Fig 1. Flavaglines stimulate TRPM6 at nanomolar concentration.

a. TRPM6 currents were evoked by a series of 500 ms voltage ramp from -100 to +100 mV applied every 2 s (0.5 Hz) from a holding potential of 0 mV (top left inset). A typical set of current-voltage curves obtained from a single cell is shown. b. Typical current-voltage curves obtained 200 s after break-in from cells pre-incubated 15 minutes with vehicle or FL23 (50 nM). c. The average time-course of TRPM6 current development with (n = 19) or without (n = 19) FL23 pre-treatment are shown for current values measured at +80 mV. d. FL23 increases TRPM6 current density in a concentration-dependent manner (n≥3 per data points). Line represents the fit of data points with a Hill equation (see Material and Methods). e. Incubation of TRPM6 expressing cells with FL3 (50 nM, n≥10), FL23 (50 nM, n≥22) or 17βE (50 nM, n≥9) significantly increased the average current densities measured at +80 mV 200 s after break-in. Mock-transfected cells showed a similar increase in current density (n≥8). TRPM6 currents were not sensitive to FL2 (n≥10). Stars indicate statistically significant difference (P<0.05) between vehicle- (white bar) and compound-treated cells (black bar). f. The chemical structures of FL2, FL3 and FL23 are shown. g. Cells were pre-incubated with vehicle, FL23 (50 nM) or 17βE (50 nM). Total lysate were subjected to Western blot analysis using an anti-HA primary antibody. FL23 and estradiol (17βE) did not significantly affect the expression of TRPM6. A representative blot of three separate experiments is shown.