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