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Promotion of Trafficking and Sensitivity to DAG are Involved in TRPC6 Channel Activation by Hydrogen Peroxide
Author(s) -
Graham Sarabeth Marie,
Ding Min,
Luchowski Rafal,
Gryczynski Zygmunt,
Ma Rong
Publication year - 2010
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.24.1_supplement.608.15
Subject(s) - trpc6 , transfection , chemistry , activator (genetics) , hek 293 cells , biophysics , microbiology and biotechnology , biochemistry , biology , receptor , gene , transient receptor potential channel
The present study was performed to examine the effect of H 2 O 2 on TRPC6 channel and the underlying mechanisms in a heterologus expression system. In the TRPC6 expressing HEK293T cells, H 2 O 2 significantly enhanced inward whole cell currents and single channel open probability (NP O ) compared with empty vector‐transfected cells. Fura‐2 fluorescence measurements revealed that H 2 O 2 stimulated Ca 2+ entry in a dose‐dependent manner in TRPC6‐expressing cells. OAG, a cell membrane permeable homologue of DAG, significantly activated TRPC6 channels in HEK293T cells. The OAG effect was significantly enhanced in the presence of H 2 O 2 (10 μM), suggesting that H 2 O 2 increased the sensitivity of TRPC6 channel to DAG, a physiological stimulus. Moreover, a biotinylation assay showed a clear increase in cell surface TRPC6 protein expression in response to H 2 O 2 (10 μM for 3 min). Consistent with this biochemical evidence, confocal microscopy revealed a significant increase in fluorescence intensity in the cell membrane or sub‐membrane in the first 3 min of H 2 O 2 treatment in TRPC6‐EGFP transfected cells, but not in EGFP‐transfected cells. Taken together, these data indicate that H 2 O 2 is an activator of TRPC6 channel and the activation mechanisms may involve both sensitization of the channel to its physiological stimulus (DAG) as well as promotion of TRPC6 trafficking to the plasma membrane.