Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)
- Dong Ho Woo†1, 2,
- Sung Jun Jung†4,
- Mei Hong Zhu†3,
- Chul-Kyu Park3,
- Yong Ho Kim3,
- Seog Bae Oh3Email author and
- C Justin Lee1, 2Email author
© Woo et al; licensee BioMed Central Ltd. 2008
Received: 03 March 2008
Accepted: 01 October 2008
Published: 01 October 2008
The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1), is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC). However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG) directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG) neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5)P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C.
The capsaicin receptor, TRPV1 (transient receptor potential channel vanilloid subtype 1), is a molecular sensor that detects a wide range of painful stimuli such as capsaicin, heat, and acid in nociceptive sensory neurons [1–4]. Since TRPV1 plays a pivotal role in thermal nociception and inflammatory hyperalgesia [2, 3] and is also widely found in the central nervous system , considerable effort has been made to identify endogenous activators for TRPV1. The products of lipoxygenases, anandamide, and other endocannabinoids [6–11] and even phosphorylation by protein kinase C (PKC)  in the absence of any other agonists have been shown to directly activate TRPV1. However, their roles under physiological condition are still debatable.
Multiple chemical mediators such as bioactive peptides or plasma proteins are generated in inflammatory sites, and many of these mediators heightens the sensitivity of nociceptive sensory neurons after binding to their respective G-protein coupled receptors (GPCR) . Indeed, many Gαq coupled receptors such as bradykinin receptor 2, prostaglandin receptor, protease activated receptor 2, histamine receptor 1, and metabotropic glutamate receptors (mGluR1 and mGluR5), are implicated in sensitization of sensory neurons via TRPV1 modulation during inflammation-induced thermal hyperalgesia [8, 14–18]. Diacylglycerol (DAG) is at the core of GPCR signaling pathway and has been shown to directly activate subfamilies of TRP channels. Mammalian homologues of TRP family (TRPC3, C6 and C7) are activated by DAG [19–21], raising the possibility that DAG directly activates TRPV1. Thus, in the present work, we set out to evaluate the possibility of TRPV1 activation by DAG.
Materials and methods
Cell preparation and transient transfection
Dorsal root ganglia (DRG) were prepared as previously described . Briefly, Sprague-Dawley rat (OrientBio, Korea) was decapitated, and DRG were rapidly removed under aseptic conditions, placed in HBSS (Gibco). DRG were digested in 0.1% collagenase and 1% collagenase/dispase (Boehringer Mammheim) in HBSS for 10 min respectively, followed by 10 min in 0.25% trypsin (Sigma), all at 37°C. DRG were washed in DMEM (Gibco) 3 times and resuspended in F 12 with 10% FBS (Gibco) and 1% penicillin/streptomycin (Sigma). DRG were then mechanically dissociated with fire-polished glass pipettes, centrifuged, resuspended in F12 media, and then plated on polyornithine (Sigma) and laminin (Sigma)-coated glass coverslips. The cells were maintained at 37°C in 5% CO2 incubator. Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) were maintained according to the supplier's recommendations. For transient transfection, cells were seeded in 12-well plates. The next day, 0.5–2 μg/well of pcDNA constructs of TRPV1 or mutants of TRPV1 were transfected into cells using lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. After 18–24 h, cells were trypsinized and used for whole cell recordings and Calcium imaging experiments.
Whole cell currents were recorded using an Axopatch 200A amplifier (Axon Instruments). Patch pipettes were made from borosilicate glass and had resistances of 3–5 MΩ when filled with standard intracellular solutions. For whole cell experiments, we used an external bath medium (normal Tyrode solution) of the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), with pH adjusted to 7.4 using NaOH. Cs+-rich external solution was made by replacing NaCl and KCl with equimolar CsCl. CaCl2 was simply omitted from the external bath medium to produce Ca2+-free PSS. The pipette solution contained (in mM) 140 CsCl, 10 HEPES, 5 EGTA, and 3 MgATP, with pH adjusted to 7.3 using CsOH. All drug solutions were applied to cells by local perfusion through a capillary tube (1.1 mm inner diameter) positioned near the cell of interest. The solution flow was driven by gravity (flow rate, ~1–5 ml/min) and controlled by miniature solenoid valves (The Lee Company, Westbrook, CT). The chamber volume was 400 μl, and the time required to reach the chamber was ~30 s. Latency was the time from arrival time of solution in the chamber to the peak activation of current. Currents were filtered at 5 kHz (-3 dB, 4-pole Bessel), digitized using a Digidata 1322 Interface (Axon Instruments), and analyzed using a personal computer equipped with pClamp 9.0 software (Axon Instruments). The calculated junction potential between the pipette and bath solutions used for all cells during sealing was 4 mV (pipette negative, using pClamp 9.0 software). No junction potential correction was applied. Experiments were performed at room temperature (18–22°C).
Ca2+ imaging experiment was performed as previously described using fura-2AM (Molecular Probes, Eugene, OR, USA) as the fluorescent Ca2+ indicator. Briefly, cells prepared loaded with fura-2 AM (5 μg) mixed with 5 μl of pluronic acid for 40 min at 37°C in a balanced salt solution [BSS; containing (in mM): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose] were plated onto poly-D-lysine-coated coverslips which were mounted onto the chamber (total volume of 500 μl), then placed onto the inverted microscope (Olympus IX70, Japan), and perfused continuously by BSS at a rate of 2 ml/min. All measurements were made at 36°C as controlled by 2-channel temperature controller PTC-20 (ALA Scientific Instrument Inc., USA). Cells were illuminated with a 175W xenon arc lamp, and excitation wavelengths (340/380 nm) were selected by a Lambda DG-4 monochromatic wavelength changer (Sutter Instrument, Novato, CA, USA). Intracellular free Ca2+ concentration ([Ca2+]i) was measured by digital video microfluorometry with an intensified CCD camera (Cascade, Roper Scientific, Trenton, NJ, USA) coupled to a microscope and a Pentium 5 computer with software (Metamorphor, Universal Imaging Corp., PA, USA).
Cobalt uptake measurement
Our cobalt uptake staining was modified from Davis . HEK 293 cells were transfected with TRPV1 and GFP and then cultured on glass coverslips. Cells were washed 3 times with uptake buffer (in mM: Sucrose 232, NaCl 5.8, MgCl2 2, CaCl2 0.25, Glucose 12, Hepes 10). The cells were pretreated for 5 min with the following blockers, 100 μM 6-Iodo nor dihydrocapsaicin (6-cap), 2 μM chelerythrine, 20 μM RHC 80267, and 20 μM capsazepine. Cells were then treated for 15 min with solution containing 5 mM cobalt and various drugs. Then the cells were washed with uptake buffer 6 times. The loaded cobalt ions were precipitated with 0.12% Ammonium Sulfide for 5 min and washed with uptake buffer 3 times. The cells were immediately fixed with 4% paraformaldehyde in PBS for 10 min and then washed 3 times in PBS. To develop the precipitated cobalt, cells were incubated with 2% sodium tungstate in uptake buffer for 10 min. During this time, 0.25% ascorbic acid was prepared freshly and mixed with silver nitrate solution. The mixture solution was exposed to the cells to develop the cobalt precipitate as a dark staining. Cells were finally washed and mounted for photography.
Whole tissue reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from HEK 293 cells and DRG neurons with the use of Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. cDNA was synthesized with the SuperscriptTM Preamplification System (Invitrogen), as previously described . PCR reaction was performed with 2 μl of the resulting cDNA, with the use of Taq DNA polymerase (Invitrogen), and primers for PCR were 'outer' primer.
Single-cell reverse transcription-polymerase chain reaction (RT-PCR)
We adopted methods described by Silbert et al. . Briefly, following Ca2+ imaging experiments, we harvested cells using patch pipettes (tip diameter, 30 μm) filled with autoclaved distilled water under visual control and then the cell was gently put into a reaction tube containing reverse transcription agents. Optionally, to avoid genomic DNA contaminations, a DNase I (for 40 min at 37°C) digest was performed before reverse transcription. After heat inactivation, RT was carried out for 50 min at 50°C (superscript III, Invitrogen RT). Subsequently, the cDNA was divided into four or five 2 μl aliquots that were used in separate PCRs. All PCR amplifications were performed with nested primers. The first round of PCR was preformed in 50 μl of PCR buffer containing 0.2 mM dNTPs, 0.2 μM 'outer' primers, 5 μl of RT product, and 0.2 μl of platinum Taq DNA polymerase (Invitrogen). The protocol included 5 min of initial denaturation at 95°C, followed by 35 cycles of 40 s of denaturation at 95°C, 40 s of annealing at 55°C, 40 s of elongation at 72°C, and was completed with 7 min of final elongation. For the second round of amplification, the reaction buffer (20 μl) contained 0.2 mM dNTPs, 0.2 μM 'inner' primers, 5 μl of the products from the first round, and 0.1 μl of platinum Taq DNA polymerase. The reaction was the same as the first round. The PCR products were then analyzed on an ethidium bromide-stained 2% agarose gel, and photographed using a digital camera.
Point mutant Y511A of TRPV1 was produced with a two step PCR approach from TRPV1 construct which was generated in our lab. In brief, it was generated by a combination of two overlapping PCR fragments, which were constructed from complimentary mutagenic primers. The following primers contained the single point mutation shown in bold (5'-GAGGAGTCGC CTTCTTCTTCC and 5'-GGAAGAAGAAGGC GACTCCTC). TRPV1 mutant of Δ747–838 was produced by one-step PCR-based method. Briefly, the PCR product containing SacII/KpnI sites was obtained with an internal forward primer (CGCTTACAGCAGCAGTGAGACCC) and a KpnI site-stop codon-tailing reverse primer (TATGGTACCTTACAGGGTGCGCTTGACGCCCTC). The pcDNA3.1 (+)/TPRV1 construct digested with SacII/KpnI was ligated with the PCR product. After mutagenesis, the sequences of the final constructs were confirmed by DNA sequencing.
Results are expressed as mean ± SE. Where appropriate, results were compared using 2-tailed Student's t-test (paired or non-paired) or ANOVA test.
OAG induces Ca2+ influx in DRG neurons
OAG-induced Ca2+ transients correlate RNA expression of TRPV1 in each DRG neuron
OAG induces Ca2+ transient and inward currents via TRPV1 in HEK 293
Next, we performed Co2+ uptake assay using TRPV1-expressing HEK 293 cell since this technique has been extensively used as a functional assay for TRPV1 channel activity . We treated 100 μM OAG and measured Co2+ uptake through TRPV1 expressed in HEK 293 cells. We found that, like capsaicin, 100 μM OAG induced Co2+ uptake although with lower intensity and less number of cells (Figure 4B). The Co2+ uptake by OAG and capsaicin was blocked by TRPV1-selective antagonists, including 50 μM 6-iodonordihydrocapsaicin or 10 μM capsazepine (Figure 4B). Taken together, our results support the idea that OAG could activate TRPV1 and OAG-induced responses in DRG neurons are mediated by TRPV1.
OAG-induced TRPV1 activation is PKC- and DAG-lipase independent
Endogenously produced DAG activates TRPV1 channels in a heterologous expression system
OAG binds to the capsaicin binding site
Our study proposes that DAG is a novel endogenous ligand of TRPV1 and regulates Ca2+ signaling in rat DRG neurons. It has been well demonstrated that the activation of TRPV1, via GPCRs for bradykinin, histamine, prostaglandin, and serotonin, is associated with sensitization of peripheral nociceptors during thermal and inflammatory hyperalgesia [8, 15, 16, 31, 32]. Although lipid metabolite products such as HPETE  and anandamide  have been suggested as candidate molecules mediating GPCR-activation of TRPV1, an endogenous ligand for TRPV1 in the physiological condition is still debated. In the present study, we found that endogenous DAG produced by GPCR-activation directly activates TRPV1 even in the presence of PKC and DAG lipase inhibitor (Figure 6a). These responses are readily inhibited by capsazepine, a selective TRPV1 blocker (Figure 1b). However, the efficacy of OAG was much less than that of capsaicin, displaying only one fifth of capsaicin induced responses, suggesting that OAG is acting as a partial agonist. Therefore, in addition to TRPC3 TRPC6, and TRPC7, which are activated by DAG , we demonstrate that TRPV1 is also activated by DAG and other DAG analogues such as OAG, SAG, and DOG. This result suggested that GPCR-coupled DAG could serve as endogenous ligand of TRPV1 in central nervous system as well as in periphery.
It was reported, using single channel studies of inside-out patches, that TRPV1 is activated by several products of lipoxygenase, but minimally by 1-Stearyl-2-arachinonyl-sn-glycerol (SAG), another analog of DAG . This discrepancy might be due to the difference in extracellular Ca2+ levels in the recording conditions and/or the patch clamp recording mode employed. Another report has provided evidence against direct activation of TRPV1 by DAG in a heterologous system utilizing the Chinese Hamster Ovary (CHO) cell line . However, other recent reports demonstrate the complexity of lipid signaling in cell membrane which includes cell-type specificity  and different channel activities depending on the mode of patch clamp recording .
Our results with whole-cell current recordings in heterologous system indicate that the binding site of DAG might be similar to that of capsaicin. In a competition assay, we found that OAG competitively and rapidly replaced the bound capsaicin, causing reduced whole-cell current amplitude (Figure 7d). These results strongly suggest that DAG binds directly to the capsaicin binding site. In addition, both OAG and capsaicin did not induce any current on the mutant form of TRPV1 with S502A/T704I, which represents the PKC and CaMKII phosphorylation sites. However, OAG and capsaicin induced inward current on the mutant with S502A/S800A, representing the PKC binding sites. These results suggest that like capsaicin, phosphorylation by CaMKII is required for the OAG-activation of TRPV1. Interestingly, in Δ774–838 deletion mutant of TRPV1, which lacks PtdIns(4,5)P2 binding site (786–828) (Figure 7b), we observed a bigger inward current by OAG than in wild type TRPV1 (Figure 5Aa) or S502A/S800A mutant (Figure 7b), which is consistent with the previous observation that PtdIns(4,5)P2 binding site displays an inhibitory effect of TRPV1 regulation on low concentration of capsaicin .
In summary, the current study demonstrates that DAG is an endogenous ligand for activating TRPV1 by binding at the capsaicin binding site but by acting independently of PKC and lipoxygenase pathway. Our findings should stimulate future investigations in this new pathway of TRPV1 activation, and promise new ways to develop novel drugs for reducing pain.
We would like to thank Dr. Uhtaek Oh in Seoul National University, Korea, for his kind gift of S502A/S800A and S502A/T704I mutants and Mi Sun Kim, who designed Δ774–838 deletion mutant of TRPV1. This work was supported by grant (S.B.O., R0A-2008-000-20101-0) from National Research Laboratory Program, grant (S.B.O., M103KV010015-08K2201-01510) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology and grants (S.B.O., R01-2004-000-10384-0; S.J.J., R01-2003-000-10737-0) from the Basic Research Program of the Korea Science & Engineering Foundation, Republic of Korea and by Korea Research Foundation (C.J.L., KRF-2005-070-C00096).
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