Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gβγ Subunits

Abstract

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gα_i/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gα_s and Gα_q Activation of the G_s-coupled adenosine 2B receptor and the G_q-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C terminus of β-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating G_s-coupled prostaglandin-EP2 and G_q-coupled bradykinin B2 (BK2) receptors. The G_i/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gα_i/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3^-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gα_i/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gα_i/o, we examined whether activation of G_s- and G_q-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of G_s- and G_q-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that G_s- and G_q-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

Keywords: DRG; G-protein beta-gamma; GPCR; TRPM3; ion channels; pain.

Figure 1.

Non–G_i/o-mediated inhibition of TRPM3. A–C, Example traces of multiple applications of PS (100 μm) in whole-cell voltage-clamp recordings (+60 mV) of TRPM3 HEK293 cells with control, GTPγS (300 μm), or GDPβS (500 μm) intracellular solutions. D, Bar chart of the average responses ± SEM of (A) (n = 7), (B) (n = 6), and (C) (n = 8) (F_(2,18) = 11.8, p = 0.0005). E, F, Example traces of multiple applications of PS (100 μm) in whole-cell voltage-clamp recordings (60 mV) of TRPM3 HEK293 cells treated with PTX (200 ng/ml) with control or GTPγS (300 μm) intracellular solutions. G, Bar chart of the average responses ± SEM of (E) (n = 7 or 8) and (F) (n = 9 or 10) (t₍₁₄₎ = 3.62, p = 0.028). *p < 0.05; **p < 0.01; compared with control, repeated-measures two-way ANOVA with Dunnett's multiple-comparisons test (D) and multiple t test (G).

Figure 2.

G_s-coupled adenosine 2B receptor activation inhibits TRPM3-mediated responses in HEK293 cells. A, Concentration response curves of intracellular cAMP production in response to adenosine or BAY 60–6583 application mean ± SEM in pGLOSensor HEK293 cells. B, Inhibitory concentration response curves using the selective A2B antagonist, PSB603, on intracellular cAMP production evoked by adenosine (50 μm) or BAY 60–6583 (5 μm) mean ± SEM. C, Concentration response curve of [Ca²⁺]_i responses in HEK293 cells following application of 2F-LIGRLO or adenosine mean ± SEM (n = 4, both). D, [Ca²⁺]_i responses in TRPM3 HEK293 cells with PS application (20 μm). E, F, [Ca²⁺]_i responses in TRPM3 HEK293 cells treated with PS (20 μm) and adenosine (100 μm) or BAY 60–6583 (20 μm). G, Scatter plot representing the mean, median, and SEM of the relative amplitude from (D) (n = 731), (E) (n = 588), and (F) (n = 1145). The minimum response amplitude during coapplication of PS and A2B agonists was compared with the maximum amplitude during PS application. H, I, Example traces of adenosine (100 μm) and BAY 60–6583 (20 μm) mediated inhibition of PS (100 μm) evoked currents in whole-cell voltage-clamp recordings (+60 mV) in TRPM3 HEK293 cells. J, Scatter plot representing TRPM3-evoked current inhibition in (H) (n = 12) and (I) (n = 9) (t₍₁₉₎ = 0.17, p = 0.867, two-tailed unpaired t-test). K, L, Example traces of adenosine (100 μm) and BAY 60–6583 (20 μm) mediated inhibition of PS (100 μm) evoked currents in whole-cell voltage-clamp recordings (60 mV) in TRPM3 and A2B transfected HEK293 cells. M, Scatter plot representing TRPM3-evoked current inhibition in K and L (n = 4, both) (t₍₆₎ = 0.902, p = 0.402, two-tailed unpaired t-test). ***p < 0.001 compared with control; ^###p < 0.001 compared with adenosine treatment; Kruskal–Wallis with Dunn's multiple-comparisons test.

Figure 3.

Prostaglandin EP2 receptor activation inhibits TRPM3-mediated responses in sensory neurons. A, [Ca²⁺]_i responses in isolated mouse DRG neurons challenged by PS (20 μm) followed by KCl (50 mm) application. B, [Ca²⁺]_i responses in mouse isolated DRG neurons treated with PS (20 μm) and PGE₂ (1 μm). C, Scatter plots representing the relative response amplitudes for control (n = 207), 1 μm PGE₂ (n = 175), and PGE₂ + PTX (200 ng/ml; n = 151) treated DRG neurons. Relative responses represent the minimum amplitudes recorded 3–6 min during PS application (corresponding to the time of drug application) expressed as a percentage of the maximum amplitude recorded in the 0–3 min period. Some reduction in response amplitude was typically seen in control conditions. D, [Ca²⁺]_i responses in isolated DRG neurons treated with PS (20 μm) and morphine (10 μm). E, [Ca²⁺]_i responses in PTX-treated (200 ng/ml) isolated DRG neurons challenged with PS (20 μm) and morphine (10 μm). F, Scatter plot representing the relative amplitudes of control (94.3 ± 1.6% SEM, n = 164), (D) (48.4 ± 2.7% SEM, n = 109), and (E) (86 ± 2.3% SEM, n = 135) neurons. G, [Ca²⁺]_i responses in isolated mouse DRG neurons treated with PS (20 μm) and butaprost (1 μm). H, Scatter plot representing the relative amplitude in control (n = 212), butaprost (n = 250), and butaprost + PTX (n = 140) treated DRG neurons. I, Scatter plot representing the relative amplitude in control (n = 409), PGE₂ (n = 185), butaprost (n = 294), and CAY10595 (30 nm; n = 207) treated DRG neurons. J, Ononetin (10 μm) mediated inhibition of PS (50 μm) evoked currents in whole-cell voltage-clamp recordings (60 mV) in isolated DRG neurons (n = 6). K, L, Example traces of PGE₂ and butaprost (both 1 μm) mediated inhibition of PS (50 μm) evoked currents in whole-cell voltage-clamp recordings (60 mV) in isolated DRG neurons. M, Scatter plot representing PGE2 and butaprost current inhibition of TRPM3-evoked currents inhibition in (K) (n = 6) and (L) (n = 7) (t₍₁₁₎ = 1.11, p = 0.289, two-tailed unpaired t-test). ***p < 0.001 compared with control; ^###p < 0.001 compared with morphine-treated cells; Kruskal–Wallis with Dunn's multiple-comparisons test.

Figure 4.

Gq-coupled muscarinic M1 receptor activation inhibits TRPM3-mediated responses in HEK293 cells. A, [Ca²⁺]_i responses in TRPM3/muscarinic M1 HEK293 cells treated with PS (20 μm) and carbachol (0.1 μm) followed by application of a high concentration of carbachol (1 μm) to confirm M1 transfection. B, Scatter plot representing the mean, median, and SEM of the relative amplitude from control cells (60 ± 0.9% SEM, n = 431) and (A) (51.7 ± 1.0% SEM, n = 329), (Mann–Whitney U = 52821, p < 10⁻⁴). ***p < 0.001. C, [Ca²⁺]_i responses in TRPM3/muscarinic M1 HEK293 cells with application of different carbachol concentrations.

Figure 5.

Bradykinin BK2 receptor activation inhibits TRPM3-mediated responses in sensory neurons. A, [Ca²⁺]_i responses in isolated mouse DRG neurons treated with PS (20 μm) and bradykinin (100 nm). B, Scatter plot representing the relative amplitudes in control (n = 293) and bradykinin (n = 293) treated DRG neurons (Mann–Whitney U = 29892, p < 10⁻⁴). ***p < 0.001. C, Scatter plot representing the relative amplitudes in control (n = 190), bradykinin (n = 157), and bradykinin + PTX (200 ng/ml; n = 165) treated DRG neurons. D, Scatter plot representing the relative amplitudes in control (n = 157), bradykinin (n = 197), and bradykinin + HOE 140 (50 nm; n = 182) treated DRG neurons. E, Scatter plot representing the relative amplitudes in control (n = 118), bradykinin (n = 106), and bradykinin + SSR240612 (50 nm; n = 144)-treated DRG neurons. ***p < 0.001 compared with control; ^###p < 0.001 compared with bradykinin-treated cells, Kruskal–Wallis with Dunn's multiple-comparisons test C–E. F, Example trace of bradykinin (100 nm) mediated inhibition of PS (50 μm) evoked currents in whole-cell voltage-clamp recordings (60 mV) in isolated DRG neurons.

Figure 6.

G_s- and G_q-coupled mediated inhibition of TRPM3 is independent of PKA and PKC activity. A, [Ca²⁺]_i responses in isolated mouse DRG neurons treated with PS (20 μm), butaprost (1 μm), and KT5720 (1 μm). B, Scatter plot representing the relative amplitudes in control (n = 112), butaprost-treated (n = 151), and butaprost + KT5720-treated (n = 151) cells. C, [Ca²⁺]_i responses in isolated mouse DRG neurons treated with PS (20 μm), butaprost (1 μm), and forskolin (10 μm). D, Scatter plot representing the relative amplitudes in control (n = 185), butaprost-treated (n = 183), forskolin-treated (n = 148), and butaprost + forskolin-treated (n = 155) cells. E, [Ca²⁺]_i responses in isolated mouse DRG neurons treated with PS (20 μm), bradykinin (100 nm), and BIM VIII (1 μm). F, Scatter plot representing the relative amplitudes in control (n = 236), bradykinin-treated (n = 257), and bradykinin + BIM VIII-treated (n = 248) cells. **p = 0.0014; ***p < 0.001 compared with control; ^###p < 0.001 compared with bradykinin-treated cells; Kruskal–Wallis with Dunn's multiple-comparisons test.

Figure 7.

G_s- and G_q-coupled GPCR inhibition of TRPM3 is reliant on Gβγ protein. A–C, [Ca²⁺]_i responses in control (top) and βARK1-ct transfected (bottom) TRPM3 HEK293 cells treated with PS (20 μm) and adenosine (100 μm, n = 852), BAY 60–6583 (20 μm, n = 553), or carbachol (0.1 μm, n = 386). D–F, Scatter plots representing the relative amplitudes of A–C with βARK1-ct untransfected control and treated TRPM3 HEK293 cells. ***p < 0.001 compared with control; ^###p < 0.001 compared with treated cells; Kruskal–Wallis with Dunn's multiple-comparisons test. G–I, Example traces of adenosine (100 μm), butaprost (1 μm), and bradykinin (100 nm) mediated inhibition of PS (100 μm for HEK cells and 50 μm for DRG neurons)-evoked currents in whole-cell voltage-clamp recordings (+60 mV) in HEK293 cells (for adenosine) and isolated DRG neurons (for butaprost and bradykinin) in control (top) and GRK2i (10 μm, bottom) intracellular solutions. J–L, Scatter plots representing TRPM3-evoked current inhibition in (G) (t₍₁₅₎ = 3.32, p = 0.0047, control n = 10, GRK2i n = 7, two-tailed unpaired t-test), (H) (t₍₁₀₎ = 3.06, p = 0.012, control n = 7, GRK2i n = 5, two-tailed unpaired t-test), and (L) (t₍₉₎ = 2.67, p = 0.026, control: n = 5, GRK2i: n = 6, two-tailed unpaired t-test). *p < 0.05. **p < 0.01.

Figure 8.

G_s- and G_q-coupled GPCR activation prevents mouse nociceptive behavior in response to TRPM3 agonists and reverses heat hyperalgesia in FCA-treated mice. A–C, Scatter plots representing the duration of pain responses in mice with intraplantar hindpaw injections of PGE₂ (t₍₁₈₎ = 2.86, p = 0.0103, n = 10, two-tailed unpaired t-test), butaprost (Mann–Whitney U = 10.5; p = 0.0258, n = 8, two-tailed), and bradykinin (Mann–Whitney U = 15.5, p = 0.0062, n = 10, two tailed) (all at a dose of 0.3 nmol) or with vehicle before PS (5 nmol)/CIM0216 (0.5 nmol) administration. *p < 0.05; **p < 0.01. D–F, Scatter plots representing the duration of pain responses in mice administered with intraplantar hindpaw injections of PGE₂ (t₍₁₀₎ = 1.11, p = 0.29, two-tailed unpaired t-test), butaprost (Man–Whitney U = 16, p = 0.82, two-tailed), and bradykinin (t₍₁₀₎ = 0.69, p = 0.5, two-tailed unpaired t-test) (0.3 nmol, n = 6, all) or with vehicle before capsaicin (5 nmol) administration. G, Bar chart comparing heat withdrawal latencies of mice from 50°C hotplate before and 72 h after intraplantar FCA (15 μl) injection in Trpm3^+/+ (t₍₁₄₎ = 5.49, p < 0.0001, two-tailed unpaired t-test) and Trpm3^−/− mice (t₍₁₄₎ = 1.31, p = 0.21, two-tailed unpaired t-test) (n = 8, both) ***p < 0.001. H, Bar chart comparing heat withdrawal latencies of mice from 50°C hotplate before and 72 h after injection with FCA (15 μl), vehicle or ononetin (10 mg/kg; F_(2,30) = 7.2, p = 0.0028) application (n = 6, both). I, Bar chart comparing heat withdrawal latencies of mice from 50°C hotplate before and 72 h after injection with FCA (15 μl) or vehicle, PGE₂ (0.3 nmol; F_(2,30) = 4.7, p = 0.0168), or bradykinin (0.3 nmol; F_(2,30) = 6.21, p = 0.0055) (n = 6, each). *p < 0.05; **p < 0.01, compared with baseline; ^#p < 0.05; ^##p < 0.01, compared with 72 h after FCA; two-way ANOVA followed by Tukey's multiple-comparisons test.