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
• Fuente: Journal of the American Society of Nephrology. August 2019:ASN.2018070729
• Materia: NEFROLOGIA; MUTACION; GENES; TRASTORNOS GENETICOS; ENFERMEDADES RENALES; RIÑON; PROTEINAS
• Institución: Pontificia Universidad Católica Argentina

Fil: Farmer, Louise K. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Fil: Rollason, Ruth. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Fil: Whitcomb, Daniel J. University of Bristol. Bristol Medical School; Gran Bretaña
Fil: Ni, Lan. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Fil: Goodliff, Alexander. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Fil: Lay, Abigail C. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Research Triangle Park. Neurobiology Laboratory; Estados Unidos
Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas
Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Heesom, Kate J. University of Bristol. Proteomics Facility; Gran Bretaña
Fil: Xu, Shang-Zhong. University of Hull. Hull York Medical School. Centre for Cardiovascular and Metabolic Research; Gran Bretaña
Fil: Saleem, Moin A. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Fil: Welsh, Gavin I. University of Bristol. Bristol Medical School. Briston Renal; Gran Bretaña
Background: Mutations in transient receptor potential channel 6 (TRPC6) are associated with an inherited form of focal segmental glomerulosclerosis (FSGS). Despite widespread expression, patients with TRPC6 mutations do not present with any other pathological phenotype suggesting that this protein has a unique but yet unidentified role within the target cell for FSGS, the kidney podocyte. Methods: A stable TRPC6 knock out podocyte cell line was generated from TRPC6 knockout mice. These cells were engineered to express wild type, dominant negative or either G109S or K874* disease causing mutants of TRPC6. These cells were extensively characterised via motility, detachment and calpain activity assays, immunofluorescence and confocal or Total Internal Reflection fluorescence (TIRF) microscopy, and western blotting. Results: TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton compared to wild type cells. Mechanistically, we show that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon and calpain 1 and 2. Calpains are calcium dependent cysteine proteases, which control the podocyte cytoskeleton, cell adhesion and motility via cleavage of paxillin, focal adhesion kinase and talin. Knockdown or expression of the truncated K874*, but not the gain of function G019S or dominant negative mutant of TRPC6 results in the mislocalization of calpain 1 and 2 and significant down-regulation of calpain activity leading to altered podocyte cytoskeleton, motility and adhesion, a phenocopy of TRPC6 -/- podocytes. Conclusions: Our data demonstrates that the physical interaction between TRPC6 and calpain in the podocyte is important in disease, independent of TRPC6 channel activity.