TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility

Significance Statement Mutations in the transient receptor potential channel 6 ( TRPC6 ) gene are associated with an inherited form of FSGS. Emerging evidence has linked TRPC6 activity with calpain activation and podocyte injury. In this study, the authors generated a TRPC6 knockout podocyte cell line from TRPC6 knockout mice, engineering these cells to express wild-type and various mutations of TRPC6. They show that TRPC6 binds to both ERK 1/2 and calpain, and is important for the localization of calpain to the cell membrane, independent of TRPC6 calcium influx. This interaction is vital for cell motility and detachment and demonstrates a scaffolding role of TRPC6. These findings suggest that calpain activation and trafficking may be novel therapeutic targets in the treatment of FSGS. 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 role within 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 TRPC6 mutation, 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, TRPC6−/− 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- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility 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 −/− 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.