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Comparative Study
. 2008 Apr;153(8):1697-705.
doi: 10.1038/bjp.2008.38. Epub 2008 Feb 25.

9-phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels

T Grand  1 , M Demion, C Norez, Y Mettey, P Launay, F Becq, P Bois, R Guinamard
Affiliations
Comparative Study

9-phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels

T Grand et al. Br J Pharmacol. 2008 Apr.

Abstract

Background and purpose: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5.

Experimental approach: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique.

Key results: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC(50) in the range of 2 x 10(-5) M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC(50). Channel inhibition was voltage independent. The IC(50) determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family.

Conclusions and implications: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.

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Figures

Figure 1
Figure 1
Biophysical properties of TRPM4 in HEK-293 cells. (a) Single-channel tracings recorded at various voltages from an inside-out patch from a TRPM4-transfected HEK-293 cell. Pipette and bath contained 145 mM NaCl solution (Ca2+: bath, 10−6 M; pipette, 10−3 M). Vm corresponds to the membrane potential. Bold dotted lines indicate the current level of closed channels. Channel activity is higher at +40 mV than at −40 mV. (b) Current–voltage relationship determined from the patch-clamp recording presented in (a). Data points were fitted by linear regression, yielding a value for slope conductance (g) of 25 pS. (c) Current tracing recorded in the whole-cell condition using the ramp protocol from −100 to +100 mV, as shown under the trace. Pipette contains mainly 156 mM CsCl and bath mainly 156 mM NaCl (see Methods). Current tracings were recorded at several time points after membrane break. (d) Time course of the maximum whole-cell current after membrane break from the cell shown in (c). Maximal current is determined as the mean current recorded during the ending step of 20 ms at +100 mV of the ramp protocol. Note the current rundown followed by a strong rise and a stabilization within few minutes. These properties are consistent with a TRPM4 current. HEK, human embryonic kidney; TRPM4, transient receptor potential melastatin 4.
Figure 2
Figure 2
TRPM4 inhibition by MPB-104. (a) Current recorded from an inside-out patch from a TRPM4-transfected HEK-293 cell showing the reversible inhibition of channel activity by several concentrations of MPB-104 (Vm=−80 mV). Magnification allows observing single-channel currents. Label ‘c' indicates the current level corresponding to the closed state of all channels. (b) Averaged currents (in % of control) in the presence of several concentrations of MPB-104 at Vm=+40, −40 and −80 mV (n=4–9 for each point). Error bars indicate s.e.mean. Data points were fitted to a Hill equation providing similar IC50 in the three conditions. (c) Single-channel currents in control conditions and in the presence of 10−4 M MPB-104 (Vm=−80 mV) on the same membrane patch. Current traces and corresponding amplitude histograms provided on the left of the panel indicate a decrease in channel activity but not in single-channel current amplitude. HEK, human embryonic kidney; MPB-104, 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride; TRPM4, transient receptor potential melastatin 4.
Figure 3
Figure 3
Effect of MPB-104 and 9-phenanthrol on iodide efflux in CHO cells stably expressing CFTR chloride channels. (a) Chemical structure of MPB-104 and 9-phenanthrol. (b) Representative curves of iodide efflux as function of time for cells treated with MPB-104 or 9-phenanthrol (2.5 × 10−4 M each) during the period indicated by the horizontal line. (c) Histograms showing rate of iodide efflux (mean±s.e.mean, n=4 for each condition) for cells stimulated by MPB-104 and 9-phenanthrol compared with non-stimulated cells (noted basal). ***P<0.001. CFTR, cystic fibrosis transmembrane conductance regulator; CHO, Chinese hamster ovary; MPB-104, 5-butyl-7-chloro-6-hydroxybenzo[c]quinolizinium chloride; ns, nonsignificant difference.
Figure 4
Figure 4
TRPM4 inhibition by 9-phenanthrol in inside-out conditions. (a) Current recorded from an inside-out patch from a TRPM4-transfected HEK-293 cell showing the reversible inhibition of channel activity by several concentrations of 9-phenanthrol (Vm=+40 mV). The zero current level was determined by lowering [Ca2+]i to a level estimated at 10−9 M that produced a total inhibition of the channel. (b) Averaged currents (in % of control) in the presence of several concentrations of 9-phenanthrol at Vm=+40, −40 and −80 mV (n=4–9 for each point). Data points were fitted to a Hill equation providing an IC50 in the range of 2 × 10−5 M and a Hill coefficient close to 1 in the three conditions (see Table 1). HEK, human embryonic kidney; TRPM4, transient receptor potential melastatin 4.
Figure 5
Figure 5
TRPM4 inhibition by 9-phenanthrol in whole-cell conditions. (a and b) Time course after membrane break of the maximal whole-cell current recorded from a TRPM4-transfected HEK-293 cell showing the reversible inhibition of the current by 9-phenanthrol (9-phe) (a). Maximal current was determined using the ramp protocol as indicated in legends of Figure 1d. Zero current level was determined by perfusion of 10 μM flufenamic acid (FA). Labels ‘a–d' indicate the values corresponding to the current recordings provided in (b). (c) Dose–response curve of 9-phenanthrol in the whole-cell condition determined using the ramp protocol (n=4–6 for each point). Data points were fitted to a Hill equation providing an IC50 of 1.67±0.45 × 10−5 M. (d) Current/voltage (I/V) relationship of the whole-cell TRPM4 current determined using a step protocol of 100 ms pulse performed every 300 ms with increasing steps of 10 mV from −100 to +100 mV (holding potential 0 mV). I/V were performed in control condition (n=4) and in the presence of 9-phenanthrol at 100 μM (n=4). Note that to construct I/V curves, the current was expressed as current density (current/cell capacitance; pA pF−1). Current tracings on the right provide an example of currents recorded using the step protocol with or without 9-phenanthrol, on the same cell. HEK, human embryonic kidney; TRPM4, transient receptor potential melastatin 4.
Figure 6
Figure 6
Biophysical properties of TRPM5 in HEK-293 cells. (a) Single-channel tracings recorded at various voltages from an inside-out patch from a TRPM5-transfected HEK-293 cell. Pipette and bath contained 145 mM NaCl solution (Ca2+: bath, 10−6 M; pipette, 10−3 M). (b) Current–voltage relationship determined from the patch-clamp recording presented in (a). Data points were fitted by linear regression, yielding a value for slope conductance (g) of 20.3 pS. (c) Current tracing recorded in the whole-cell condition using the ramp protocol from −100 to +100 mV as shown under the trace (same ionic conditions as in Figure 1c). Note the characteristic outward rectification (d) I/V determined using the step protocol as described in Figure 5d from a whole-cell recording of a TRPM5-transfected cell. Outward rectification is similar as the one observed in (c). These properties are consistent with a TRPM5 current. HEK, human embryonic kidney; TRPM5, transient receptor potential melastatin 5.
Figure 7
Figure 7
Lack of effect of 9-phenanthrol on TRPM5 current. (a) Current tracing recorded from an inside-out patch from a TRPM5-transfected HEK-293 cell (Vm=+40 mV). Although reducing [Ca2+]i to 10−9 M totally inhibits channel activity, perfusing 9-phenanthrol at 10−4 M has no significant effect. The magnification in the inset shows single-channel currents and corresponds to the period indicated by an asterisk in the whole trace. (b) Mean current (Imean) in the presence of 9-phenanthrol at 10−4 M normalized to mean current in control (ctrl) conditions at Vm=+40 and −80 mV for TRPM4- and TRPM5-transfected cells. Values are from inside-out recordings. Numbers above bars indicate the number of trials. (c) Time course of the maximal current recorded in the whole-cell conditions using the ramp protocol (see legend of Figure 1d) from a TRPM5-transfected HEK-293 cell. Perfusion of 9-phenanthrol had no effect. The inset shows the current traces used to determine the points indicated by arrows. HEK, human embryonic kidney; TRPM4, transient receptor potential melastatin 4; TRPM5, transient receptor potential melastatin 5.
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