Canonical Transient Receptor Potential 6 (TRPC6), a redox-regulated cation channel

Sarabeth Graham, Min Ding, Yanfeng Ding, Sherry Sours-Brothers, Rafal Luchowski, Zygmunt Gryczynski, Thomas Yorio, Haiying Ma, Rong Ma

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Abstract

This study examined the effect of H2O2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H2O2 significantly stimulated Ca2+ entry in a dose-dependent manner. Electrophysiological experiments showed that H2O2 significantly increased TRPC6 channel open probability and whole-cell currents. H2O2 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 Ca2+ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H 2O2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H2O2-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-snglycerol effect on TRPC6 was significantly greater in the presence of H2O2. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H2O 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 H2O2 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.

Original languageEnglish
Pages (from-to)23466-23476
Number of pages11
JournalJournal of Biological Chemistry
Volume285
Issue number30
DOIs
StatePublished - 23 Jul 2010

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Arginine Vasopressin
Oxidation-Reduction
Cations
Vasopressin Receptors
Ethylmaleimide
Catalase
Fluorescence
Thimerosal
Confocal microscopy
Dithiothreitol
Diglycerides
Cell membranes
Sulfhydryl Compounds
Small Interfering RNA
Glutathione
Cysteine
Muscle
Assays
Esters
Chemical activation

Cite this

Graham, Sarabeth ; Ding, Min ; Ding, Yanfeng ; Sours-Brothers, Sherry ; Luchowski, Rafal ; Gryczynski, Zygmunt ; Yorio, Thomas ; Ma, Haiying ; Ma, Rong. / Canonical Transient Receptor Potential 6 (TRPC6), a redox-regulated cation channel. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 30. pp. 23466-23476.
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abstract = "This study examined the effect of H2O2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H2O2 significantly stimulated Ca2+ entry in a dose-dependent manner. Electrophysiological experiments showed that H2O2 significantly increased TRPC6 channel open probability and whole-cell currents. H2O2 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 Ca2+ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H 2O2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H2O2-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-snglycerol effect on TRPC6 was significantly greater in the presence of H2O2. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H2O 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 H2O2 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.",
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Canonical Transient Receptor Potential 6 (TRPC6), a redox-regulated cation channel. / Graham, Sarabeth; Ding, Min; Ding, Yanfeng; Sours-Brothers, Sherry; Luchowski, Rafal; Gryczynski, Zygmunt; Yorio, Thomas; Ma, Haiying; Ma, Rong.

In: Journal of Biological Chemistry, Vol. 285, No. 30, 23.07.2010, p. 23466-23476.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Canonical Transient Receptor Potential 6 (TRPC6), a redox-regulated cation channel

AU - Graham, Sarabeth

AU - Ding, Min

AU - Ding, Yanfeng

AU - Sours-Brothers, Sherry

AU - Luchowski, Rafal

AU - Gryczynski, Zygmunt

AU - Yorio, Thomas

AU - Ma, Haiying

AU - Ma, Rong

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AB - This study examined the effect of H2O2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H2O2 significantly stimulated Ca2+ entry in a dose-dependent manner. Electrophysiological experiments showed that H2O2 significantly increased TRPC6 channel open probability and whole-cell currents. H2O2 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 Ca2+ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H 2O2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H2O2-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-snglycerol effect on TRPC6 was significantly greater in the presence of H2O2. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H2O 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 H2O2 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.

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