TRPC6 Binds to and Activates Calpain, Independent of Its... : Journal of the American Society of Nephrology (original) (raw)

Basic Research

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

Farmer, Louise K.1; Rollason, Ruth1; Whitcomb, Daniel J.2; Ni, Lan1; Goodliff, Alexander1; Lay, Abigail C.1; Birnbaumer, Lutz3,4; Heesom, Kate J.5; Xu, Shang-Zhong6; Saleem, Moin A.1; Welsh, Gavin I.1

1Bristol Renal, Bristol Medical School,

2Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Bristol Medical School, and

5Proteomics Facility, University of Bristol, Bristol, United Kingdom;

3Neurobiology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina;

4Faculty of Medical Sciences, Institute of Biomedical Research, Catholic University of Argentina, Buenos Aires, Argentina; and

6Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull, United Kingdom

Correspondence: Dr. Gavin I. Welsh, Bristol Renal, Bristol Medical School, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK. Email: [email protected]

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Abstract

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.

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