Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels

Abstract

Background and purpose: Transient receptor potential melastatin 7 (TRPM7) is a unique channel kinase which is crucial for various physiological functions. However, the mechanism by which TRPM7 is gated and modulated is not fully understood. To better understand how modulation of TRPM7 may impact biological processes, we investigated if TRPM7 can be regulated by the phospholipids sphingosine (SPH) and sphingosine-1-phosphate (S1P), two potent bioactive sphingolipids that mediate a variety of physiological functions. Moreover, we also tested the effects of the structural analogues of SPH, N,N-dimethyl-D-erythro-sphingosine (DMS), ceramides and FTY720 on TRPM7.

Experimental approach: HEK293 cells stably expressing TRPM7 were used for whole-cell, single-channel and macropatch current recordings. Cardiac fibroblasts were used for native TRPM7 current recording.

Key results: SPH potently inhibited TRPM7 in a concentration-dependent manner, whereas S1P and other ceramides did not produce noticeable effects. DMS also markedly inhibited TRPM7. Moreover, FTY720, an immunosuppressant and the first oral drug for treatment of multiple sclerosis, inhibited TRPM7 with a similar potency to that of SPH. In contrast, FTY720-P has no effect on TRPM7. It appears that SPH and FTY720 inhibit TRPM7 by reducing channel open probability. Furthermore, endogenous TRPM7 in cardiac fibroblasts was markedly inhibited by SPH, DMS and FTY720.

Conclusions and implications: This is the first study demonstrating that SPH and FTY720 are potent inhibitors of TRPM7. Our results not only provide a new modulation mechanism of TRPM7, but also suggest that TRPM7 may serve as a direct target of SPH and FTY720, thereby mediating S1P-independent physiological/pathological functions of SPH and FTY720.

Figure 1

Effects of SPH on TRPM7 whole-cell currents recorded in HEK-293 cells over-expressing TRPM7. Cells were perfused with Tyrode solution before and after exposure to SPH. Currents were elicited by a ramp protocol ranging from −100 to +100 mV. (A) Representative recordings of TRPM7 in the absence and presence of SPH at 0.3, 0.5 and 0.8 μM. Note the large outward current and small inward current, a feature of outward-rectifying TRPM7. (B) Illustration of changes of inward currents with enlarged y-axis for the recordings shown in (A). Note the inhibition of TRPM7 inward currents by 0.3, 0.5 and 0.8 μM SPH. (C) Time-dependent changes of TRPM7 outward current measured at +100 mV and inward current measured at −100 mV before and after 0.5 μM SPH. (D) Time-dependent changes of TRPM7 outward and inward currents before and after 1 μM SPH. Note the slow recovery of TRPM7 after being completely blocked by 1 μM SPH. (E) Analysis of voltage-dependent effects of SPH on TRPM7. The inhibitory effects at all the tested voltages were calculated by subtracting a TRPM7 control trace (Ctl) from a trace recorded after 1 μM SPH, and then dividing by the Ctl trace: Inhibition = (I_Ctl–I_SPH)/I_Ctl. Data points at 0 mV were not included in the ‘E’. Note that the inhibitory effects at all the voltages are similar. Inset: average inhibition at −100, −80, −60, −40, −20, +20, +40, +60, +80 and +100 mV, respectively (n = 6). No statistical difference was observed. (F) Concentration-dependent effects of SPH on TRPM7. The best fit of dose–response curve yielded IC₅₀ = 0.59 ± 0.02 μM (n = 6 at each concentration).

Figure 2

Chemical structure of SPH analogues. SPH: D-erythro-sphingosine; S1P: D-erythro-sphingosine-1-phosphate; DMS: N,N-dimethyl-D-erythro-sphingosine; C2-Cer: N-acetyl-D-erythro-sphingosine; C8-Cer: N-octanoyl-D-erythro-sphingosine; FTY720: 2-amino-2-propane-1,3-diol hydrochloride; FTY720-P: 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, mono dihydrogen phosphate ester.

Figure 3

Effects of C2-Cer, C8-Cer and S1P on TRPM7 whole-cell currents. (A, C, E) Representative recordings of TRPM7 recorded in HEK-293 cells over-expressing TRPM7 before and after application of 10 μM C2-Cer (A), C8-Cer (C) and S1P (E). (B, D, F) Time-dependent changes of outward current amplitude measured at +100 mV before and after 10 μM C2-Cer (B), C8-Cer (D) and S1P (F). There was no noticeable change observed after application of C2-Cer, C8-Cer or S1P (n = 5 cells were tested for each compound).

Figure 4

DSM potently inhibited TRPM7 channel activity in whole-cell current recordings in the over-expressing HEK-293 cells. (A) Representative recordings of TRPM7 in the absence and presence of 0.3, 0.5 and 0.8 μM DMS. Cells were perfused with Tyrode solution before and after exposure to DMS. (B) Changes of inward currents by DMS using an enlarged y-axis. (C) Time-dependent changes of TRPM7 outward current measured at +100 mV and inward current measured at −100 mV before and after 1 μM DMS. (D) Concentration-dependent effects of DSM on TRPM7. The inhibition effects were evaluated using the current amplitude measured at +100 mV. The best fit of dose–response curve yielded IC₅₀ = 0.59 ± 0.02 μM (n = 6).

Figure 5

Effects of FTY720 and FTY720-P on TRPM7 channel activity evaluated by whole-cell current recording. (A) Blockade effects of 1 μM FTY720 on TRPM7 whole-cell current recorded in the HEK-293 cells over-expressing TRPM7. Current recorded at 120 s represents whole-cell current before FTY720 treatment, and current recorded at 370 s was after FTY720. (B) Time-dependent changes of TRPM7 outward current amplitude measured at +100 mV and inward current measured at −100 mV before and after 1 μM FTY720. Currents were completely blocked by 1 μM FTY720 and fully recovered after washout. (C) Inhibitory effects of FTY720 on TRPM7 at various concentrations (n = 4–6). (D) Dose–response curve of FTY720 on TRPM7. The average inhibition at each concentration of FTY720 was obtained by measuring the changes of current amplitude at +100 mV. Best fit of the dose–response curve yielded an IC₅₀ of 0.72 ± 0.04 μM. (E) FTY720-P at 10 μM has no effect on TRPM7 currents. (F) TRPM7 current amplitude measured at +100 mV before and after 10 μM FTY720-P. Similar results were reproduced in another five cells.

Figure 6

Effects of SPH on single-channel properties of TRPM7 recorded in HEK293 cells over-expressing TRPM7. (A) Single-channel current of TRPM7 recorded at different voltages in an inside-out excised patch. (B) Single-channel conductance of TRPM7. Linear regression of the single-channel current as a function of voltage yielded single-channel conductance of 38.3 ± 0.9 pS. (C) Effects of SPH on single-channel current. MgCl₂ (50 μM) was applied as a control in inhibiting TRPM7 current. (D) Average NP_o before and after application of SPH, and after washout.

Figure 7

Effects of SPH on TRPM7 currents recorded by macropatches in HEK293 cells over-expressing TRPM7. (A) TRPM7 currents recorded in macropatches using a ramp protocol ranging from −100 to +100 mV. SPH at 1 μM applied in the bath solution (inside of the cell) completely inhibited TRPM7 currents. (B) Time-dependent changes of TRPM7 current amplitude (measured at +100 mV) before and after 1 μM SPH. The effects of SPH are reversible and reproducible. (C) Average TRPM7 current amplitude before and after 1 μM SPH (n = 6).

Figure 8

Effects of SPH, DMS and FTY720 on endogenous TRPM7 current in human atrial fibroblasts. (A, C, E) TRPM7 current elicited by a ramp protocol was blocked by 1 μM SPH (A), DMS (C) and FTY720 (E). (B, D, F) Changes of TRPM7 outward currents before and after application of SPH (B), DMS (D) and FTY720 (F).

Figure 9

Inhibition of TRPM7 whole-cell currents by SPH and DMS in mouse cardiac fibroblasts. (A, C) TRPM7 currents before and after SPH (A) and DMS (C). (B–D) TRPM7 current amplitude before and after 1 uM SPH (B) and DMS (D). Similar results were obtained in five other fibroblasts.

Figure 10

Effects of FTY720 on mobility and proliferation of HEK-293 cells. (A) Top: original images taken at 0 h after wounding in control, 2 μM FTY-720-treated and 2 μM FTY720-P-treated HEK-293 cells. Bottom: outlines of the wound determined using the program ImageJ. (B) Images taken 24 h after wounding (top), and outlines of the wound (bottom) in control, 2 μM FTY-720-treated and 2 μM FTY720-P-treated HEK-293 cells. (C) Average percentage of area closure assessed 24 h after wounding (n = 6, ***P < 0.001). (D) Effects of FTY720 and FTY720-P on proliferation of HEK293 cells over-expressing TRPM7. Cells were treated with control, 2 μM FTY720 and 2 μM FTY720-P for 48 h. Average data were from six independent experiments (n = 6, ***P < 0.001).

Figure 11

Effects of SPH on TRPM2, TRPM4 and TRPM6. (A–B) Effects of SPH on TRPM2 whole-cell currents recorded in HEK-293 cells. (A) Representative TRPM2 currents elicited by voltage ramps ranging from −100 to +100 mV under the following conditions: (a) when Tyrode solution was replaced by NMDG-Cl; (b) when current reached steady state (before SPH); (c) in the presence of SPH; (d) after block by 30 μM ACA. (B) Time-dependent changes of outward and inward currents of TRPM2 after replacement of Tyrode solution with NMDG (a), before (b) and after (c) 10 μM SPH, and in the presence of 30 μM ACA (d). NMDG was used to ensure that there were no leak currents. The time points labelled with ‘a’, ‘b’, ‘c’ and ‘d’ represent the time points when the representative recordings in ‘A’ were taken. (C–D) Effects of SPH on TRPM4 whole-cell currents recorded in HEK-293 cells by voltage ramp protocols. (C) Typical TRPM4 currents recorded after initial phase of activation (a), after second phase of activation, and when the current reached steady state (b) in the presence of 10 μM SPH (c), and with NMDG-Cl to replace Tyrode solution (d). (D) Time-dependent changes of outward and inward currents of TRPM4 in the absence and presence of 10 μM SPH. NMDG-Cl was applied twice to eliminate the possibility of involvement of leak currents. The time points labelled with ‘a’, ‘b’, ‘c’ and ‘d’ represent the time points when the representative recordings in ‘C’ were taken. (E–H) Effects of SPH on TRPM6 whole-cell currents. TRPM6 plasmids were transiently transfected into CHOK1 cells and the currents were elicited by voltage ramps ranging from −120 to +100 mV. (E) Typical TRPM6 recordings in the absence and presence of 200 μM 2-APB and 1 μM SPH, and after washout SPH. (F) Illustration of the changes in TRPM6 inward currents by the treatments shown in ‘E’. (G) Time course of TRPM6 activation, potentiation by 2-APB, and inhibition by 1 μM SPH. The inward currents were measured at −100 mV and outward currents were measured at +100 mV. (H) Average inhibition of TRPM6 at various concentrations of SPH. The best fit of the dose–response curve yielded an IC₅₀ of 0.63 ± 0.1 μM (n = 4–6 cells). Note that the effect of SPH was reversible. The dashed traces in (E) and (F) were recorded after washout for 7 min.