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, 148 (3), 264-73

Inhibition of TRPM2 Cation Channels by N-(p-amylcinnamoyl)anthranilic Acid

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Inhibition of TRPM2 Cation Channels by N-(p-amylcinnamoyl)anthranilic Acid

Robert Kraft et al. Br J Pharmacol.

Abstract

1. TRPM2 is a Ca2+ -permeable nonselective cation channel activated by intracellular ADP-ribose (ADPR) and by hydrogen peroxide (H2O2). We investigated the modulation of TRPM2 activity by N-(p-amylcinnamoyl)anthranilic acid (ACA). ACA has previously been reported to inhibit phospholipase A2 (PLA2). 2. Using patch-clamp and calcium-imaging techniques, we show that extracellular application of 20 microM ACA completely blocked ADPR-induced whole-cell currents and H2O2-induced Ca2+ signals (IC50 = 1.7 microM) in HEK293 cells transfected with human TRPM2. Two other PLA2 inhibitors, p-bromophenacyl bromide (BPB; 100 microM) and arachidonyl trifluoromethyl ketone (20 microM), had no significant effect on ADPR-stimulated TRPM2 activity. 3. Inhibition of TRPM2 whole-cell currents by ACA was voltage independent and accelerated at decreased pH. ACA was ineffective when applied intracellularly. The single-channel conductance was not changed during ACA treatment, suggesting a reduction of TRPM2 open probability by modulating channel gating. 4. ACA (20 microM) also blocked currents through human TRPM8 and TRPC6 expressed in HEK293 cells, while BPB (100 microM) was ineffective. TRPC6-mediated currents (IC50 = 2.3 microM) and TRPM8-induced Ca2+ signals (IC50 = 3.9 microM) were blocked in a concentration-dependent manner. 5. ADPR-induced currents in human U937 cells, endogeneously expressing TRPM2 protein, were fully suppressed by 20 microM ACA. 6. Our data indicate that ACA modulates the activity of different TRP channels independent of PLA2 inhibition. Owing to its high potency and efficacy ACA can serve, in combination with other blockers, as a useful tool for studying the unknown function of TRPM2 in native cells.

Figures

Figure 1
Figure 1
Activation of human TRPM2 currents by ADP-ribose and inhibiton by ACA and FFA. (a) Currents were evoked after obtaining the whole-cell (w.c.) configuration due to infusion of a pipette solution containing 1 mM ADPR. The time course of currents at −80 and +80 mV were obtained from responses during voltage ramps from −100 to +100 mV. (b) Current–voltage relationships from the TRPM2-expressing cell were obtained at different time points following addition of ACA. (c) Nontransfected HEK293 cells showed no development of TRPM2-like, symmetric current pattern upon infusion with ADPR-containing pipette solution. (d) Current–voltage relationships from the nontransfected cell were obtained at different time points following addition of ACA. (e) Chemical structure of ACA. (f) Chemical structure of another TRPM2 antagonist FFA. (g) Extracellular application of 20 μM FFA to a TRPM2-expressing cell partially inhibited inward and outward currents (left trace). After washout of FFA, addition of 20 μM ACA induced a complete block of currents. The cationic nature of inward currents was checked by the exchange of Na+- and Ca2+-containing bath solution by an NMDG+-containing solution. Application of 20 μM FFA following block by ACA also induced a partial inhibition (right trace).
Figure 2
Figure 2
Effects of different phospholipase A2 inhibitors on TRPM2-mediated currents in HEK293 cells. (a) Chemical structures of the phospholipase A2 inhibitors AACOCF3 and BPB. (b) Currents were evoked by obtaining the whole-cell (w.c.) configuration and infusion of a pipette solution containing 1 mM ADPR. Currents at −80 and +80 mV were only slightly affected by application of 20 μM AACOCF3 but completely inhibited by subsequent application of 20 μM ACA. (c) ADPR-induced currents were unchanged in the presence of 100 μM BPB but completely inhibited by application of 20 μM ACA.
Figure 3
Figure 3
Concentration-dependent inhibition of ADPR-induced currents and H2O2-evoked increases in [Ca2+]i in TRPM2-transfected HEK293 cells by ACA. (a) Currents were evoked after obtaining the whole-cell (w.c.) configuration due to infusion of a pipette solution containing 1 mM ADPR. Currents at −80 and +80 mV were slowly inhibited by application of 1 μM ACA. (b) Concentration dependence of ACA-induced block (means±s.e.m. from n=6 each) was calculated from current amplitudes at −80 and +80 mV immediately before and 1 min (20 μM) or 4 min (1 μM) after addition of ACA. (c) Effect of 5 mM H2O2 on [Ca2+]i in TRPM2-transfected cells in the absence (control; n=8) and presence of 20 μM ACA (n=4). Shown are mean values from n independent experiments with at least 25 cells each. (d) Concentration-inhibition curve for ACA on H2O2-evoked increases in [Ca2+]i in TRPM2-transfected cells. Data points (mean±s.e.m. of n=3–4 independent experiments with at least 25 cells each) were calculated from the H2O2-induced responses 450 s after application of ACA. The curve was fitted to the logistic function Rnorm=1/{1+([ACA]/IC50)n}, where Rnorm is the fluorescence ratio F340/F380 in the presence of ACA normalized to that in control conditions, IC50 the concentration resulting in half-maximal inhibiton and nH the Hill coefficient.
Figure 4
Figure 4
Block of TRPM2 by ACA occurs via an extracellular site and is pH dependent. (a) Currents at −80 and +80 mV were evoked by obtaining the whole-cell (w.c.) configuration and infusion of a pipette solution containing 1 mM ADPR. Extracellular application of 20 μM ACA completely inhibited ADPR-induced inward and outward currents, whereas intracellular (i.c.) application of 50 μM ACA had no effect. (b) ADPR-induced currents were completely blocked by 1 μM ACA at pH 6.0. During washout, ADPR-induced currents reappeared immediately after switching to pH 7.4. The restored current was again inhibited after application of pH 6.0.
Figure 5
Figure 5
Effect of ACA on TRPM2-mediated single-channel currents in whole-cell (w.c.) recordings. (a) Currents at −70 mV were evoked by obtaining the w.c. configuration and infusion of a pipette solution containing 1 mM ADPR. Extracellular application of 20 μM ACA decreased inward currents. (b) Currents at −70 mV recorded from another cell showed a reduction of channel open states (downward deflections) but no obvious reduction of single-channel amplitude. (c) Current traces of 10 s duration were recorded at −70 mV during maximal current activation (0 s) and following application of ACA for 50 s or in the absence of ACA (control, 50 s). Mean inward current amplitudes (Imean) were calculated from 10 s traces as shown on the left side. Imean at 50 s was normalized to the values at 0 s and is shown as mean±s.e.m. of n=6 cells, each. (d) Amplitude histograms were calculated from 2 s intervals shown in (c) (lower panel, marked by bars). The resulting single-channel currents for this recording were −4.7 pA (ACA, 0 s) and −5.2 pA (ACA, 50 s).
Figure 6
Figure 6
Inhibition of human TRPM8 channels by ACA. (a) Currents through TRPM8 were activated by 1 mM menthol. The time course of currents at −80 and +80 mV yielded from responses during voltage ramps from −100 to +100 mV. Extracellular application of 20 μM ACA inhibited both inward and outward currents. Current–voltage relationships were recorded at the time points a and b. (b) Application of the PLA2-inhibitor BPB did not inhibit menthol-induced currents. Current–voltage relationships were recorded at the time points a and b. (c) Concentration-inhibition curve for ACA on menthol-evoked increases in [Ca2+]i in TRPM8-transfected cells. [Ca2+]i responses were either measured as changes in fluorescence intensity (F) before and 30 min after the addition of ACA, 1 mM menthol and Ca2+ (mean±s.e.m. of n=6–8 independent experiments for each concentration) or as changes in F before and 30 min after addition of ACA and Ca2+ to a Ca2+-free solution (mean±s.e.m. of n=4 independent experiments for each concentration). Data points for menthol-induced responses were calculated by subtracting mean F values in the presence of ACA and the absence of menthol from the respective values in the presence of both ACA and 1 mM menthol. The curves were fitted to the logistic function F=Fmin+(FmaxFmin)/{1+([ACA]/IC50)n}. The concentrations of ACA giving a half-maximal inhibitory effect (IC50) were 5.4 and 3.9 μM for menthol/ACA-induced and menthol-induced responses, respectively. The respective Hill coefficients nH were 2.3 and 1.9.
Figure 7
Figure 7
Inhibition of human TRPC6 channels by ACA. (a) Currents through TRPC6 were evoked by obtaining the whole-cell (w.c.) configuration and infusion of a pipette solution containing 30 μM AlF4. The time course of currents at −80 and +80 mV yielded from responses during voltage ramps from −100 to +100 mV. Extracellular application of 20 μM ACA led to a reduction of both inward and outward currents. Exchange of the Na+- and Ca2+-containing bath solution by an NMDG+-containing solution abolished inward currents. Current–voltage relationships were recorded at the time points a and b. (b) Application of the PLA2-inhibitor BPB did not inhibit w.c. currents, whereas subsequent application of ACA blocked TRPC6 activity. Current–voltage relationships were recorded at the time points a and b. (c) Concentration-inhibition curve for ACA on AlF4-evoked inward currents in TRPC6-transfected cells. Data points (mean±s.e.m. of n=5–7 cells for each concentration) were calculated from current responses before and after application of 1, 5, 20 and 50 μM ACA. The curve was fitted to the logistic function Inorm=1/{1+([ACA]/IC50)n}, where Inorm is the current amplitude at −80 mV in the presence of ACA normalized to that in control conditions, IC50 the concentration resulting in half-maximal inhibiton and nH the Hill coefficient.
Figure 8
Figure 8
ACA inhibits TRPM2 currents in human U937 cells. (a) Membrane proteins (4 μg) from nontransfected cells (Blank), hTRPM3-transfected and hTRPM2-transfected HEK293 cells were used for characterization of the TRPM2 antibody. The specificity of the antibody reaction was verified by incubating parallel lanes with the antibody in the presence of the peptide used for immunization. (b) Membrane proteins from U937, HL-60, MEG-01 cells (20 μg each) and from hTRPM2-transfected HEK293 cells (4 μg) were used for Western blot analysis. (c) Currents were evoked after obtaining the whole-cell (w.c) configuration due to infusion of a pipette solution containing 1 mM ADPR. Application of 20 μM ACA inhibited both inward and outward currents. Exchange of the Na+- and Ca2+-containing bath solution by an NMDG+-containing solution suppressed inward currents. Current–voltage relationships were obtained at different time points following addition of ACA. (d) The graph shows the mean times±s.e.m. required to produce a 50% reduction of ADPR-induced currents by 20 μM ACA in hTRPM2-transfected HEK293 cells (n=6) and U937 cells (n=4).

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