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.

FIGURE 1.

TRPC6 channel response in TRPC6-expressing HEK293T cells. A, Western blot, showing the expression level of TRPC6 protein in untransfected (UT) HEK293T cells and the HEK293T cells with transient transfection of vector (Mock), rat TRPC6 expression plasmid (rTRPC6), and rTRPC6 plus RNAi constructs for rat TRPC6 (rTRPC6+rTRPC6-shRNA). TRPC6 was probed with rabbit anti-TRPC6 antibody. L, ladder of a protein marker. Actin was used as a loading control. B, fura-2 fluorescence measurement of Ca²⁺ entry response stimulated by 100 μm OAG in untransfected (UT), vector-transfected (Mock), and TRPC6-expressing (TRPC6) cells, and TRPC6-expressing cells treated with 1 μm calphostin C (TRPC6+Cal) or 2 μm La³⁺ (TRPC6+La³⁺). C, cell-attached patch clamp, showing channel open probability (NP_o) in response to 100 μm OAG in untransfected (UT), vector-transfected (Mock), TRPC6 expression plasmid (TRPC6)-transfected, and TRPC6 expression plasmid plus its RNAi construct-transfected (TRPC6+RNAi) HEK293T cells. B and C, *, p < 0.05, between the groups as indicated. n indicates the number of cells analyzed from at least six different transfections.

FIGURE 2.

Effect of H₂O₂ on the intracellular Ca²⁺ signal in TRPC6-expressing and empty vector-transfected HEK293T cells. A, representative traces, showing the changes in intracellular Ca²⁺ level upon 5 mm Ca²⁺ readdition to a Ca²⁺ free bathing solution in cells treated with different concentrations of H₂O₂. The Ca²⁺ concentration in the bathing solution is indicated by the numbers inside the top open bar. Application of H₂O₂ is indicated by the top solid bar. [Ca²⁺]_B indicates the Ca²⁺ concentration in the bathing solution. The numbers next to each trace indicate the concentration of H₂O₂ (μm) applied. B, representative traces, displaying intracellular Ca²⁺ response to Ca²⁺ readdition in vector-transfected (Mock) and rTRPC6-transfected (TRPC6) cells in the presence of 10 μm H₂O₂. The Ca²⁺ concentration in the bathing solution is indicated by the numbers inside the top open bar. Application of H₂O₂ is indicated by the top solid bar. [Ca²⁺]_B indicates the Ca²⁺ concentration in the bathing solution. C, summary data from five sets of TRPC6-transfected cells and two sets of empty vector-transfected cells, showing a dose-dependent response of Ca²⁺ entry on H₂O₂ treatments in TRPC6-expressing but not in vector-transfected cells. Δ340:380 ratio indicates the difference in the values before addition and the peak values after addition of 5 mm Ca²⁺ solution, representing the Ca²⁺ entry response. The numbers of cells in each group are from 16 to 25. *, p < 0.05, compared with 10⁻⁸ m in TRPC6-transfected cells; †, p < 0.05, comparison between TRPC6-transfected and Mock-transfected cells at corresponding doses. D, percent increases in the ratios of 340:380 in response to 10 μm H₂O₂ in Mock- and TRPC6-transfected cells. n indicates the number of cells analyzed. *, p < 0.05, TRPC6 versus Mock.

FIGURE 3.

Effect of H₂O₂ on whole-cell currents (A and B, whole-cell patch) and single channel currents (C–F, cell-attached patch) in TRPC6-expressing HEK293T cells. A, representative whole-cell current measurements obtained by a Gap-free protocol in a Mock (open circles)- and a TRPC6-transfected (solid circles) cell at a holding potential of −60 mV. The dashed lines indicate zero currents. Application and removal of H₂O₂ are indicated by the vertical lines. B, summary data, showing the H₂O₂ effect on the whole-cell currents in the Mock control and TRPC6-expressing cells. The responses were measured by the difference between the membrane current before application of H₂O₂ and the peak current after application of H₂O₂. The whole-cell currents are expressed as current density (pA/pF), normalized to the cell membrane capacitance. n indicates the number of cells analyzed. Asterisk indicates statistically significant difference compared with Mock (Student's t test). C, representative traces of single channel currents in a cell-attached patch. Left panel, a real time continuous recording. The arrowhead indicates the closed state of the channels. Downward deflections indicate inward currents. Right panel, the time-expanded portions of the selected regions in the upper trace, indicated by a (Pre-H₂O₂) and b (Post-H₂O₂). D, amplitude histograms (upper panels) and open time distributions (bottom panels) of the currents before (left panels) and after (right panels) application of H₂O₂ in the traces shown in C. The amplitude histograms were fitted to Gaussian function. M in the bottom panels indicates mean open time. E, single channel amplitudes before and after application of 10 μm H₂O₂. n indicates the number of cells analyzed. F, effect of H₂O₂ on the NP_o of TRPC6 channels in 12 cells. *, p < 0.05, compared with pre-H₂O₂ (paired Student's t test). The cells in each group of B, E, and F were from at least four sets of transfection.

FIGURE 4.

A–D, Ca²⁺ imaging experiments, showing involvement of endogenous H₂O₂ in AVP-stimulated Ca²⁺ entry. A, representative traces, showing the AVP-evoked Ca²⁺ response in HEK293T cells expressing TRPC6 and an empty vector (T6+vector) or in cells co-transfected with TRPC6 and AVP V1R in the presence (T6+V1R+Catalase) or absence (T6+V1R) of PEG-catalase (250 units/ml for 30 min). Ca²⁺ concentration in the bathing solution is indicated by the numbers inside the top open bar. Application of 100 nm AVP is indicated by the top solid bar. [Ca²⁺]_B indicates the Ca²⁺ concentration in the bathing solution. B, summary data, showing Ca²⁺ entry responses in different groups. *, p < 0.05, compared with T6 + vector; †, p < 0.05, compared with T6 + vector and T6 + V1R. C, effect of catalase on AVP-induced Ca²⁺ release in cells co-transfected with TRPC6 and AVP V1R. D, effect of catalase on normalized Ca²⁺ entry (ratio of Ca²⁺ entry to Ca²⁺ release) in response to AVP stimulation. *, p < 0.05, compared with T6 + V1R. B–D, n indicates the number of cells analyzed from 4 to 5 sets of transfection. E–G, in A7r5 cells. E, whole-cell patch clamp, showing inward currents in response to 100 μm H₂O₂ in A7r5 with (siRNA-T6) and without (Scramble) knockdown of TRPC6 at a holding potential of −60 mV. The dashed lines indicate zero currents. Application and removal of H₂O₂ were indicated by the vertical lines. F, Western blot, showing TRPC6 expression levels in A7r5 cells transfected with TRPC6 siRNA sequence (siRNA-T6) and scrambled sequence (Scramble). Actin served as a loading control. UT, untransfected. G, summary data, showing the H₂O₂ effect on whole-cell currents in A7r5 cells with (SiRNA-T6) and without (Scramble) knockdown of TRPC6. The responses were measured by the difference between the membrane current before application of H₂O₂ and the peak current after application of H₂O₂. The whole-cell currents were expressed as current density (pA/pF), normalized to the cell membrane capacitance. n indicates the number of cells analyzed from three sets of transfection. Asterisk indicates statistically significant difference compared with Scramble (Student's t test).

FIGURE 5.

Effect of thiol group oxidation and reduction on TRPC6-mediated Ca²⁺ response. A, Ca²⁺ entry response in TRPC6-expressing cells with treatments of NEM (300 μm) and thimerosal (Thim, 100 μm), and their vehicles (DMSO for NEM and H₂O for thimerosal). *, p < 0.05, compared with corresponding vehicle. n indicates the number of cells analyzed. B, percent increases in the intracellular Ca²⁺ levels upon Ca²⁺ readdition in TRPC6-expressing cells with the treatments of H₂O₂ (10 μm), H₂O₂ plus DTT (1 mm), or H₂O₂ plus GSH-MEE (1 mm). DTT was applied ∼2 min prior to H₂O₂ application. *, p < 0.05, compared with H₂O₂ group. n indicates the number of cells analyzed. Each group in A and B represents the cells from 5 to 6 sets of transfection.

FIGURE 6.

Patch clamp experiments, analyzing the sites of thiol groups involved in TRPC6 channel activation. All experiments were carried out in TRPC6-expressing HEK293T cells. A, representative traces of single channel recordings in a cell-attached patch. Upper panel, a real time continuous recording. Arrowhead, the closed state of channels. Bottom panel, the time-expanded portions of the selected regions in the upper trace. B, summary data, showing the effect of NEM (300 μm) on NP_o of TRPC6 channels in the cell-attached and inside-out mode of patch clamp recordings. *, p < 0.05, compared with pre-NEM (paired Student's t test). n indicates the number of analyzed cells with four to five sets of transfection. C, representative traces in an inside-out patch recording, showing the channel activity before and after application of NEM and NEM+OAG. Arrowhead, the closed state of channels. The bottom trace is a time-expanded portion of the region in the upper trace, as indicated. D, summary data, showing the effect of NEM, H₂O₂, and OAG on TRPC6 channel activity in the inside-out patch clamp mode. Asterisk indicates p < 0.05, compared with other groups (one-way repeated measures analysis of variance). n indicates the number of analyzed cells with four sets of transfection.

FIGURE 7.

Cell-attached patch clamp, showing the OAG effect on channel activity in TRPC6 expressing HEK293T cells with and without pretreatment with H₂O₂ (10 μm for 3 min). A and B, cells were stimulated with 0.1 μm OAG. C and D, cells were stimulated with 100 μm OAG. A and C indicate the channel activity (NP_o) before and after OAG treatment in the presence and absence of H₂O₂. B and D show the difference in OAG-induced increase in channel activity (ΔNP_o) with and without existence of H₂O₂. n indicates the number of cells analyzed, and each group represents at least four sets of transfection. *, p < 0.05, versus pre-OAG in C, and versus “Without H₂O₂” in D.

FIGURE 8.

Analysis of TRPC6 trafficking to the cell surface in response to H₂O₂. A, biotinylation of TRPC6 protein in HEK293T cells transfected with TRPC6 (T6) or empty vector (Mock) with and without H₂O₂ stimulation. L, a protein ladder; T6+UT, TRPC6-expressing cells without H₂O₂ treatment; T6+H₂O₂, TRPC6-expressing cells treated with 10 μm H₂O₂ for 3 min; Mock+H₂O₂, empty vector-transfected cells treated with 10 μm H₂O₂ for 3 min; Inputs, the whole-cell lysates (unbiotinylated), ∼1:10 of the lysates used for biotinylation. B, quantitative analysis of the cell surface TRPC6 protein with and without H₂O₂ stimulation. Data were expressed as percentages of integrated optical density of TRPC6 immunoblots in biotinylated fractions to the corresponding inputs, calculated by (integrated optical density of biotinylated TRPC6 blot/(integrated optical density of TRPC6 blot in input × 10)) × 100. n indicates the number of independent experiments. The optical density of immunoblots was measured with a software provided by AlphaEaseFC Imaging System (Alpha Innotech). Asterisk indicates significant difference compared with T6+UT. C, representative confocal microscopy photographs, showing TRPC6 trafficking to the region of the plasma membrane in response to 10 μm H₂O₂ stimulation in the cells transfected with TRPC6-EGFP (T6-EGFP), but not in the cells transfected with EGFP alone. Images were consecutively captured before application, immediately, 2, and 4 min after application of H₂O₂ (labeled with 0, 1′, 3′, and 5′, respectively). The duration of scanning one image was ∼1 min. The rainbow bars on the right indicates the fluorescence intensity in arbitrary units. Arrows indicate the sites of an increase in fluorescence intensity compared with before application of H₂O₂ (time 0). The horizontal bars in the left panels represent 20 μm. D, time course changes in fluorescence intensity on the cell surface in response to H₂O₂ in TRPC6-EGFP (T6-EGFP) or EGFP alone (EGFP) transfected cells. Data were expressed as percent changes from the values before application of H₂O₂ (time 0). The fluorescence intensity was measured using the software SymPhoTime (version 5.0). Asterisk indicates significant difference between T6-EGFP and EGFP at the corresponding time point. n indicates the number of analyzed cells from four sets of transfection. E, percent changes in fluorescence intensity on the cell surface 3 min after application of AVP (100 nm) in HEK293 cells cotransfected with EGFP or TRPC6-EGFP (T6-EGFP) and V1R with and without pretreatment of PEG-catalase (250 units/ml for 30 min). See D for the approach for data analysis. Asterisk indicates significant difference between the indicated groups. n indicates the number of analyzed cells from four sets of transfection.