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Inhibition of human TRPC5 activity by PIP2
Author(s) -
Zeng Fanning,
McHugh Damian,
Beech David
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a329-d
Subject(s) - trpc5 , phosphatidylinositol , wortmannin , hek 293 cells , microbiology and biotechnology , chemistry , mutant , transfection , transient receptor potential channel , biochemistry , trpc , receptor , kinase , biology , gene
Phosphatidylinositol 4,5‐bisphosphate (PIP2) has important regulatory roles in many cellular process. Several members of the TRPM and TRPV ion channel families have been shown to be regulated by PIP2. Using a HEK293 cell line stably expressing human TRPC5, we tested whether the function of TRPC5 is also regulated by PIP2. Wortmannin at micromolar concentrations blocks the activity of most PI4 kinases, and therefore, interferes with replenishment of PIP2 levels. When the cells were pre‐treated with 30 μM wortmannin, the TRPC5 response to extracellular gadolilium (Gd 3+ ) increased more than 7‐fold, as determined by Ca 2+ ‐imaging. Furthermore, if TRPC5 was pre‐activated by Gd 3+ , application of DiC8 PIP2, a short chain synthetic form of PIP2, inhibited channel activity by more than 60%. There is a highly positively charged, 15 amino acids stretch just down‐stream the sixth transmembrane segment of TRPC5, which can putatively bind to the negatively charged head group of PIP2. A deletion mutant was generated, transiently transfected in HEK 293 and tested against the wild type TRPC5: Channel activity increased by 4‐fold upon the deletion of the putative PIP2 binding site. These data suggested that PIP2 might directly bind to TRPC5 and play an inhibitory role in its function. This work is supported by Wellcome Trust.