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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.

SUBMITTER: Graham S

PROVIDER: S-EPMC2906337 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.

Graham Sarabeth S Ding Min M Ding Yanfeng Y Sours-Brothers Sherry S Luchowski Rafal R Gryczynski Zygmunt Z Yorio Thomas T Ma Haiying H Ma Rong R

The Journal of biological chemistry 20100525 30

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 kno ...[more]

PMID: 20501650

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Project description:BackgroundFibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca(2+)-permeable transient receptor potential canonical-3 (TRPC3) channels.Methods and resultsFreshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (I(NSC)) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca(2+) influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca(2+) removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5' promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression.ConclusionsTRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca(2+) influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

Project description:Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na+ and Ca2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na+ and Ca2+ free solution, we found an increased decay of the cytosolic Ca2+ concentration [Ca2+]i and increased SR Ca2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca2+ leak through SR TRPC channels increased the systolic and diastolic [Ca2+]i with only minor effects on the action potential and SR Ca2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca2+]i and contractility in cardiomyocytes.

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Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.

Publications

Canonical transient receptor potential 6 (TRPC6), a redox-regulated cation channel.

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