title: Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.
abstract: This study examined the effect of H(2)O(2) on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H(2)O(2) significantly stimulated Ca(2+) entry in a dose-dependent manner. Electrophysiological experiments showed that H(2)O(2) significantly increased TRPC6 channel open probability and whole-cell currents. H(2)O(2) also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca(2+) entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H(2)O(2) response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H(2)O(2)-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H(2)O(2). Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H(2)O(2). Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H(2)O(2) activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.
authors.affiliation: Department of Integrative Physiology and Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA.
authors.forename: Sarabeth
authors.initials: S
authors.lastname: Graham
authors.forename: Min
authors.initials: M
authors.lastname: Ding
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Ding
authors.forename: Sherry
authors.initials: S
authors.lastname: Sours-Brothers
authors.forename: Rafal
authors.initials: R
authors.lastname: Luchowski
authors.forename: Zygmunt
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: Thomas
authors.initials: T
authors.lastname: Yorio
authors.forename: Haiying
authors.initials: H
authors.lastname: Ma
authors.forename: Rong
authors.initials: R
authors.lastname: Ma
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004553:Electric Conductivity; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D050052:TRPC Cation Channels / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004075:Diglycerides; D013438:Sulfhydryl Compounds; D050052:TRPC Cation Channels; C492145:Trpc6 protein, rat; D006861:Hydrogen Peroxide; D002118:Calcium