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. 2013 May 3;288(18):12818-27.
doi: 10.1074/jbc.M113.450072. Epub 2013 Mar 18.

Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine

Jonathan Y-X L Than  1 , Lin Li, Raquibul Hasan, Xuming Zhang
Affiliations

Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine

Jonathan Y-X L Than et al. J Biol Chem. .

Abstract

The sensations of pain, itch, and cold often interact with each other. Pain inhibits itch, whereas cold inhibits both pain and itch. TRPV1 and TRPA1 channels transduce pain and itch, whereas TRPM8 transduces cold. The pruritogen chloroquine (CQ) was reported to excite TRPA1, leading to the sensation of itch. It is unclear how CQ excites and modulates TRPA1(+), TRPV1(+), and TRPM8(+) neurons and thus affects the sensations of pain, itch, and cold. Here, we show that only 43% of CQ-excited dorsal root ganglion neurons expressed TRPA1; as expected, the responses of these neurons were completely prevented by the TRPA1 antagonist HC-030031. The remaining 57% of CQ-excited neurons did not express TRPA1, and excitation was not prevented by either a TRPA1 or TRPV1 antagonist but was prevented by the general transient receptor potential canonical (TRPC) channel blocker BTP2 and the selective TRPC3 inhibitor Pyr3. Furthermore, CQ caused potent sensitization of TRPV1 in 51.9% of TRPV1(+) neurons and concomitant inhibition of TRPM8 in 48.8% of TRPM8(+) dorsal root ganglion neurons. Sensitization of TRPV1 is caused mainly by activation of the phospholipase C-PKC pathway following activation of the CQ receptor MrgprA3. By contrast, inhibition of TRPM8 is caused by a direct action of activated Gαq independent of the phospholipase C pathway. Our data suggest the involvement of the TRPC3 channel acting together with TRPA1 to mediate CQ-induced itch. CQ not only elicits itch by directly exciting itch-encoding neurons but also exerts previously unappreciated widespread actions on pain-, itch-, and cold-sensing neurons, leading to enhanced pain and itch.

Keywords: Calcium Imaging; Chloroquine; Electrophysiology; G Protein-coupled Receptor (GPCR); Itch; Pain; Sensory Transduction; Signal Transduction; TRP Channels.

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Figures

FIGURE 1.
FIGURE 1.
Direct excitation of DRG neurons by CQ. A, fluorescence images of DRG neurons loaded with Fluo-4-AM under basal conditions and after stimulation with CQ (1 mm, 2 min) and AITC (100 μm, 15 s). The fourth panel is a bright-field image of the neurons. Arrows indicate neurons responding to CQ. Scale bars = 10 μm. B–D, representative traces from DRG neurons as described for A activated sequentially by CQ (1 mm), menthol (Men; 100 μm), AITC (100 μm), and capsaicin (Cap; 500 nm). Ionomycin (Iono; 10 μm) was added at the end. CQ-responsive neurons either coexpressed TRPA1 (B) or TRPV1 (C) or both TRPV1 and TRPA1 (D). E, expression of TRP channel mRNA in individual DRG neurons amplified by RT-PCR from different subpopulations categorized by calcium imaging as shown in A–D. +, neurons sensitive to CQ, menthol, AITC, or capsaicin; −, insensitive neurons. The fifth lane is the amplification from the control bath solution. F and G, summary of percentages of CQ-responsive, capsaicin-sensitive, and AITC-responsive neurons related to a total of 2693 neurons isolated from neonatal mice (F) or 1450 neurons isolated from adult mice (G) in experiments similar to those in A–C. H, the Venn diagram illustrates the mutual relationship of TRPV1+, TRPA1+, and TRPM8+ (M8) neurons with CQ-responsive neurons. The number of neurons is shown for each population and was obtained from a total of 2693 neonatal neurons.
FIGURE 2.
FIGURE 2.
CQ excites DRG neurons through both TRPA1- and TRPC3-dependent mechanisms. A and B, representative calcium responses from three DRG neurons activated by CQ (1 mm) and AITC (100 μm). The TRPA1 antagonist HC-030031 (10 μm) was added together with the second CQ application as indicated. Iono, ionomycin. C–E, typical calcium responses from DRG neurons excited by CQ followed by exposure to AITC (100 μm) and capsaicin (Cap; 500 nm). The calcium-free solution, ruthenium red (RR; 10 μm), and BTP2 (10 μm) were perfused together with the second CQ addition as indicated. F, summary of ratio percentages between the second CQ-excited response and the first CQ-induced response obtained from neonatal DRG neurons in experiments similar to those in A–E. +, CQ-sensitive, AITC-sensitive, and capsaicin-sensitive neurons; −, insensitive neurons. The TRPA1 antagonist HC-030031 (10 μm), the TRPC channel blocker BTP2 (10 μm), the TRPC3 channel inhibitor Pyr3 (10 μm), the TRPV1 antagonist capsazepine (CSZ; 10 μm), and ruthenium red were added as indicated. The numbers of neurons are given above each bar. Data are means ± S.E. ***, p < 0.001; NS, not significant. All results are compared with the first bar.
FIGURE 3.
FIGURE 3.
CQ sensitizes TRPV1 in DRG neurons. A and B, the calcium response elicited by capsaicin (Cap; 100 nm) was enhanced by CQ (1 mm) in a DRG neuron; however, the neuron did not respond to 100 μm AITC, and CQ did not elicit a calcium increase irrespective of whether CQ was applied before (B) or after (A) capsaicin. Men, menthol. C and D, representative calcium response traces from a DRG neuron in an experiment similar to that in A, but CQ induced a transient calcium increase. Note that the application sequence of AITC in D was different from that in C. AITC was added after the second application of CQ in D. E, distribution of TRPV1-dependent calcium response ratios after (fifth response) and before (fourth response) CQ in DRG neurons from experiments similar to those in A. The number of cells was 137 for the control cells and 581 for the CQ-treated cells. F, summary of experiments similar to those in E. The number of experiments was 5 for the control (Con) cells, 11 for the 1 mm CQ-treated cells, and 7 for the BIM-pretreated cells (a total of 108 cells). Data are means ± S.E. ***, p < 0.001 compared with the control; ###, p < 0.001 compared with the second bar.
FIGURE 4.
FIGURE 4.
The PLC-PKC pathway is involved in the sensitization of TRPV1 induced by CQ. A, translocation of Tubby-R332H-cYFP caused by CQ in HEK293 cells expressing MrgprA3. CQ (1 mm) was added at 70 s. Scale bars = 10 μm. B, quantification of relative membrane Tubby fluorescence signal as a function of time in A. This experiment was repeated at least four times with similar results. PM, plasma membrane. C, CQ-sensitized TRPV1 inward current activated by 50 nm capsaicin (Cap; 5 s) recorded from a HEK293 cell expressing MrgprA3 and TRPV1. The dotted line represents zero current. D, CQ did not cause sensitization of the TRPV1 inward current recorded from a HEK293 cell expressing MrgprA3, TRPV1, and PLCβ-ct. The dotted line represents zero current. E, summary of results similar to those in C and D. Sensitization of TRPV1 was blocked by 2.5 μm U73122, by coexpression with PLCβ-ct, by 1 μm BIM, or by double-mutant (DM) TRPV1 S502A/S801A. All error bars are means ± S.E. The number of experiments is given above each bar. ***, p < 0.001 compared with the control (Con); ## p < 0.01; ### p < 0.001 compared with the second bar.
FIGURE 5.
FIGURE 5.
CQ inhibits TRPM8 in DRG neurons. A and B, CQ-inhibited calcium increase elicited by the TRPM8 agonist menthol (Men; 100 μm) in a DRG neuron. 100 μm AITC and capsaicin (Cap; 500 nm) were added as indicated. C, histogram distribution of calcium response ratios after (fifth response) and before (fourth response) CQ treatment. The number of cells was 30 for the control cells and 43 for the CQ-treated cells. D, summary of results similar to those in A after pretreatment with 1 μm BIM (38 cells). All data are means ± S.E. ***, p < 0.001 compared with the first bar. Con, control. E, summary of TRPM8-mediated calcium response ratios between after and before BK or CQ treatment in experiments similar to those in A and D and those obtained previously (10), but only inhibited neurons were included. The numbers of inhibited neurons are given above each bar. All data are means ± S.E. ***, p < 0.001 compared with the first bar.
FIGURE 6.
FIGURE 6.
CQ-induced TRPM8 inhibition is caused by direct action of activated Gαq. A, inward and outward currents (at −60 and +60 mV) activated by menthol (200 μm, 5 s) in HEK293 cells expressing TRPM8 and MrgprA3 were inhibited by pretreatment with CQ (1 mm, 1 min) applied alone or together with U73122 (2.5 μm) or after coexpression with PLCβ-ct. The dotted line represents zero current. Con, control. B, summary of peak currents in experiments similar to those in A. The number of experiments is shown above each bar. C, representative traces of TRPM8 inward and outward currents (at −60 and +60 mV) activated by menthol (200 μm, 5 s) in Gαq/11−/− MEF cells transfected with MrgprA3 and TRPM8. Currents are shown before and after CQ (1 mm, 1 min) treatment. The dotted line represents zero current. D, summary of results similar to those in C. The number of experiments is shown above each bar. All data are means ± S.E. **, p < 0.01; NS, not significant. E, HEK293 cells expressing TRPM8-V5 and MrgprA3 were stimulated with 1 mm CQ for 5 min. TRPM8 was then immunoprecipitated (IP) with anti-V5 antibody, and coprecipitated Gαq was detected by anti-Gαq antibody The same blot was stripped and redetected with anti-V5 antibody. TCL, total cell lysate; IB, immunoblot. Molecular masses are shown on the right.
FIGURE 7.
FIGURE 7.
CQ excites TRPA1 and TRPC3, sensitizes TRPV1, and inhibits TRPM8 through different signaling mechanisms. Binding of CQ to the CQ receptor MrgprA3 causes activation of Gαq and release of Gβγ. The released Gβγ then activates the TRPA1 channel, leading to opening of the channel and calcium influx. Meanwhile, activated Gαq activates PLCβ, leading to excitation of the TRPC3 channel and calcium influx. Activated Gαq also stimulates PKC and increases phosphorylation of TRPV1, leading to the sensitization of TRPV1 and subsequently enhanced itch. Activated Gαq also directly inhibits TRPM8 by binding to TRPM8 without the need to involve the PLC pathway. Inhibition of TRPM8 probably also contributes indirectly to enhanced pain and itch.
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