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
Louise K Farmer
Daniel J. Whitcomb
Abigail C. Lay
Kate J Heesom
Moin A Saleem
Gavin I. Welsh
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.
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.
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.
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|
|Publisher||American Society of Nephrology|
|Peer Reviewed||Peer Reviewed|
|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|
|Keywords||Focal segmental glomerulosclerosis; Podocyte; Renal cell biology; Glomerulus|
|Related Public URLs||http://hdl.handle.net/1983/6a539044-09c8-4ee2-90d8-bfd1e71bddcc|
©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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