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

Abstract

Mutations in the transient receptor potential channel 6 (<i>TRPC6</i>) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with <i>TRPC6</i> 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. We generated a stable <i>TRPC6</i> knockout podocyte cell line from <i>TRPC6</i> knockout mice. These cells were engineered to express wild-type <i>TRPC6</i>, a dominant negative <i>TRPC6</i> mutation, or either of two disease-causing mutations of <i>TRPC6</i>, 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. Compared with wild-type cells, <i>TRPC6^-/-</i> 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 <i>via</i> 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 <i>TRPC6</i>) 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 <i>TRPC6</i>^-/- podocytes. 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.