Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinases

Abstract

Background and purpose: TRPC5 is a Ca(2+)-permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved.

Experimental approach: Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca(2+) imaging and electrophysiological techniques.

Key results: In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca(2+) influx, concentration dependently (EC(50)= 93 microM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 microM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 microM), PP2 (20 microM) and lavendustin A (10 microM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 microM) or a phospholipase C inhibitor U73122 (10 microM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 microM) stimulated TRPC5-mediated Ca(2+) influx. In patch clamp studies, both genistein (50 microM) and daidzein (50 microM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors.

Conclusions and implications: The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells.

Figure 1

Effect of genistein on TRPC5 channel activity in HEK cells. (A) Representative immunoblot probed with anti-TRPC5 and anti-β-tubulin (n= 3). Left panel, pcDNA3-transfected HEK cells (vector); middle panel, TRPC5-transfected HEK cells; right panel, BAECs. (B and C) Representative time-course (n= 5–6) of whole-cell currents in TRPC5-over-expressing HEK cells. Currents were sampled every 5 s at +60 and −60 mV. Genistein (50 µM), LaCl₃ (100 µM) and 2-APB (75 µM) were applied to the bath at the time-points indicated by arrows. (D) Summary of maximal whole-cell currents before and after genistein addition at +60 and −60 mV in TRPC5-over-expressing cells. +LaCl₃, with 100 µM LaCl₃; −LaCl₃, without LaCl₃. Mean ± SEM (n= 5–6). (E) Representative traces showing voltage protocol (top) and corresponding whole-cell currents immediately before (middle) and 5 min after (bottom) bath application of 50 µM genistein in TRPC5-over-expressing HEK cells. (F) Current–voltage relationships as in (E). (G and H) Current–voltage relationships in TRPC5-over-expressing (G) or wild-type HEK cells (H) immediately before and 5 min after bath application of 50 µM genistein (H) or 0.1% DMSO (G). Mean ± SEM (n= 3–8). *P < 0.05; **P < 0.01 compared to ‘before’.

Figure 2

Single-channel recording of genistein-modulated channels in excised inside-out patches from TRPC5-over-expressing HEK cells. (A and B) Single-channel traces of membrane patches excised from TRPC5-over-expressing HEK cells; 0.1% DMSO (A) or 50 µM genistein (B) was applied as indicated by the horizontal bar. The patch potential was held at −60 mV. C, close state; O, open state. (C) Channel open probability (Po) values of the patch (as in A and B) in 3 s intervals. (D) Event histogram of a representative experiment before and after 50 µM genistein application at −60 mV. (E) Single-channel current–voltage relationship of the genistein-modulated channel. Mean ± SEM (n= 3–5).

Figure 3

Effect of genistein on TRPC5-mediated [Ca²⁺]_i rise. (A–D) Representative time-course of [Ca²⁺]_i change in vector-transfected (B, D) and TRPC5-transfected HEK cells (A, C). (C and D) Cells were pretreated with genistein (50 µM) or vehicle (0.1% DMSO) for 10 min in 0Ca²⁺–PSS. In (A–D), 100 µM La³⁺ was included in the bath to block other non-selective cation channels and to select for TRPC5-mediated Ca²⁺ influx. Each trace represents mean ± SEM of at least 20 cells in one representative experiment (n= 4–7 experiments).

Figure 4

Concentration-dependent stimulation of TRPC5 channels by genistein and synergy between genistein and La³⁺. (A and B) Representative time-course of [Ca²⁺]_i change in TRPC5-transfected HEK cells in response to different concentrations of genistein. Cells were bathed in NPSS (A) or NPSS + 100 µM LaCl₃ (B). (C) Plot of concentration–response with curve fitting to determine the genistein concentration for half-maximal response (EC₅₀). [Ca²⁺]_i response at each concentration was subtracted by the background given by vehicle control; means ± SEM (n= 4–12). (D) Representative time-course of La³⁺ (100 µM)-induced [Ca²⁺]_i change in TRPC5-expressing HEK cells in the absence or presence of 50 µM genistein. (E) Summary of data showing the maximal [Ca²⁺]_i rise in response to La³⁺. For A, B and D, each trace represents means ± SEM of at least 20 cells from one representative experiment (n= 4–10 experiments). Vehicle contained 0.1% DMSO. **P < 0.01, compared to vehicle.

Figure 5

Effect of ICI-182780, U73122, a panel of tyrosine kinase inhibitors and an inactive genistein analogue, daidzein, on TRPC5-mediated [Ca²⁺]_i rise. (A and B) Representative time-course of [Ca²⁺]_i change in response to 100 µM genistein in TRPC5-over-expressing HEK cells in the absence (A, left) or the presence (A, right) of 50 µM ICI-182780, or in the presence of 10 µM U73343 (B, left) or 10 µM U73122 (B, right). Cells were pretreated with ICI-182780 for 5 min, or with U73122 or U73343 for 30 min. (C and D) Representative time-course of [Ca²⁺]_i change in response to 100 µM daidzein (C), 20 µM PP2 (D, left), 2 µM herbimycin (D, middle) or 10 µM lavendustin A (D, right) in TRPC5-over-expressing HEK cells. Each trace in A–D represents mean ± SEM of at least 20 cells from one representative experiment (n= 3–8). Cells were bathed in NPSS in the presence of 100 µM LaCl₃. (E) Summary of data showing the maximal [Ca²⁺]_i rise as in A–D. Mean ± SEM (n= 3–8). **P < 0.01, compared to vehicle.

Figure 6

Effect of daidzein on whole-cell currents in TRPC5-over-expressing HEK cells. (A) Representative time-course of whole-cell currents. Currents were sampled every 5 s at +60 and −60 mV. Daidzein (50 µM) was applied to the bath at the time-point indicated by arrow. (B) Summary of maximal whole-cell currents before (before) and after daidzein (+daidzein) addition at +60 and −60 mV. Mean ± SEM (n= 7). (C) Current–voltage relationships of whole-cell currents immediately before and 5 min after bath application of 50 µM daidzein. Voltage protocol is the same as in Figure 1E. Mean ± SEM (n= 7). *P < 0.05; **P < 0.01 compared to ‘before’.

Figure 7

Effect of genistein on TRPC5-mediated [Ca²⁺]_i rise in native vascular endothelial cells. (A) Immunocytochemical detection of TRPC5 proteins in BAECs using confocal microscopy. Shown were the paired images of TRPC5 fluorescent signal (red, left panel) and the BrightView image merged with fluorescent signal. The control images (right panel) were from samples without addition of primary antibodies. Scale bar represents 20 µm. (B and D) Representative time-course of [Ca²⁺]_i change in response to vehicle (0.1% DMSO) or genistein in BAECs. (D) Pre-incubated with pre-immune IgG (4 µg·mL⁻¹) or T5E3 (antibody to TRPC5 channels; 4 µg·mL⁻¹) for 10 min. Each trace represents mean ± SEM of at least 15 cells from one representative experiment (n= 6–12). (C) Summary of data showing the maximal [Ca²⁺]_i changes in response to 0.1% DMSO or various concentrations of genistein. Mean ± SEM (n= 6–12). (E) Summary of data showing [Ca²⁺]_i changes 10 min after genistein application in the presence of pre-immune IgG (4 µg·mL⁻¹) or T5E3 (4 µg·mL⁻¹) or Ca²⁺-free HBS (0[Ca²⁺]_o). Mean ± SEM (n= 3–8). **P < 0.01.