Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels

Abstract

Background and purpose: Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation.

Experimental approach: Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances.

Key results: Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels.

Conclusions and implications: Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.

Keywords: ClC-3; PAORAC; cation membrane channel; dihydropyridine; enantiomer; neurosteroid; pregnenolone sulphate; proton-activated outwardly rectifying anion channel; transient receptor potential; transient receptor potential melastatin.

Figure 1

PS and nifedipine (Nif) activate TRPM3 channels supra-additively. (A) Ca²⁺-imaging experiment with TRPM3-expressing cells stimulated with 50 μM PS, and 50 μM Nif together with 50 μM PS (n = 20). The addition of Nif increased the intracellular Ca²⁺-concentration. (B) Representative whole-cell patch-clamp experiment during which PS and Nif (at indicated concentrations) were applied to a TRPM3-expressing cell. The current–voltage relationships observed during this recording were highly similar to the outwardly rectifying curves typical for TRPM3 currents published previously (Wagner et al., 2008) and are depicted in Supporting Information Figure S2A. (C) Statistical analysis of currents elicited by application of PS (at concentrations indicated; blue bars), Nif (20 μM; red bars) and a combination of both substances (turquoise bars). Please note the different scale of the Y axes. The bars with two colours indicate the numerical sum of the currents obtained during application of PS and Nif alone. Inward currents (at −80 mV) at different concentrations of PS were evaluated separately. For quantitative analysis, all currents were normalized to the responses to 35 μM PS applied alone at the beginning and the end of each recording. Statistical tests were performed between the sum of the currents obtained during the separate application of a single substance (two-coloured bars) and the currents measured during co-application of both substances ( n = 7–11).

Figure 2

Effects of a variety of 1,4-dihydropyridines on TRPM3 channels. (A) During a Ca²⁺-imaging experiment TRPM3-expressing cells were stimulated with 50 μM photo-inactivated nifedipine (Nif p.i.) and 50 μM not inactivated Nif as indicated (n = 56). (B) Similar experiment, using PS and nimodipine (both at 50 μM, n = 20). (C) Whole-cell patch-clamp measurement of a TRPM3-expressing cell obtained during similar experimental conditions as in (B), but using 21 μM PS and 21 μM nimodipine. The current–voltage relationships of this recording are given in Supporting Information Figure S2B. (D) Statistical analysis (n = 7) of currents measured in experiments performed as shown in (C). (E,F) Similar Ca²⁺-imaging experiments as in (B), but using nicardipine (Nic) in (E) and nitrendipine (Nit) in (F) at 50 μM (n = 20 for each of the two panels).

Figure 3

The two enantiomers of PS affect TRPM3 channels differentially. (A) TRPM3-expressing cells were superfused with ent-PS and nat-PS (both at 50 μM) in a Ca²⁺-imaging experiment (n = 19). (B) Representative whole-cell patch-clamp recording from a TRPM3-expressing cell stimulated with ent-PS and nat-PS at the indicated concentrations. Upper panels show the current amplitude at +80 and −80 mV, lower panel depicts the apparent electrical capacitance. (C) Current–voltage relationships from the cell shown in (B). (D) Statistical analysis of cells (n = 12–38 per data point) recorded in similar experiments to those shown in (B). Inward and outward currents were normalized separately to the current amplitude measured with 10 μM nat-PS (arrow). (E) Dose-response curve for capacitance increase found for ent-PS and nat-PS during experiments conducted similarly to those shown in (B).

Figure 4

PAORAC are inhibited by PS and dehydroepiandrosterone (DHEA) sulphate. (A) Current traces of HEK293 cells at membrane potentials of −80 and +80 mV during application of acidic solution (pH 4) and PS. Arrowheads designate quickly inactivating currents presumably caused by the activation of acid-sensing ion channels known to be expressed in HEK293 cells (Gunthorpe et al., 2001). These currents were not further investigated. Current–voltage relationships obtained in this recording were typical for PAORAC currents and are displayed in Supporting Information Figure S2C. (B) Statistical analysis of the inhibition of the pH 4-evoked current induced by the indicated substances at a concentration of 50 μM (n = 5–6, for each substance). Outward currents (at +80 mV) were analysed from experiments performed as shown in (A). (C) Normalized dose-response curves established from experiments comparable to those shown in (A) at a membrane potential of +80 mV. The continuous lines were obtained by fits to the Hill function, which yielded an IC₅₀ = 5.1 ± 1.1 μM and a Hill coefficient = 1.8 ± 0.4 for PS and an IC₅₀ = 25.7 ± 1.1 μM and a Hill coefficient = 1.4 ± 0.1 for DHEA sulphate (n = 5–8, for each data point).

Figure 5

Both enantiomers of PS inhibit PAORAC with similar potency. (A) Current traces obtained from a HEK293 cell at membrane potentials of −80 and +80 mV. The lower panel shows a capacitance trace of this recording. The application of acidic solution (pH 4) and nat-PS or ent-PS (both at 50 μM) is indicated. (B) Statistical analysis (n = 7) of the inhibition of the pH 4-evoked current at +80 mV. (C) Statistical comparison (n = 7) of membrane capacitance changes during application of nat-PS or ent-PS. (D) Similar experiment as in (A) but employing a lower concentration (5 μM) of steroids. (E) Statistical analysis (n = 5) of the experiments performed as shown in (D). Arrowheads in (A) and (D) indicate Acid-sensing ion channel-like inward currents at the beginning of exposure to acidic solutions. Current–voltage relationships of the recordings shown in panel (A) and (D) are depicted in Supporting Information Figure S2D and E.

Figure 6

The effects of the configuration at the C3 and C5 positions on the activity of TRPM3 channels. (A) Changes in intracellular calcium measured in TRPM3-transfected cells are shown during the application of PS and epipregnanolone sulphate (3β5βPregnanS), both at 50 μM (n = 55). (B) Similar experiment with 50 μM epiallopregnanolone sulphate (3β5αPregnanS), which evokes a similar response to that of PS (n = 36). (C) Summary of stimulatory effects of the indicated substances on TRPM3 channels. Increases in the 340/380 ratio were evaluated, averaged (n = 20–55) and normalized to the response to PS (same concentration as test compound) of the same cell. Untransfected HEK293 cells did not respond to these substances (not shown). (D) Electrophysiological recording of a TRPM3-expressing cell (at +80 and −80 mV) stimulated with PS or epiallopregnanolone sulphate (3β5αPregnanS) at the indicated concentration. The current–voltage relationships of this recording are shown in Supporting Information Figure S2F. (E) Dose-response curves obtained from experiments (n = 8–11) similar to those shown in (D). Amplitudes of outward currents (+80 mV, left panel) and of inward currents (−80 mV, right panel) were independently normalized to the response to 10 μM PS (arrows).

Figure 7

A negative charge at the C3 position of steroids is necessary to activate TRPM3 channels. (A) Summary of Ca²⁺-imaging experiments on TRPM3-expressing cells with PS-analogues in which the sulphate group had been substituted either with acetate (PAc), methyl ether (POMe), glucuronic acid (PGlucur) or hemisuccinate (PHemisuc). Increases in fluorescence ratio values were normalized to the response to PS at the same concentration as the test substance (n = 20–43). Pregnenolone hemisuccinate also induced a small signal in untransfected HEK293 cells indicating a minor TRPM3-independent effect (data not shown). (B) Electrophysiological recording of a TRPM3-expressing cell stimulated with 3β,5β-pregnanolone-acetate (3β5βPregnanAc) or PS at the indicated concentration. Current–voltage relationships from this recording are plotted in Supporting Information Figure S2G. (C) Summary of electrophysiological experiments (n = 6) showing that neither 3β,5α-pregnanolone acetate (5αPregnanAc) nor 3β,5β-pregnanolone acetate was capable of stimulating TRPM3 channels, even at high concentrations (100 μM).