Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound

Abstract

Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

Fig. 1.

Selective inhibition of TRPC3-mediated Ca²⁺ influx by Pyr3. (A) Chemical structure of Pyr3. (B–E) Concentration-dependent inhibitory action of Pyr3 on ATP receptor-induced (B and C) or mAChR-induced (D and E) induced Ca²⁺ influx via TRPCs. (B and D) Average time courses of Ca²⁺ responses induced by 100 μM ATP in HEK293 cells (B) or by 100 μM CCh in HEK293T cells (D) transfected with TRPCs at indicated Pyr3 concentrations. (C and E) Percentage peak [Ca²⁺]_i rises in Ca²⁺-free, 0.5 mM EGTA-containing (Upper) or 2 mM Ca²⁺-containing (Lower) external solution compared with control responses without Pyr3 (n = 33–104). (F) Pyr3 Inhibition of Ca²⁺ influx via OAG-activated TRPC3. Average time courses of Ca²⁺ responses induced by 10 μM OAG at indicated Pyr3 concentrations in TRPC3-transfected HEK293 cells (Left). Percentage peak [Ca²⁺]_i rises in 2 mM Ca²⁺ solution (Right) (n = 19–37).

Fig. 2.

Inhibition of TRPCs by Pyr2, Pyr4, or Pyr5. (A–C) Concentration-dependent inhibitory action of Pyr2 (A), Pyr4 (B), or Pyr5 (C) on Ca²⁺ influx induced by 100 μM ATP via TRPCs. Percentage peak [Ca²⁺]_i rises in Ca²⁺-free, 0.5 mM EGTA- (Upper) or 2 mM Ca²⁺-containing (Lower) external solution compared with control responses without drugs (n = 18–66).

Fig. 3.

mAChR-activated TRPC3 current is suppressed by extracellular application of Pyr3. (A–C) Traces of ionic currents induced by 60 μM CCh at a holding potential of −50 mV in TRPC3-transfected HEK293 cells (Left). I–V relationships obtained by subtracting the currents evoked by the voltage-ramps before activation of channels (a and c) from those after activation (current traces b and d) (Right). (B) Three μM Pyr3 is added 1.5 min before second stimulation of CCh into the external solution. (C) Three μM Pyr3 is added in internal solution before the recordings, and then external Pyr3 is also applied 1.5 min before second CCh stimulation. (D) Average current amplitudes of the first response at −50 mV in the presence (n = 7) or absence (n = 8) of 3 μM Pyr3 in the internal solution. (E) Concentration-dependent inhibitory action of Pyr3, using the testing paradigm depicted in B and C. The amplitude of the second response was normalized to that of the first (peak2/peak1) (n = 4–8). **, P < 0.01 and ***, P < 0.001 vs. 0 μM Pyr3.

Fig. 4.

Photochemical cross-linking of TRPC3 with Pyr-PP. (A) Chemical structure of Pyr-PP. (B) Pyr-PP directly binds TRPC3. After P-PALM, TRPC3-GFP proteins are detected with anti-GFP antibody by Western blot analysis (WB) in avidin pull-down samples. The photochemical Pyr-PP cross-linking of TRPC3 is inhibited by 3-min preincubation and subsequent coincubation with Pyr3 (10 or 100 μM) (Left). After P-PALM, the incorporation of the Pyr-PP-ARP adduct is detected with anti-biotin antibody by WB in immunoprecipitated (IP) samples with anti-TRPC3 antibody (Right). (C) Electron microscopic visualization of negatively stained TRPC3 after P-PALM with gold nanoparticles. Streptavidin-gold conjugate is attached to labeled-TRPC3 via biotin-labeling site.

Fig. 5.

Pyr3 inhibits native TRPC3 channels and downstream responses in DT40 B lymphocytes. (A and B) Inhibitory action of 0.3 μM Pyr3 on Ca²⁺ oscillation upon BCR stimulation with 1 μg/mL anti-IgM. (A) Representative time courses with (Right) or without of Pyr3 (Left). (B) Peak [Ca²⁺]_i rises at initial and sustained phases (30–40 min). (C) Inhibitory action of 1 μM Pyr3 on BCR-induced Ca²⁺ influx. Average time courses (Left). Peak [Ca²⁺]_i rises in Ca²⁺-free, 0.5 mM EGTA- or 2 mM Ca²⁺-containing solution (n = 42–49) (Right). (D) Confocal fluorescence images indicating PM translocation of PLCγ2-EYFP upon BCR-stimulation with 10 μg/mL anti-IgM. Three μM Pyr3 is applied 10 min before BCR stimulation. (E) Effects of Pyr3 on ERK activation induced by BCR stimulation (5 μg/mL anti-IgM). Three μM Pyr3 is applied 10 min before BCR stimulation. Cells are analyzed by WB, using anti-phospho-ERK2 antibody. ***, P < 0.001 vs. DMSO.

Fig. 6.

Potent suppressive effects of Pyr3 on cardiac hypertrophy induced by pressure overload in mice. (A–C) Effects of Pyr3 on 1-week TAC-induced concentric hypertrophy. (A) H&E-stained mid transverse sections of hearts isolated from sham- and TAC-operated mice. (B and C) Effects of Pyr3 on the increase in cross-sectional areas (CSA) (B) and ANP mRNA expressions (C). *, P < 0.05, **, P < 0.01, and ***, P < 0.001. (D and E) Effects of Pyr3 on 6-weeks TAC-induced dilated hypertrophy. Scattergram of systolic pressure gradient vs. FS, a surrogate of systolic function (D) and LVW/TL (E) in TAC-operated mice with (red) and without (black) treatment with Pyr3. Pyr3 significantly shifts relationships upward in FS (P < 0.001) and downward in LVW/TL (P < 0.01).