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. 2006 Oct 27;281(43):32879-90.
doi: 10.1074/jbc.M603220200. Epub 2006 Sep 5.

Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons

Nathaniel A Jeske  1 , Amol M Patwardhan, Nikita Gamper, Theodore J Price, Armen N Akopian, Kenneth M Hargreaves
Affiliations

Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons

Nathaniel A Jeske et al. J Biol Chem. .

Abstract

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.

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Figures

FIGURE 1
FIGURE 1. Western blot and mass spectrometric data illustrating the specificity of the antibodies used to identify TRPV1 and TRPA1
Western blot data were generated utilizing several antibodies and sources, including anti-TRPV1 Ab-2 on 50µg of TG cell lysate (A), anti-TRPV1 Ab-1 on 50µg of TG cell lysate (B), anti-TRPV1 Ab-1 preincubated with 25µg of antigenic peptide (Calbiochem) on 50µg of TG cell lysate (C), anti-TRPV1 Ab-1 on 25 µg of cell lysate from CHO cells rat transiently transfected with TRPV1 (D), antibody against the TRPA1 N terminus on 50 µg of TG cell lysate (E), antibody against the TRPA1 N terminus preincubated with 100 µg of antigenic peptide on 50 µg of TG cell lysate (F), and antibody against the TRPA1 N terminus on 25 µg of cell lysate from CHO cells transiently transfected with mouse TRPA1 (G). Black arrows indicate size markers (in kilodaltons), and gray arrows indicate the immunoreactive band of interest. Western blot results are representative of two independent experiments. TRPV1 was immunoprecipitated from 500 mg of TG cell lysate, resolved on 15% SDS-polyacrylamide gel, stained with Coomassie Blue, excised from the gel, and analyzed for trypsin digestion by mass spectrometry. 13 unique peptides were identified (H, highlighted in yellow), providing 15% coverage of the amino acid sequence for rat TRPV1 (GenBank™ accession number AF029310). Mascot analytical software made a 100% match between the cumulative peptides and rat TRPV1.
FIGURE 2
FIGURE 2. Cannabinoids reduce TRPV1 phosphorylation
TG neurons were treated with ACEA (25 µM), anandamide (AEA; 25 µm), and WIN (25 µM) and analyzed by SDS-PAGE and Western blotting (WB) for [32P]phosphate incorporation by TRPV1 (A). Autoradiographic (AutoRad) results were normalized to total immunoprecipitated (IP) TRPV1 (B), with band densities quantified and expressed as a percentage of vehicle (Veh)-treated cells (C). *, p < 0.05 (ANOVA; n = 3). TG neurons were treated with ACEA (25µm), anandamide (25 µm), and WIN (25µm), and immunoprecipitated TRPV1 was analyzed for phosphoserine (D) and phosphothreonine (E) immunoreactivities. Black arrows indicate size markers (in kilodaltons), and gray arrows indicate the immunoreactive band of interest. Western blot results are representative of three to four independent trials. Phospho results were normalized to total immunoprecipitated TRPV1, with band optical densities quantified and expressed as a percentage of vehicle-treated cells (F). S, serine phosphorylation; T, threonine phosphorylation. *, p < 0.05; **, p < 0.01 (significant compared with vehicle; ANOVA; n = 4).
FIGURE 3
FIGURE 3. WIN dephosphorylates TRPV1 through calcineurin
A, FKBP12 expression in TG neurons was analyzed by Western blotting (WB) in the absence and presence of antibody-blocking peptide (BP). The results are representative of three independent trials. B, FKBP12 expression was analyzed by immunofluorescence in a TG neuron coexpressing TRPV1. The results are representative of two independent trials. Scale bars = 25µm. C, calcineurin activity from TG neurons treated with Me2SO vehicle (Veh), capsaicin (CAP; 100 nm), WIN (25µm), and FK506 (100 µm) and WIN was analyzed by RII phosphopeptide assay. *, p < 0.05 (ANOVA and one-tailed t test; n=3). D, TG neurons were treated with WIN (25µm) or FK506 (100µm) and WIN, and immunoprecipitated TRPV1 was analyzed for phosphothreonine immunoreactivity. Phosphothreonine results were normalized to total immunoprecipitated TRPV1, with band optical densities quantified and expressed as a percentage of vehicle-treated cells. **, p < 0.01 (ANOVA; n = 4). The results are representative of four independent trials.
FIGURE 4
FIGURE 4. Current traces from transfected CHO cells
CHO cells were transfected with GFP, TRPV1, or TRPA1 vector and treated with WIN (25µm) with or without capsaicin (100 nm) or mustard oil (MO; 20µm). Recordings were made in the perforated patch voltage-clamp configuration. Cells were analyzed for currents, and the results are representative of five to nine trials. Drug application durations are indicate by horizontal bars.
FIGURE 5
FIGURE 5. TRPA1 mediates WIN-induced dephosphorylation of TRPV1 in TG neurons
TG neurons were transfected with siRNA directed against TRPA1 (A-1), Silencer negative siRNA ((−)), siRNA directed against Drosophila TRPA1 (Adr-1), or no siRNA (mock) and analyzed for TRPV1 phosphothreonine by Western blotting (WB) following WIN (25 µm) treatment (Trtmt). Lanes 1, mock transfection, Me2SO vehicle (Veh) treatment; lanes 2, mock transfection, WIN treatment; lanes 3, A-1 transfection, WIN treatment; lanes 4, Silencer negative siRNA control, WIN treatment; lanes 5, Adr-1 control, WIN treatment. Phospho results (A) were normalized to total immunoprecipitated (IP) TRPV1 (B), and TRPA1 expression (C) was normalized to TRPV1 expression (D), with band optical densities quantified and expressed as a percentage of vehicle-treated cells (E). *, p < 0.05 (significant compared with vehicle/mock transfection). Gray bars indicate threonine phosphorylation (T), and white bars indicate TRPA1 protein expression (A) by Western blotting. Black arrows indicate size markers (in kilodaltons), and gray arrows indicate the immunoreactive band of interest. The results are representative of four independent trials. F shows mustard oil (MO; 20µm)-evoked calcium imaging of mock-, Adr-1 (fluorescein isothiocyanate-labeled)-, and A-1-transfected TG cultures over a 4-day period of post-transfection (Post-Trans). **, p < 0.01 (ANOVA; n = 4).
FIGURE 6
FIGURE 6. WIN directly activates TRPA1 to increase [Ca2+]i through a Gαq-independent mechanism
TG neurons were transfected with the GFP-PHD PLC sensor, whereas CHO cells were transiently transfected with the GFP-PHD PLC sensor and different combinations of TRPA1, the muscarinic type 1 receptor (m1), or the bradykinin type 2 receptor (B2). Translocation of GFP-PHD from the plasma membrane to the cytosol and accumulation of intracellular Ca2+ (Δ[Ca2+]i) were concurrently imaged by a set of filters. Data for analysis were collected every 5 s after exposure to WIN (25µm, 3 min), oxotremorine (Oxo; 10µm, 5 min), or bradykinin (BK; 200 nm, 5 min) with 5-min washout period. A, translocation of GFP-PHD (F/F0) was calculated from the measurement of F470 nm within a predetermined area of the cytosol using MetaFluor software. *, p < 0.05 (ANOVA, with n values indicated). B, isΔ[Ca2+]i was calculated from measurements of changes in the F340/380 nm ratio using MetaFluor software. **, p < 0.005 (ANOVA, with n values indicated). C and D, shown are typical traces of GFP-PHD translocation and calcium influx on the same temporal scale for muscarinic type 1 receptor/GFP-PHD-transfected CHO cells and TRPA1 (A1)/muscarinic type 1 receptor/GFP-PHD-transfected CHO cells, respectively. GFP images of a cell within the indicated time points are presented. E, shown is the time course of GFP-PHD translocation in a TG neuron stimulated by WIN (25 µM) or bradykinin (1 µm) plotted against GFP fluorescence density (F470 nm) in the cytosol.
FIGURE 7
FIGURE 7. TRPV1 desensitization of ICAP by WIN is dependent on Thr144 and Thr370 phosphorylation
CHO cells were transiently transfected with TRPA1 and wild-type TRPV1 (WT), TRPV1(T144A), TRPV1(T370A), TRPV1(T704A), or TRPV1(T144A/T370A); treated with vehicle (Veh; 0.1% Me2SO) or WIN (25µm) for 10 min; washed for 5 min; and patched. ICAP was recorded during a 30-s application of 300 nm capsaicin (CAP). Data were normalized to the mean peak of ICAP measured from vehicle-treated cells (n = 8–15/treatment, noted inside each bar). A, ICAP measured from CHO cells expressing wild-type TRPV1, TRPV1(T144A), TRPV1(T370A), TRPV1(T704A), or TRPV1(T144A/T370A). pF, picofarads. *, p < 0.05; ***, p < 0.001 (paired t test); NS, not significant. B, tachyphylaxis after re-application of capsaicin to cells expressing phosphorylation site mutants. **, p < 0.005 (ANOVA). C, normalized data on desensitization of ICAP by WIN in TRPV1 phosphorylation site mutants. *, p < 0.05; **, p < 0.005 (ANOVA). D–G, typical ICAP traces of capsaicin- and WIN-induced desensitization in wild-type TRPV1 and the phosphorylation site mutants. Durations of drug application are indicated by horizontal bars.
FIGURE 8
FIGURE 8. Acute TRPV1 desensitization is modulated by phosphorylation at Thr144 and Thr370
A, current traces of five separate transiently transfected CHO cells expressing wild-type TRPV1 (WT), TRPV1(T704A), TRPV1(T370A), TRPV1(T144A), or TRPV1(T144A/T370A) upon a 30-s application of 300 nm capsaicin (CAP). Current amplitudes were normalized to that of the largest response to demonstrate differences in desensitization kinetics. B, summary graph of the percentage of acute desensitization measured 10 s after the peak of ICAP was reached. The number of neurons in each trial is indicated within each bar. *, p < 0.05; **, p < 0.01; ***, < 0.005 (ANOVA).
FIGURE 9
FIGURE 9. TRPA1coexpression is necessary for WIN-induced dephosphorylation of TRPV1
CHO cells were transiently transfected with the indicated cDNAs and analyzed for [32P]phosphate incorporation by TRPV1 following WIN (25 µM) or vehicle treatment. Autoradiographic (AutoRad) results (A) were normalized to total immunoprecipitated (IP) TRPV1 (B), with band optical densities quantified and expressed as a percentage of vehicle-treated cells (C). *, p < 0.05 (paired t test; n = 3). Black arrows indicate size markers (in kilodaltons), and gray arrows indicate the immunoreactive band of interest. The results are representative of three independent trials. TRPV1 M, TRPV1(T144A/T370A) mutant; WB, Western blot.
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