Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone

Abstract

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.

Fig. 1

Inhibition of TRPM3-mediated Ca²⁺ influx by progesterone. Data were generated by intracellular Ca²⁺ measurement. (a) Structures of pregnenolone sulphate (PregS) and progesterone (prog). (b) Example responses to 1 μM PregS in cells induced to express TRPM3 (Tet+) in the presence of vehicle (open circles) or 10 μM prog (filled black circles). Control (Tet−) cell data (filled gray circles) are shown for comparison (no response to PregS) (N = 8 for each). (c) Cells were treated with 10 μM progesterone and its effect on PregS-activated TRPM3 was determined after 2 min (labelled ‘2’) and at 5 min intervals thereafter. For every time-point, the response in the presence of progesterone is indicated as a percentage of the control response (N/n = 32/4). (d) Mean normalised data for the effect of different concentrations of prog (in μM) at room temperature (Room temp., N/n = 24/3) or at 37 °C (N/n = 22/3). (e) Concentration–response curves for PregS in the absence (vehicle) or presence of 10 μM prog (+10 prog). The fitted Hill equation gave EC₅₀ and slope values of 1.56 μM and 1.74 (vehicle) and 16.1 μM and 1.06 (prog), respectively (N/n = 6/3 for each concentration of PregS). (f) Mean TRPM2 activity stimulated by 1 mM hydrogen peroxide, comparing vehicle (control) with 10 μM prog (N/n = 24/3 each). (g) Mean 1 μM thapsigargin (TG) responses in non-induced (Tet−) cells in Ca²⁺-free SBS, comparing vehicle (control) with 10 μM prog (N/n = 24/3 each).

Fig. 2

Effects of progesterone metabolites, oestradiol and corticosteroids. Data were generated by intracellular Ca²⁺ measurement in cells over-expressing TRPM3 (Tet+). (a) Mean normalised data for the effect of 10 μM each of pregnanolone (5β), allopregnanolone (5α), 17-hydroxy progesterone (17-OH), 21-hydroxy progesterone (21-OH) or 17β-oestradiol (oest) on responses to 1 μM PregS (N/n = 24/3 each). (b) As for (a), but tests of 50 μM aldosterone or cortisol on responses to 5 μM PregS (N/n = 24/3 each).

Fig. 3

Inhibition of TRPM3-mediated ionic current by progesterone. Data were generated by whole-cell patch-clamp. (a) Example time-series showing the effect of 10 μM prog on current evoked by 5 μM PregS in TRPM3-expressing (Tet+) cells (DMSO vehicle was constant throughout). (b) For the experiment of (a), PregS-evoked I–V in vehicle (5 PregS) and then plus 10 μM prog (+10 prog). (c) As for (a), but mean currents normalised to pre-prog amplitudes (n = 8).

Fig. 4

Inhibition of TRPM3 by dihydrotestosterone. Data are from intracellular Ca²⁺ measurements in cells over-expressing TRPM3 (Tet+) (b, d, e) or control (Tet−) cells (c). (a) Structure of dihydrotestosterone (DhT). (b) Example responses to 1 μM PregS in the presence of vehicle or 50 μM DhT (N = 8 for each). (c) Mean 1 μM thapsigargin (TG) responses in non-induced (Tet−) cells in Ca²⁺-free SBS, comparing vehicle (control) with 50 μM DhT (N/n = 24/3 each). (d) Mean normalised data for the effect of DhT at two concentrations on responses to 1 μM PregS (N/n = 24/3 each). (e) Mean data for the effect of 50 μM DhT on TRPM3-dependent responses elicited by 1 μM or 100 μM PregS (N/n = 6/3).

Fig. 5

TRPM3 activity evoked by nifedipine. Data are from intracellular Ca²⁺ measurements. (a) Example demonstration of the effect of 20 μM nifedipine (nif) in cells expressing TRPM3 (Tet+) but not control (Tet−) cells. (b) In Tet+ cells, example responses to 20 μM nif in the presence of vehicle or 10 μM progesterone (prog) (N = 8 for each). (c), (d) Comparisons of the inhibitory effects of 10 μM prog (c) and 50 μM DhT (d) on TRPM3 stimulated by 1 μM PregS or 20 μM nif (N/n = 24/3 for each). PregS data are from the same experiments as those underlying Figs. 1d and 4d.

Fig. 6

Steroid effects on agonist-independent TRPM3 activity. Data are from intracellular Ca²⁺ measurements. (a), (b) Example traces showing the effects of 10 μM progesterone (a) or 50 μM dihydrotestosterone (b) on the Ca²⁺ signals elicited by addition of 5 mM Ca²⁺ in cells over-expressing TRPM3 (Tet+). Cells were incubated in Ca²⁺-free SBS for 30 min and then Ca²⁺ was ‘added-back’. Responses observed in control (Tet−) cells are shown for comparison (N = 8 for each condition). (c), (d) Mean responses to 5 mM Ca²⁺ add-back in the presence of vehicle, 10 μM progesterone (+10 prog) (c) or 50 μM dihydrotestosterone (+50 DhT) (d) (N/n = 24/3 for each).

Fig. 7

Insensitivity of the progesterone effect to mifepristone. Data were generated by Ca²⁺ measurement in cells over-expressing TRPM3 (Tet+). (a) Cells were treated with 2 μM mifepristone (mfp) or vehicle for 30 min prior to experiments and mfp was maintained during the recordings. Pre-treatment with 10 μM progesterone (prog) was as described in Fig. 1b, and PregS was applied at 1 μM. (b) Mean data for experiments exemplified in (a), showing the effect of prog alone (+10 prog), mfp alone (+2 mfp), or prog with mfp (+10 prog + 2 mfp) on the PregS-induced response (N/n = 40/5 for each condition).

Fig. 8

Chemical interaction with TRPM3. Typical result for lysate from HEK 293 cells expressing GFP alone (control) or TRPM3-YFP (TRPM3) incubated with membranes that had been spotted with the indicated chemicals at 30 or 60 nmol (left panel). Detection occurred using anti-GFP antibody. The results for dihydrotestosterone (DhT) and cortisol are duplicated with enhanced contrast in the right panel. The data are representative of three independent experiments.

Fig. 9

Competition with a non-steroidal look-alike chemical. (a) 3-Dimensional overlay of PregS with the look-alike chemical, 2-CMNPBC. (b), (d) In Tet+ cells, example responses to 1 μM PregS (b) or 20 μM nifedipine (nif) (d) in the presence of vehicle or 10 μM 2-CMNPBC (N = 4 for each). (c), (e) Mean data for experiments exemplified in (b) and (d) (N/n = 12/3).

Fig. 10

Inhibition by progesterone of Ca²⁺-entry evoked by PregS in vascular smooth muscle cells (VSMCs). Data were generated by intracellular Ca²⁺ measurement from human saphenous vein VSMCs. Shown are mean data for the effect of 15-min treatment with 1, 10, 30 or 100 μM prog on the Ca²⁺ response elicited by 25 μM PregS (N/n = 9/3 for each concentration).

Fig. 11

Progesterone resistant ionic currents in granulosa cells. Data were generated by whole-cell patch-clamp performed in freshly isolated bovine granulosa cells. (a) Example time-series plot showing the effect of 10 μM prog on current evoked by 100 μM PregS. 100 μM lanthanum (La³⁺) was applied at the end as a quality control for the whole-cell recording. (b) For the experiment of (a), I–Vs in the presence of 100 μM PregS, with 10 μM prog (+10 prog), or 100 μM La³⁺. (c) As for (a), but mean currents normalised to pre-prog amplitudes (n = 4). (d) Example time-series plot showing the effect of 10 μM prog on current evoked by 10 μM PregS. (e) For the experiment of (d), I–Vs in the presence of 10 μM PregS, or with 10 μM prog (+10 prog). (f) As for (d), but mean currents normalised to pre-prog amplitudes (n = 5).