Abstract: SA-PO227

Novel Methods to Analyze Mechanisms of TRPC6 Activation

Session Information

  • Glomerular: Cell Biology
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Glomerular

  • 1003 Glomerular: Cell Biology

Authors

  • Dey, Asim Bikash, Eli Lilly and Company, Indianapolis, Indiana, United States
  • Li, Fang, Eli Lilly and Company, Indianapolis, Indiana, United States
  • Atkinson, Simon J., Indiana University - Purdue University Indianapolis, Indianapolis, Indiana, United States
  • Kowala, Mark, Eli Lilly and Company, Indianapolis, Indiana, United States
  • Rekhter, Mark, Eli Lilly and Company, Indianapolis, Indiana, United States
Background

TRPC6 is a calcium channel activated by diacylglycerol (DAG) and reactive oxygen species (ROS). TRPC6 mutations are associated with proteinuria in human focal segmental glomerulosclerosis. New data suggest that circulating factors in diabetes induce kidney injury via TRPC6-mediated calcium flux. It is unknown how different factors activate TRPC6. The goal of this study was to develop a platform for analysis of DAG and ROS in TRPC6 activation.

Methods

Full length human TRPC6 cDNA was cloned into pcDNA5/TO (Thermo Fischer) under CMV promoter and transfected into HEK293 cells. Stable clones were selected based on the TRPC6 expression. Calcium signaling was analyzed using FLIPR membrane potential assay.

DAG synthesis was monitored using recombinant circularly permutated probe, Downward DAG (Montana Molecular). Cells were infected with baculovirus carrying the biosensor construct. Fluorescent signal was captured using FLIPR.

For ROS detection, cells were stained with Cell-ROX dye and analyzed using confocal microscope.

Results

The stable cell line, C11, showed >7000x increase in hTRPC6 gene expression. Calcium signaling was stimulated in a dose-dependent manner by angiotensin II (AngII), 1-oleoyl-2-acetyl-sn-glycerol (OAG), endothelin 1 and hyperforin 9 (Hyp9), specific activator of TRPC6. DAG signal was increased by Hyp9 and endothelin 1, but not by AngII. However, AngII but neither Hyp9 nor endothelin 1 induced ROS formation.

Conclusion

We generated novel tools to investigate mechanisms of TRPC6 activation and demonstrated differential involvement of DAG and ROS in TRPC-induced calcium flux.