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. 2011 Apr;162(7):1509-20.
doi: 10.1111/j.1476-5381.2010.01136.x.

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids

Y Majeed  1 , M S Amer, A K Agarwal, L McKeown, K E Porter, D J O'Regan, J Naylor, C W G Fishwick, K Muraki, D J Beech
Affiliations

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids

Y Majeed et al. Br J Pharmacol. 2011 Apr.

Abstract

Background and purpose: Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.

Experimental approach: Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca2+ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells.

Key results: Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1-2min. Dehydroepiandrosterone sulphate or 17β-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid.

Conclusions and implications: Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.

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Figures

Figure 1
Figure 1
Inhibition of Gd3+-stimulated TRPC5 channels by pregnenolone sulphate (PregS). All data were generated from cells over-expressing TRPC5 (Tet+) except for the control (Tet-) data shown in panel A, and by intracellular Ca2+ measurement (A,C,D) or whole-cell patch-clamp (E–G). A. Effect of application of 20 µM Gd3+. (B) Chemical structure of pregnenolone sulphate, including labelling of rings and carbon atom positions. (C) Effect of a 15 min treatment with 10 or 50 µM pregnenolone sulphate on responses to 20 µM Gd3+ (N= 4 for each concentration). (D) Concentration-dependent inhibition by pregnenolone sulphate (n/N= 3/12) with a fitted Hill equation (IC50 19 µM). Responses to Gd3+ were measured 80 s after its application, as indicated in panel C. (E) Time-series plot showing the effect of extracellular application of 1 or 10 µM pregnenolone sulphate on the outward (+95 mV) and inward currents (−95 mV) stimulated by 20 µM Gd3+. (F) Typical current-voltage relationships (I-Vs) in the presence of Gd3+ alone (20 Gd3+), and then plus 100 µM pregnenolone sulphate. (G) Mean data for effects of pregnenolone sulphate (µM) on outward and inward currents elicited by 20 µM Gd3+ (n= 3–4 for each concentration). TRPC5, transient receptor potential canonical 5.
Figure 2
Figure 2
General inhibition of TRPC5 channels and selectivity. Data were generated by intracellular Ca2+ measurement from TRPC5-expressing (Tet+) cells (A–D), TRPM2-expressing (Tet+) cells (E,F), or control cells not induced to express TRPC5 channels (Tet-) (G,H). (A) Typical effect of 10 µM pregnenolone sulphate on TRPC5 channels stimulated by 5 µM sphingosine-1-phosphate (S1P). (B) Mean data for experiments shown in panel A (n/N= 3/18). (C) Typical effect of 10 µM pregnenolone sulphate on TRPC5 stimulated by 5 µM lysophosphatidylcholine (LPC). (D) Mean data for experiments in panel C (n/N= 3/18). (E) Typical effect of 10 µM pregnenolone sulphate on TRPM2 stimulated by 1 mM hydrogen peroxide (H2O2). (F) Mean data for experiments in panel E (n/N= 3/24). (G) Typical effect of 10 µM pregnenolone sulphate on Ca2+ release elicited by 100 µM ATP. (H) Mean data for experiments in (G) (n/N= 3/12). Ctrl indicates the solvent control. TRPC5, transient receptor potential canonical 5; TRPM, transient receptor potential melastatin.
Figure 3
Figure 3
Steroid structure-activity relationships at TRPC5 channels. All data were generated from cells over-expressing TRPC5 channels (Tet+) by intracellular Ca2+ measurement (B,F,J,M) or whole-cell patch-clamp. (A,E,I,L) 2-dimensional structures of pregnenolone (preg), pregnanolone sulphate (pregnas), pregnanolone (3α5β), allopregnanolone (3α5α) and dehydroepiandrosterone sulphate (DHEAS). (B) Mean data for the effect of 10 µM pregnenolone on TRPC5 channels stimulated by 20 µM Gd3+ (n/N= 3/12). (C) Time-series plot showing the effect of 10 µM pregnenolone on current elicited by 20 µM Gd3+. (D) Mean data for experiments in panel C (n= 3). (F) Mean data for the effect of 10 or 50 µM pregnanolone sulphate on TRPC5 channels stimulated by 20 µM Gd3+ (n/N= 3/12). (G) Time-series plot from a whole-cell recording showing the effect of 100 µM pregnanolone sulphate on the current elicited by 20 µM Gd3+. (H) Mean data for experiments in panel G (n= 3). (J) Mean data for the effect of 10 µM pregnanolone or allopregnanolone on TRPC5 channels stimulated by 20 µM Gd3+ (n/N= 3/12). (K) Mean data for the effect of 10 µM pregnanolone on current elicited by 20 µM Gd3+ (n= 3). (M) Mean data for the effect of 50 µM dehydroepiandrosterone sulphate on TRPC5 channels stimulated by 20 µM Gd3+ (n/N= 3/24). Ctrl indicates the solvent control. TRPC5, transient receptor potential canonical 5; TRPM, transient receptor potential melastatin.
Figure 4
Figure 4
Inhibition of Gd3+-stimulated TRPC5 channels by progesterone. All data were generated from cells over-expressing TRPC5 channels (Tet+) by intracellular Ca2+ measurement (B,C) or whole-cell patch-clamp. (A) Two-dimensional structure of progesterone. (B) Mean data for the inhibitory effect of progesterone on TRPC5 channel-mediated Ca2+ signals elicited by 20 µM Gd3+, fitted using the Hill equation giving an IC50 of 5 µM (n/N= 6/30 for 1 µM, and 3/12 each for 10 and 50 µM progesterone). (C) Typical time-series plot showing the effects of application of 1 and 10 µM progesterone on current elicited by 20 µM Gd3+. (D) Typical I-Vs in the presence of Gd3+ alone (20 Gd3+), and then plus 1 and 10 µM progesterone. (E) Mean data for experiments in (C,D) (n= 13 and 7 for 1 and 10 µM progesterone, respectively). TRPC5, transient receptor potential canonical 5.
Figure 5
Figure 5
General TRPC5 channel inhibition by progesterone and potency against S1P-evoked activity. Data were generated by intracellular Ca2+ measurement from TRPC5-expressing (Tet+) cells or cells not induced to express TRPC5 channels (Tet-) (H,J). (A) Experimental record showing the effect of 10 µM progesterone on TRPC5 channels stimulated by 5 µM sphingosine-1-phosphate (S1P). (B) Mean data for experiments in panel A (n/N= 3/18). (C) Typical trace showing the effect of 10 µM progesterone on TRPC5 channels stimulated by 5 µM lysophosphatidylcholine (LPC). (D) Mean data for experiments in panel C (n/N= 3/18). (E) Typical trace showing the effects of 0.01 and 0.1 µM progesterone (prog) on TRPC5 channels stimulated by 5 µM S1P. (F) Mean data for experiments in panel E (n/N= 4/18). (G,H) Typical traces showing the effect of 0.1 µM progesterone (prog) on Ca2+ signals elicited by 100 µM ATP in cells. Note the more sustained response in Tet+ cells, reflecting TRPC5 channel activity. (I,J) Mean data for the effect of 0.1 µM progesterone (+prog) on the transient (Trans.) and sustained (Sust.) responses evoked by ATP in TRPC5 channel-expressing or control (Tet-) cells (n/N= 3/16). Ctrl indicates the solvent control. S1P, sphingosine-1-phosphate. TRPC5, transient receptor potential canonical 5.
Figure 6
Figure 6
Inhibition of TRPC5 channels by dihydrotestosterone. (A) Chemical structures of dihydrotestosterone (DhT), 17β-oestradiol (17β) and cortisol (cort). (B–D) Data were generated by intracellular Ca2+ measurement (B) or whole-cell patch-clamp (C,D) from TRPC5-expressing (Tet+) cells B. Mean data for the effect of 15 min treatment with 10 µM each of dihydrotestosterone, 17β-oestradiol or cortisol on TRPC5 channels stimulated by 20 µM Gd3+ (n/N= 3/24). (C) Typical time-series plot showing the effect of extra-cellular application of 10 µM cortisol, dihydrotestosterone or 17β-oestradiol on currents at −95 mV and +95 mV elicited by 20 µM Gd3+. The durations of compound application are indicated by horizontal bars. (D) Mean normalized data for experiments in panel C (n= 4–5 for each steroid, each compared with its own control). TRPC5, transient receptor potential canonical 5.
Figure 7
Figure 7
Inhibition of TRPC5 channels in excised outside-out patches. Data were generated from patches excised from cells over-expressing TRPC5 channels (Tet+). (A) Typical time-series plot from an outside-out patch recording showing the effects of extra-cellular application of 10 µM dihydrotestosterone (DhT) or progesterone (prog) on current elicited by 20 µM Gd3+. Arrows indicate the time points (i–iv) at which I-Vs were sampled for panels B and C. (B,C) Typical I-Vs in the presence of Gd3+ alone (20 Gd3+), and then plus 10 µM dihydrotestosterone (B) and progesterone (C). (D) Mean data for experiments in (A–C) (n= 4–6). TRPC5, transient receptor potential canonical 5.
Figure 8
Figure 8
Inhibition by progesterone of Ca2+-entry through endogenous TRPC5-containing channels. Data were generated by intracellular Ca2+ measurement from human saphenous vein vascular smooth muscle cells (VSMC). (A) Effect of 15 min treatment with 50 µM progesterone (prog) on the Ca2+ response elicited by 3 µM 1-palmitoyl-2-glutaroyl-phosphatidylcholine (PGPC). (B,C) Mean data for the effects of progesterone on the amplitude of the PGPC response (B, normalized to control) and basal Ca2+ level (C, absolute fura-2 ratio) in VSMC (n/N= 5/54). TRPC5, transient receptor potential canonical 5.
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