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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

Authors

Louise Farmer

Ruth Rollason

Daniel Whitcomb

Lan Ni

Alexander Goodlif

Abigail Lay

Lutz Birnbaumer

Kate Heesom

Shang-Zhong Xu

Moin Saleem

Gavin Welsh



Contributors

Louise K Farmer
Composer

Ruth Rollason
Composer

Daniel J. Whitcomb
Composer

Lan Ni
Composer

Alexander Goodliff
Composer

Abigail C. Lay
Composer

Lutz Birnbaumer
Composer

Kate J Heesom
Composer

Shang-Zhong Xu
Supervisor

Moin A Saleem
Supervisor

Gavin I. Welsh
Supervisor

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.

Journal Article Type Article
Publication Date Sep 30, 2019
Journal JASN
Print ISSN 1046-6673
Publisher American Society of Nephrology
Peer Reviewed Peer Reviewed
Volume 30
Issue 10
Pages 1910-1924
APA6 Citation (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
DOI https://doi.org/10.1681/ASN.2018070729
Keywords Focal segmental glomerulosclerosis; Podocyte; Renal cell biology; Glomerulus
Publisher URL https://jasn.asnjournals.org/content/30/10/1910
Related Public URLs http://hdl.handle.net/1983/6a539044-09c8-4ee2-90d8-bfd1e71bddcc

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Copyright Statement
©2019 The authors. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder





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