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Abstract: TH-PO060

Calpain Activation by TRPC6 in the Podocyte

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology


  • Farmer, Louise K., University of Bristol, Bristol, United Kingdom
  • Welsh, Gavin Iain, University of Bristol, Bristol, United Kingdom
  • Saleem, Moin, University of Bristol, Bristol, United Kingdom

TRPC6 mutations have been shown to cause Focal Segmental Glomerulosclerosis (FSGS). Previously the pathology of these mutations was thought to be due to an increased calcium conductance of this membrane channel. However, several disease-causing mutations have been reported to have no change in, or decreased, calcium conductance. We have investigated whether these mutations affect protein interactions.


Conditionally immortalised podocyte cell lines were generated from TRPC6 KO C57Bl/6 mice. GFP tagged WT/mutant TRPC6 was stably reintroduced into the KO cell line using a lentiviral construct. Cell lines were then characterised to determine motility and adhesion using scratch and adhesion assays. GFP TRAP beads were used to immunoprecipitate TRPC6 and proteomics was performed to identify novel binding partners. Calpain assays were performed using a commercially available kit.


TRPC6 KO (T6K) podocytes are less motile and more adhesive than those expressing WT TRPC6. Calpain 1 and 2, ERK 1/2 and caldesmon were identified as novel TRPC6 binding partners using GFP-TRAP pull down, proteomics and verified through co-immunoprecipitation experiments. Calpain is a protease with a variety of targets including focal adhesion kinase (FAK). T6K cells have decreased FAK cleavage and increased FAK phosphorylation compared to cells containing WT TRPC6. The cleavage of the calpain targets talin-1 and caldesmon was also decreased in T6K cells. Calpain assays demonstrated a loss of calpain activity in T6K cells. This suggests that in WT cells TRPC6 is responsible for calpain activation. The disease causing mutant form of TRPC6, K874*, has normal calcium conductance, however, as with T6K cells, cells expressing this mutant have decreased calpain activity and decreased cleavage of calpain targeted proteins. Co-IP experiments have shown that there is decreased binding of TRPC6 K874* to calpain compared to WT.
We have also shown that treatment of WT cells with plasma from nephrotic syndrome patients during relapse, but not remission, causes an increase in calpain activity. This suggests that TRPC6 mediated activation of calpain could be playing an important role in disease.


TRPC6 mediated activation of calpain plays an important role in podocyte motility and detachment. Disease causing mutations in TRPC6 could be acting by preventing the binding of calpain 1/2 to TRPC6, decreasing calpain activation