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

Farmer, Louise; Rollason, Ruth; Whitcomb, Daniel; Ni, Lan; Goodlif, Alexander; Lay, Abigail; Birnbaumer, Lutz; Heesom, Kate; Xu, Shang-Zhong; Saleem, Moin; Welsh, Gavin

Abstract

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.

Citation

Farmer, L., Rollason, R., Whitcomb, D., Ni, L., Goodlif, A., Lay, A., Birnbaumer, L., Heesom, K., Xu, S.-Z., Saleem, M., & Welsh, G. (2019). TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility. Journal of the American Society of Nephrology : JASN, 30(10), 1910-1924. https://doi.org/10.1681/ASN.2018070729

• Journal Article Type: Article
• Acceptance Date: Jul 17, 2019
• Online Publication Date: Sep 30, 2019
• Publication Date: Sep 30, 2019
• Deposit Date: Oct 2, 2019
• Publicly Available Date: Oct 3, 2019
• Journal: JASN
• Print ISSN: 1046-6673
• Publisher: American Society of Nephrology
• Peer Reviewed: Peer Reviewed
• Volume: 30
• Issue: 10
• Pages: 1910-1924
• DOI: https://doi.org/10.1681/ASN.2018070729
• Keywords: Focal segmental glomerulosclerosis; Podocyte; Renal cell biology; Glomerulus
• Public URL: https://hull-repository.worktribe.com/output/2838830
• Publisher URL: https://jasn.asnjournals.org/content/30/10/1910
• Related Public URLs: http://hdl.handle.net/1983/6a539044-09c8-4ee2-90d8-bfd1e71bddcc
• Contract Date: Oct 2, 2019