Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

• Published in: Cell calcium (Edinburgh) 2009-04, Vol.45 (4), p.384-390
• Main Authors: Foster, Rebecca R, Zadeh, Maryam A.H, Welsh, Gavin I, Satchell, Simon C, Ye, Yi, Mathieson, Peter W, Bates, David O, Saleem, Moin A
• Format: Article
• Language: English
• Subjects: Adenovirus; Advanced Basic Science; Boron Compounds - pharmacology; Calcium - metabolism; Calcium Signaling - drug effects; Calcium signalling; Cell Line, Transformed; Flufenamic acid; Flufenamic Acid - pharmacology; HEK293; Humans; Imidazoles - pharmacology; Indomethacin - pharmacology; Intracellular Space - drug effects; Intracellular Space - metabolism; Ion Channel Gating - drug effects; Niflumic Acid - pharmacology; Podocytes; Podocytes - cytology; Podocytes - drug effects; Podocytes - metabolism; Transfection; TRPC Cation Channels - metabolism; TRPC6; TRPC6 Cation Channel
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/j.ceca.2009.01.003

Abstract Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca2+ ]i ) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod). Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca2+ ]i ) were calculated. 200 μM FFA induced an increase in [Ca2+ ]i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca2+ ]i , which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca2+ ]i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca2+ ]i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6. In conclusion, FFA activates TRPC6 [Ca2+ ]i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.