TRPC6 Binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility
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 r...
| Autores: | , , , , , , , , , , |
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| Formato: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2019 |
| País: | Argentina |
| Recursos: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Idioma: | inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/120497 |
| Acesso em linha: | http://hdl.handle.net/11336/120497 |
| Access Level: | acceso abierto |
| Palavra-chave: | focal segmental glomerulosclerosis glomerulus podocyte renal cell biology https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| Resumo: | 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. |
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