daniel J.; Ni, Lan; Goodliff, Alexander; et al.; TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility; Synthesis-Stuttgart; Journal of the American Society of Nephrology; 30; 10; 10-2019; 1910-19241046-6673CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1681/ASN.2018070729info:eu-repo/semantics/altIdentifier/url/https://jasn.asnjournals.org/content/30/10/1910info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:47:28Zoai:ri.conicet.gov.ar:11336/120497instacron: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 09:47:29.167CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
title TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
spellingShingle TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
Farmer, Louise K.
focal segmental glomerulosclerosis
glomerulus
podocyte
renal cell biology
title_short TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
title_full TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
title_fullStr TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
title_full_unstemmed TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
title_sort TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
dc.creator.none.fl_str_mv Farmer, Louise K.
Rollason, Ruth
Whitcomb, Daniel J.
Ni, Lan
Goodliff, Alexander
Lay, Abigail C.
Birnbaumer, Lutz
Heesom, Kate J.
Xu, Shang Zhong
Saleem, Moin A.
Welsh, Gavin I.
author Farmer, Louise K.
author_facet Farmer, Louise K.
Rollason, Ruth
Whitcomb, Daniel J.
Ni, Lan
Goodliff, Alexander
Lay, Abigail C.
Birnbaumer, Lutz
Heesom, Kate J.
Xu, Shang Zhong
Saleem, Moin A.
Welsh, Gavin I.
author_role author
author2 Rollason, Ruth
Whitcomb, Daniel J.
Ni, Lan
Goodliff, Alexander
Lay, Abigail C.
Birnbaumer, Lutz
Heesom, Kate J.
Xu, Shang Zhong
Saleem, Moin A.
Welsh, Gavin I.
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv focal segmental glomerulosclerosis
glomerulus
podocyte
renal cell biology
topic focal segmental glomerulosclerosis
glomerulus
podocyte
renal cell biology
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Background Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified rolewithin the target cell for FSGS, the kidney podocyte. Methods We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. Results Compared with wild-type cells, TRPC62/2 podocytes are less motile and more adhesive, with an altered actin cytoskeleton.We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- A nd actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, andmotility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton,motility, and adhesion-characteristics of TRPC6 2/2 podocytes. Conclusions Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.
Fil: Farmer, Louise K.. Bristol Medical School; Reino Unido
Fil: Rollason, Ruth. Bristol Medical School; Reino Unido
Fil: Whitcomb, Daniel J.. Bristol Medical School; Reino Unido
Fil: Ni, Lan. Bristol Medical School; Reino Unido
Fil: Goodliff, Alexander. Bristol Medical School; Reino Unido
Fil: Lay, Abigail C.. Bristol Medical School; Reino Unido
Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina
Fil: Heesom, Kate J.. No especifíca;
Fil: Xu, Shang Zhong. University of Hull; Reino Unido
Fil: Saleem, Moin A.. Bristol Medical School; Reino Unido
Fil: Welsh, Gavin I.. Bristol Medical School; Reino Unido
description Background Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified rolewithin the target cell for FSGS, the kidney podocyte. Methods We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. Results Compared with wild-type cells, TRPC62/2 podocytes are less motile and more adhesive, with an altered actin cytoskeleton.We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- A nd actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, andmotility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton,motility, and adhesion-characteristics of TRPC6 2/2 podocytes. Conclusions Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.
publishDate 2019
dc.date.none.fl_str_mv 2019-10
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/11336/120497
Farmer, Louise K.; Rollason, Ruth; Whitcomb, Daniel J.; Ni, Lan; Goodliff, Alexander; et al.; TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility; Synthesis-Stuttgart; Journal of the American Society of Nephrology; 30; 10; 10-2019; 1910-1924
1046-6673
CONICET Digital
CONICET
url http://hdl.handle.net/11336/120497
identifier_str_mv Farmer, Louise K.; Rollason, Ruth; Whitcomb, Daniel J.; Ni, Lan; Goodliff, Alexander; et al.; TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility; Synthesis-Stuttgart; Journal of the American Society of Nephrology; 30; 10; 10-2019; 1910-1924
1046-6673
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1681/ASN.2018070729
info:eu-repo/semantics/altIdentifier/url/https://jasn.asnjournals.org/content/30/10/1910
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 Synthesis-Stuttgart
publisher.none.fl_str_mv Synthesis-Stuttgart
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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