Protein kinase C phosphorylation sensitizes but does not activate the receptor transient receptor potential vanilloid 1 (TRPV1)

Gautam Bhave*, Hui-Juan Hu*, Kathi S. Glauner*, Weiguo Zhu†‡, Haibin Wang†, D. J. Brasier*, Gerry S. Oxford†‡, and Robert W. Gereau IV*§

*Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030; and †Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599

Edited by Charles F. Stevens, The Salk Institute for Biological Studies, La Jolla, CA, and approved August 8, 2003 (received for review April 16, 2003) Protein kinase C (PKC) modulates the function of the capsaicin nociceptive sensory neurons and integrates noxious thermal and receptor transient receptor potential vanilloid 1 (TRPV1). This chemical stimuli analogous to polymodal nociceptors (9, 10). In modulation manifests as increased current when the channel is response to these stimuli, TRPV1 depolarizes sensory neurons activated by capsaicin. In addition, studies have suggested that and either directly or indirectly initiates peptide release from phosphorylation by PKC might directly gate the channel, because afferent terminals (13). These properties suggest that TRPV1 PKC-activating esters induce TRPV1 currents in the absence plays a critical role in noxious thermal transduction and in of applied ligands. To test whether PKC both modulates and gates neurogenic components of inflammation. Consistent with this, the TRPV1 function by direct phosphorylation, we used direct TRPV1 knock-out mice exhibit reduced detection of strong sequencing to determine the major sites of PKC phosphorylation thermal stimuli and reduced inflammatory thermal hypersensi- on TRPV1 intracellular domains. We then tested the ability of the tivity (14, 15). PKC-activating phorbol 12-myristate 13-acetate (PMA) to potenti- Given the important roles of PKC and TRPV1 in inflamma- ate capsaicin-induced currents and to directly gate TRPV1. We tory hypersensitivity, TRPV1 may act as a PKC substrate after found that mutation of S800 to alanine significantly reduced the tissue injury. In cultured dorsal root ganglion neurons, PKC PMA-induced enhancement of capsaicin-evoked currents and the potentiates heat and capsaicin responses (1, 7, 16–19). In het- direct activation of TRPV1 by PMA. Mutation of S502 to alanine erologous expression systems, studies have shown that PKC reduced PMA enhancement of capsaicin-evoked currents, but had activation enhances TRPV1 function (16, 18, 20, 21). A recent no effect on direct activation of TRPV1 by PMA. Conversely, study has suggested that PKC-activating phorbol esters may mutation of T704 to alanine had no effect on PMA enhancement of directly bind to TRPV1 (22), and a number of studies have capsaicin-evoked currents but dramatically reduced direct activa- suggested that PKC-mediated phosphorylation may cause tion of TRPV1 by PMA. These results, combined with pharmaco- TRPV1 activation (16, 18, 23). In this study, we show that PKC logical studies showing that inactive phorbol esters also weakly activation results in direct phosphorylation of TRPV1. By using activate TRPV1, suggest that PKC-mediated phosphorylation mod- in vitro phosphorylation and protein sequencing techniques, we ulates TRPV1 but does not directly gate the channel. Rather, identified several potential PKC phosphorylation sites on currents induced by phorbol esters result from the combination of TRPV1 intracellular domains. Functional studies suggest that a weak direct ligand-like activation of TRPV1 and the phosphory- activation of TRPV1 by phorbol esters is phosphorylation- lation-induced enhancement of the TRPV1 function. Furthermore, independent but modulated by PKC-mediated phosphorylation. modulation of the TRPV1 function by PKC appears to involve Further, we show that distinct phosphorylation sites are involved distinct phosphorylation sites depending on the mechanism of in the modulation of TRPV1 currents activated by capsaicin and channel activation. phorbol esters.

rotein kinase C (PKC) in peripheral sensory afferents plays Materials and Methods Pa prominent role in hypersensitivity to thermal and mechan- 32P Metabolic Labeling and Immunoprecipitation. COS7 cells were ical stimuli after tissue injury. PKC sensitizes heat responses and transfected with pcDNA3 TRPV1 by using Lipofectamine 2000 potentiates peptide release from cultured dorsal root ganglion (Invitrogen) according to the manufacturer’s protocol 12–24 h neurons (1, 2) and sensitizes nociceptive afferent neurons to after plating onto six-well plates. Cells were rinsed with phos- thermal and mechanical stimuli in intact peripheral nerve prep- phate-free Eagle’s minimal essential medium with Earle’s salts arations (3, 4). Diabetic neuropathic hyperalgesia and epineph- (P-EMEM; Sigma) and incubated with P-EMEM containing 250 rine-induced hyperalgesia are attenuated by PKC inhibitors in ␮Ci͞ml (1 Ci ϭ 37 GBq) [32P]orthophosphate for 4 h. Phorbol vivo (5, 6). Recently, several studies have focused on the role of 12-myristate 13-acetate (PMA; Biomol, Plymouth Meeting, PA) ␧ ␧ the PKC isoform. Specific blockade of PKC diminishes PKC- was added to the medium at 100 nM and incubated for 15 min, mediated enhancement of heat currents in sensory neurons and after which 100 nM PMA dissolved in standard electrophysiol- ␧ epinephrine-induced hypersensitivity in vivo (7, 8). PKC knock- ogy external solution (see Electrophysiology) was applied. Cells out mice exhibit reduced hyperalgesia after intracutaneous were lysed in 500 ␮l of lysis buffer (50 mM NaCl͞50 mM NaF͞25 injection of epinephrine and nerve growth factor (8). Whereas mM sodium phosphate, pH 7.4͞2.5 mM EDTA͞1% Triton a role of PKC in peripheral sensitization is well established, PKC-mediated phosphorylation and modulation of specific sub- strates during peripheral sensitization is not fully understood. This paper was submitted directly (Track II) to the PNAS office. Transient receptor potential vanilloid 1 [TRPV1; formerly Abbreviations: TRPV1, transient receptor potential vanilloid 1; PMA, phorbol 12-myristate known as vanilloid receptor 1 (VR1)] is an attractive PKC 13-acetate. effector in peripheral nociceptors. TRPV1 was cloned as a ‡Present address: Stark Neurosciences Research Institute, Indiana University School of capsaicin receptor and is a ligand-gated ion channel, which is also Medicine, Indianapolis, IN 46202. activated by heat, protons, leukotrienes, and (9– §To whom correspondence should be addressed. E-mail: [email protected]. 12). TRPV1 is specifically localized to small-diameter, primarily © 2003 by The National Academy of Sciences of the USA

12480–12485 ͉ PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2032100100 Downloaded by guest on September 29, 2021 X-100͞10 mM sodium pyrophosphate͞2 mM sodium orthovana- Mutagenesis. TRPV1 mutants were created by using primer date͞0.1 mM PMSF͞10 ␮g/ml leupeptin͞5 ␮g/ml aprotinin͞10 extension by Pfu DNA polymerase (Stratagene) and DpnIto ␮g/ml pepstatin͞1 ␮M microcystin-LR), and the resulting lysate digest the wild-type template as described (26). The entire ORF was centrifuged at 20,800 ϫ g in a microcentrifuge for 15 min at of all constructs was sequenced. 4°C. After preclearing with 25 ␮l of protein A-Sepharose (Pierce), 2–3 ␮g of affinity-purified rabbit anti-TRPV1 C- Electrophysiology. COS7 cells were maintained in DMEM plus ͞ terminal peptide antibody (raised against the epitope CLKPE- 10% FBS at 37°C, under 5% CO2 95% O2, and transfected with DAEVFKDSMVPGEK) was added to the lysate and incubated pCDNA3 TRPV1 or various mutants with pEGFP (9:1) by using overnight at 4°C. Twenty-five microliters of protein A-Sepharose Lipofectamine 2000 according to the manufacturer’s instruc- was incubated with the lysate for3htoprecipitate antigen– tions. Whole-cell patch-clamp recordings were conducted by antibody complexes. Beads were rinsed three times with 1 ml of using standard techniques with an Axopatch 200B amplifier and lysis buffer, twice with high ionic strength buffer (lysis buffer but PCLAMP 8 software at room temperature Ϸ24–36 h after trans- with 500 mM NaCl), and twice with low ionic strength buffer fection. The external solution consisted of (in mM) 140 NaCl, 5 (lysis buffer without NaCl and with sodium phosphate replaced KCl, 2 MgCl2, 5 EGTA, 10 Hepes, and 10 at pH 7.4; by 20 mM Tris–Cl, pH 6.8). Two percent SDS sample buffer whereas the pipette solution contained (in mM) 140 CsCl, 5 was added to the beads and heated for 10 min at 40°C. Samples EGTA, and 10 Hepes at pH 7.4. Cells were voltage-clamped at Ϫ were electrophoresed on SDS͞8% polyacrylamide gels. Radio- 60 mV. Dose–response curves shown in Fig. 4 C–E were labeled TRPV1 was detected by phosphorimaging (Packard obtained from CHO-K1 cells maintained in Ham’s F12 medium Instruments). with other aspects of culture maintenance and transfection as above. Fusion Protein Production. N- and C-terminal cytoplasmic frag- ments (amino acids 1–432 and 686–838, respectively) of TRPV1 Calcium Imaging. COS7 cells grown on poly-D-lysine-coated cov- were cloned into pGEX-4T1 by using PCR with XhoI ends. erslips were transfected 18 h before imaging with TRPV1 and enhanced GFP (9:1). Transfected cells were loaded just before These constructs were transformed into BL21-RP Escherichia ␮ coli bacteria (Stratagene), and fusion protein production was imaging with 250 l of 10 mM fura 2-AM (Molecular Probes) froma1Mstock prepared with 20% Pluronic F-127 (Molecular induced with 0.1 mM isopropyl ␤-D-thiogalactoside for4hat room temperature. Cells were converted to spheroplasts with Probes) in dimethyl sulfoxide (Sigma) diluted in Hanks’ bal- lysozyme (24) and lysed by sonication. The resulting lysate was anced salt solution (Invitrogen) with 10 mM Hepes, pH 7.3, for NEUROSCIENCE clarified by centrifugation, and fusion proteins were purified 1 h at room temperature then rinsed three times with Hanks’ with GSTrap FF columns essentially according to the manufac- balanced salt solution plus Hepes. Cells were then equilibrated for 30 min. Imaging was carried out by using a Hamamatsu Orca turer’s instructions (Amersham Biosciences). After elution with cooled charge-coupled device camera on an Olympus IX70 10 mM reduced glutathione, fusion proteins were concentrated inverted microscope with an Olympus UPlanF1 ϫ10 lens using to 0.5–4mg͞ml by ultrafiltration and centrifuged at 100,000 ϫ SIMPLEPCI software (Compix Imaging Systems, Cranberry Town- g for 60 min to remove protein aggregates and debris. ship, PA). Coverslips containing transfected cells were contin- uously perfused at a rate of 3–4ml͞min. GFP-positive cells were In Vitro Phosphorylation. For stoichiometry estimates, reaction ␮ defined as transfected cells and imaged at room temperature by mixtures consisted of 25 M first intracellular loop peptide ͞ ␮ Ϸ ͞ using ratio imaging with a 380 357 filter set. After acquisition of (CLQRRPSLKSLF) or 1.8 M fusion protein ( 0.1 mg ml), 50 2 min of baseline, cells were perfused with 1 ␮M4␣-PMA mM Tris–Cl at pH 7.5, 1 mM NaEDTA, 12 mM magnesium (Biomol) for 1 min, followed by a 5-min rinse and then by a 1-min acetate, 0.25 mM NaATP, 1 ␮Ci͞␮l[␥-32P]ATP (Ϸ6,000–8,000 ␮ ͞ ͞␮ application of 1 M PMA. (ammoniated ruthe- cpm pmol ATP), and 0.8 ng l catalytically active PKC (Cal- nium oxychloride, 10 ␮M; Sigma), when used, was present in the biochem; Biomol). Reaction mixtures were incubated at 30°C for bath and phorbol ester-containing solutions. Ratio measure- various durations and stopped by spotting onto P81 phospho- ϫ ments were taken for each individual cell with SIMPLEPCI and the cellulose paper (peptide) or by adding an equal volume of 2 peak responses to 4␣-PMA and PMA were determined. These Laemmli sample buffer (fusion proteins). For kinetic analysis, data were analyzed with MICROCAL ORIGIN Version 5.0 (Micro- reaction mixtures were assembled in a similar fashion, but fusion cal Software, Northampton, MA). protein reaction mixtures contained 0.16 ng͞␮l activated PKC and peptide reactions contained 0.1 ng͞␮l activated PKC. Re- Results 32 actions were incubated at 30°C and stopped after 5 min. P To determine whether PKC directly phosphorylates TRPV1, we Ͻ incorporation was linear at time points 10 min. immunoprecipitated TRPV1 from metabolically 32P-labeled P81 paper squares were washed four times with large volumes COS7 cells transfected with TRPV1 to examine TRPV1 phos- of 75 mM phosphoric acid, rinsed quickly with methanol, air- phate incorporation. TRPV1 32P incorporation significantly dried, and placed into 10 ml of scintillation fluid. Fusion proteins increased after a brief incubation with a phorbol ester, PMA (15 ͞ were electrophoresed on SDS PAGE gels and quantified by min, 100 nM), suggesting that PKC directly phosphorylates using Coomassie blue staining and nearby BSA standards. 32P TRPV1 in cultured cells. incorporation was determined by scintillation counting of blot- Given that TRPV1 may act as a PKC substrate, we in vitro ted peptides or phosphorimaging and densitometry of fusion phosphorylated engineered TRPV1 cytoplasmic domains with protein bands (Packard Instruments). PKC to identify candidate phosphorylation sites. Bacterially produced GST fusion proteins of the large N and C termini (Fig. Phosphorylation Site Determination. In-gel digests, HPLC, and 1B) were incubated with and without activated PKC, and phos- Edman sequencing were conducted essentially as described (25). phorimaging revealed that both proteins acted as in vitro sub- For electrospray ionization mass spectroscopy (API 3000 LC͞ strates (Fig. 1C). With our reaction conditions, the N-terminal MS͞MS System, PE Sciex, Thornhill, ON, Canada), peptides fusion protein showed a stoichiometry of Ϸ40% (Fig. 1C), from in-gel digests were desalted by using a C18 ZipTip column whereas the C-terminal protein exhibited a saturating stoichi- (Millipore). Phosphopeptides were initially delineated by using ometry of Ϸ55% (Fig. 1D). a Ϫ79 precursor ion scan and sequenced with tandem mass We used classic HPLC peptide mapping and Edman sequenc- spectrometry. ing to delineate PKC in vitro phosphorylation sites located on the

Bhave et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12481 Downloaded by guest on September 29, 2021 Fig. 2. T704 and S800 identified as in vitro PKC phosphorylation sites on the TRPV1 C terminus. (A) Cerenkov counting of HPLC fractions from an in-gel Lys-C digest of PKC-phosphorylated TRPV1 C-terminal fusion protein reveals two major phosphopeptides. (B) Edman sequencing and scintillation counting Fig. 1. TRPV1 acts as a PKC substrate in cultured cells and in vitro.(A) Bands of the sequencing cycles from the first phosphopeptide fraction delineates represent TRPV1 immunoprecipitated from transiently transfected, metabol- T704 as a phosphorylated site. (C) Scintillation counting of the Edman se- 32 32 ically P-labeled COS7 cells. PMA treatment (right lane) increases TRPV1 P quencing cycles from the second phosphopeptide fraction reveals S800 as an incorporation compared with control (left lane). (B) Coomassie blue staining in vitro PKC phosphorylation site. (D) PKC phosphorylation velocity of the of GST N-terminal (Nterm) and C-terminal (Cterm) TRPV1 fusion proteins TRPV1 C-terminal fusion protein plotted against protein concentration, show- without (Ϫ) or with (ϩ) added PKC (Left) and the corresponding phospho- ing a relatively strong PKC substrate. (E) Lineweaver–Burk plot (inverse veloc- rimage (Right) showing 32P incorporation into fusion proteins only with added ity versus inverse concentration) and linear regression delineates a Km of 7.73 PKC. Reactions were incubated at 30°Cfor1h.(C) Time course of phosphor- Ϫ1 Ϫ1 ␮M and Vmax of 30 nmol⅐min ⅐mg .(F) Kinetic analysis of various mutants ylation for GST TRPV1 N-terminal fusion protein. Phosphorylation stoichiom- plotted alongside wild-type kinetics from D. The decrement observed in the etry is at least 5 pmol͞␮g fusion protein or Ϸ40%. (D) GST TRPV1 C-terminal S800 to alanine mutant is similar to that seen with a mutant fusion protein fusion protein phosphorylation time course showing saturating stoichiometry with all identified PKC phosphorylation sites (T704, S774, S800, and S820) of Ϸ12 pmol͞␮g fusion protein or Ϸ55%. converted to alanine, suggesting that S800 is the predominant substrate.

engineered cytoplasmic domains. Cerenkov counting of the GST PKC phosphorylation sites on the TRPV1 C-terminal fusion C-terminal fusion protein, which was digested with endopro- protein. teinase Lys-C and fractionated by HPLC, revealed two major In-gel digestion with Lys-C followed by HPLC of the N- phosphopeptides (Fig. 2A). Sequencing of these fractions cou- terminal fusion protein revealed a single major phosphopeptide pled with scintillation counting of the sequencing cycles revealed peak (Fig. 3A). Edman sequencing and scintillation counting T704 and S800 as in vitro PKC phosphorylation sites (Fig. 2 B and delineated T144 as the in vitro PKC phosphorylation site (Fig. C). In addition, electrospray ionization mass spectroscopy of 3B). Mutating T144 to alanine eliminates the major phosphor- peptides from an in-gel trypsin digest of the C-terminal fusion ylation peak observed in the wild-type fusion protein, whereas a protein delineated S774 and S820 as minor PKC phosphoryla- small peptide in the HPLC flow-through exhibits increased tion sites (data not shown). phosphorylation (Fig. 3C). This indicates that the major phos- To determine the relative importance of the C-terminal phorylation site was correctly identified as T144. The increased phosphorylation sites, we analyzed the phosphorylation kinetics phosphorylation in the flow-through may be compensatory and of the wild-type and various mutant fusion proteins. Wild-type may explain the relatively high phosphorylation stoichiometry of C-terminal TRPV1 fusion protein acted as a relatively strong the T144A mutant (data not shown). ␮ ⅐ Ϫ1⅐␮ Ϫ1 substrate with a Km of 7.73 M and a Vmax of 30 nmol min g The first intracellular loop contains two serines, and a corre- (Fig. 2 D and E). Significant deficits in reaction velocity occurred sponding peptide exhibited a phosphorylation stoichiometry of Ϸ ␮ ␮ ⅐ Ϫ1⅐ Ϫ1 with S800 mutated to alanine (S800A) and T704A mutants (Fig. 50% with a Km of 2.77 M and Vmax of 7.26 mol min mg 2F). However, little difference in phosphorylation kinetics was (Fig. 3 D and E). Edman sequencing and scintillation counting found between the single S800A mutant and the multiply of the phosphorylated peptide confirmed S502 as the major mutated fusion protein with T704A, S774A, S800A, and S820A phosphorylation site rather than nearby S505 (Fig. 3F). mutations (Fig. 2F). These data suggest that S800 is the most Taken together, the in vitro phosphorylation data identified efficacious or rate-limiting substrate amongst the four identified T144, S502, T704, and S800 as major PKC phosphorylation sites

12482 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2032100100 Bhave et al. Downloaded by guest on September 29, 2021 NEUROSCIENCE

Fig. 3. Identification of phosphorylation sites on the TRPV1 N-terminal fusion protein and first intracellular loop. (A) Cerenkov counting of the HPLC fractions after an in-gel endoproteinase Lys-C digestion of the PKC- phosphorylated N-terminal fusion protein reveals one major phosphopep- tide. (B) Edman sequencing correlated with scintillation counting pinpoints T144 as the phosphorylated site. (C) Cerenkov counting of the HPLC fractions for the T144A mutant TRPV1 N-terminal fusion protein. The major phos- phopeptide is eliminated, indicating correct identification of the major site in the wild-type protein, but the radioactivity peak in the flow-through (fraction 83) is dramatically increased. (D) Time course of 32P transfer to TRPV1 first intracellular loop peptide indicates a saturating stoichiometry of Ϸ50%. (E) Edman sequencing correlated with scintillation counting clearly demarcates S502 as the phosphorylated site rather than S505. (F) Phosphorylation kinetics of the loop peptide with Lineweaver–Burk analysis (Inset) reveals a highly Fig. 4. S502 and S800 mediate PKC modulation of capsaicin-evoked currents. efficacious PKC substrate with a K of 2.77 ␮M and V of 7.26 m max (A) Representative current responses to consecutive applications of 20 nM ␮mol⅐minϪ1⅐mgϪ1. capsaicin (black dots; 10 s) without or with an intervening 1-min application of PMA (100 nM, gray bars) in transiently transfected COS7 cells. (B) Ratio of the second to the initial peak capsaicin response (mean Ϯ SEM) is plotted for most likely to possess functional value. The other sites may wild-type TRPV1 and TRPV1 PKC site mutants in the absence (Ϫ) or presence represent minor in vitro phosphorylation sites with potential (ϩ) of an intervening PMA application. None of the mutants show a significant functional significance. difference compared with wild type without PMA (ANOVA, P ϭ 0.20). Only We constructed alanine or valine mutants of these sites in S502A, S800A, and the double mutant S502A͞S800A show a decrease in PMA TRPV1 and first examined PKC modulation of capsaicin-evoked potentiation compared with wild type (ANOVA followed by post hoc Dun- currents in these mutants by using whole-cell patch-clamp tech- nett’s test; *, P Ͻ 0.05), whereas all of the constructs, except S502A and S502͞800A, exhibit some form of PMA potentiation (ϪPMA versus ϩPMA, niques. As shown by other studies (16, 18, 20, 23), a short ϩ Ͻ treatment of TRPV1-expressing cells with phorbol ester (PMA, unpaired t test; , P 0.05). (C and D) Dose–response relationships for capsaicin are shown under control conditions (F) and after a 3-min pretreat- 100 nM, 1 min) potentiates nondesensitizing TRPV1 currents E Ϸ ment with the PKC activator, PMA (300 nM, ). Data are plotted as a percent- 2-fold (Fig. 4 A and B). When all of the inactivating phos- age of the maximal response elicited by capsaicin (n ϭ 6). C shows represen- phorylation site mutants were treated with PMA in a similar tative traces of two cells showing the currents induced by various doses fashion, only the S502A and S800A mutants exhibited dimin- of capsaicin (indicated below the traces, in nM). (E) PMA enhancement of ished PMA potentiation (Fig. 4 A and B). Whereas the S800A capsaicin-evoked currents is PKC-mediated. Two successive applications of mutant continues to exhibit some PMA potentiation, the S502A 30 nM capsaicin were performed. The second response is potentiated by the mutant shows little, if any, PMA potentiation. Mutating both PKC-activating phorbol ester PMA but not by the inactive phorbol ester ␣ ␣ S502 and S800 to alanine eliminates PMA potentiation similar 4 -phorbol 12,13-didecanoate (4 PDD; 300 nM each). The PMA modulation of capsaicin responses was blocked by the PKC inhibitor bisindolylmaleimide to mutating S502 alone (Fig. 4B). These data suggest that PKC (BIM; 1 ␮M). directly phosphorylates TRPV1 on S502 and S800 to enhance nondesensitizing capsaicin-evoked currents. Essentially identical results were obtained when capsaicin-evoked currents were ϩ (23), we found that activation of PKC by PMA led to a leftward recorded in the presence of external Ca2 and under modestly shift in the capsaicin dose–response curve for activation of rat desensitizing conditions (data not shown). As has been previ- TRPV1 expressed in mammalian cells (Fig. 4 C and D). This ously reported for human TRPV1 expressed in Xenopus oocytes effect of PMA was clearly mediated by PKC activation because

Bhave et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12483 Downloaded by guest on September 29, 2021 were not observed in untransfected cells or cells transfected with GFP alone (Fig. 5A). The activation of TRPV1 by PMA was largely caused by PKC activation, because the response to PMA was dramatically reduced by preincubation with the PKC inhib- itor Ro31-8220 (Fig. 5B). Interestingly, when the non-PKC- activating PMA analogue 4␣-PMA was applied, responses were also observed. Although responses to 4␣-PMA were much smaller than those observed in response to PMA, they were statistically indistinguishable from responses to PMA in the presence of Ro31-8220. By using whole-cell recording, we also observed responses to 4␣-PMA in three of nine cells tested. Again, the average response to 4␣-PMA was smaller than that seen with PMA; the average PMA response was 11.8 ϩ 4.6 pA͞pF (n ϭ 6), whereas responses to 4␣-PMA averaged 4.7 ϩ 1.3 pA͞pF (n ϭ 3). Another inactive analogue of PMA, 4␣- phorbol, did not induce Ca2ϩ influx in TRPV1-transfected cells (not shown). Whereas 4␣-phorbol is frequently used as a neg- ative control analogue for phorbol esters, this compound lacks the fatty acid group seen in PKC-activating phorbol esters. 4␣-PMA most closely approximates the structure of the PKC- activating PMA. Our data suggest that the fatty acid moiety is necessary for the PKC-independent activation of TRPV1 by phorbol esters. It is important to note that Chuang et al. (22) have shown that PMA decreases binding of [3H] to TRPV1, sug- gesting that PMA directly interacts with TRPV1. Taken together Fig. 5. PKC-dependent and -independent effects of phorbol esters on TRPV1. (A) Averaged traces of wild-type TRPV1 responses to 4␣-PMA (1 ␮M) with our data, this finding suggests that there may be two and PMA (1 ␮M) under control conditions or in the presence of Ro31-8220 (1 components involved in the activation of TRPV1 by phorbol ␮M, present in all perfusion solutions) or of ruthenium red (10 ␮M). Lower esters: (i) a direct ligand-like action that accounts for our finding trace shows the response of vector-transfected cells to the same concentra- that 4␣-PMA can activate TRPV1 and (ii) a second phospho- tions of 4␣-PMA and PMA. (B) Population data showing the average peak rylation-dependent enhancement of TRPV1 currents that ac- responses to 4␣-PMA and PMA under control conditions and in the presence counts for a PMA response that is much larger than the response of Ro31-8220 [n ϭ 189 (control) and 145 (Ro31-8220)]. (C) Mutation of T704 elicited by 4␣-PMA. To test these possibilities, we performed a and S800 to alanine residues reduces the response of TRPV1 to PMA to the series of studies examining the activation of TRPV1 PKC ␣ level of the 4 -PMA response, whereas mutation of S502A statistically in- phosphorylation site mutants by 4␣-PMA and PMA. We found, creases the responses to both 4␣-PMA and PMA (n ϭ 103–233 cells). , P Ͻ 0.05. * rather surprisingly, that the enhanced response to PMA com- Asterisks above the bars indicate a significant difference between the 4␣-PMA ␣ and PMA response for a given construct. Asterisks within the bars indicate a pared with 4 -PMA was not dependent on the same amino acid significant difference for the response to 4␣-PMA or PMA compared with the residues that mediate enhancement of capsaicin-evoked re- response of wild-type TRPV1 to the same compound. (D) Capsaicin responses sponses by PKC (Fig. 5C). Specifically, the S800A mutant, which of wild-type and TRPV1 PKC-phosphorylation site mutants are not statistically blocked PMA-induced enhancement of capsaicin responses, also different (n ϭ 38–161). A wild-type control was done on each experimental eliminated the relative enhancement of the response to PMA day to eliminate variability resulting from day-to-day changes in expression. compared with 4␣-PMA. However, the S502A mutant, which The responses of each mutant were then normalized to the size of the also abrogated almost all PMA-induced potentiation of capsaicin ␣ wild-type 4 -PMA response within each day’s experiment. Statistical analyses responses, did not eliminate the relative enhancement of the were performed by using a one-way ANOVA followed by a Tukey’s post hoc ␣ test. response to PMA compared with 4 -PMA. Furthermore, the T704A mutant, which had no effect on the modulation of capsaicin responses by PMA, also completely eliminated the an inactive phorbol ester, 4␣-phorbol 12,13-didecanoate, did not relative enhancement of the response to PMA compared with ␣ enhance capsaicin-evoked currents (Fig. 4E). Furthermore, the 4 -PMA. These findings suggest that PKC regulates the activa- tion of TRPV1 differentially depending on the ligand used for enhancement of capsaicin-evoked currents by PMA was blocked ␣ by the PKC inhibitor bisindolylmaleimide (1 ␮M; Fig. 4E)orby activation. The responses to both 4 -PMA and PMA in the inclusion of the PKC-inhibiting peptide PKC in the record- S502A mutant were significantly larger than those for wild-type 19–31 TRPV1 or any of the other mutants (Fig. 5C), although the Ca2ϩ ing pipet (data not shown). responses to capsaicin for S502A did not differ from wild type Previous studies have shown that TRPV1 can be directly or the other mutants (Fig. 5D). The reason for this discrepancy activated by PKC-activating phorbol esters (16, 18, 23). In is not clear. TRPV1-transfected cells, we observed inward currents in re- sponse to application of PMA in only 6 of 13 cells (data not Discussion shown). Because of the low percentage of cells that responded PKC modulation of TRPV1 function has recently become an to phorbol ester application, we chose to measure phorbol ester active, yet confusing, field with several proposed mechanisms. 2ϩ activation of TRPV1 by using Ca imaging, with which we can Premkumar and Ahern (16) reported that PMA not only po- measure the responses of many individual cells simultaneously. tentiates capsaicin-activated TRPV1 currents but also activates We have previously shown that native TRPV1 responses in TRPV1 itself in TRPV1-expressing Xenopus oocytes. Neither of dorsal root ganglion neurons can be faithfully recorded by using these effects was replicated by the inactive 4␣-phorbol, and both calcium imaging (19). We found that cells transfected with effects were inhibited by a PKC inhibitor. However, subsequent TRPV1 responded to application of PMA with a rise in intra- studies have disputed and extended some of the initial findings. ϩ ϩ cellular Ca2 (Fig. 5). Ca2 responses to PMA were completely First, direct activation of TRPV1 by phorbol esters is quite blocked by the TRPV1 antagonist ruthenium red (10 ␮M) and variable. One group reported direct activation in transiently

12484 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2032100100 Bhave et al. Downloaded by guest on September 29, 2021 transfected HEK293 cells, but not in stably transfected cells (18). both phorbol ester binding and PKC-mediated phosphorylation Second, direct activation of TRPV1 appears to have a higher are required to activate and͞or potentiate TRPV1 currents. EC50 than phorbol ester-mediated potentiation of TRPV1- Our studies using PKC phosphorylation site mutants may help mediated currents. Whereas 100 nM phorbol 12,13-dibutyrate or clarify the multiple mechanisms involved in the activation of 200 nM PMA maximally potentiates TRPV1 currents, at least 1 TRPV1 by phorbol esters. First, we show that TRPV1 can be ␮M phorbol 12,13-dibutyrate or PMA appears to be required to activated by 4␣-PMA, which does not activate PKC, suggesting observe direct activation (data not shown) (16, 18, 20, 23). Third, that phorbol esters can gate TRPV1 either directly or through phorbol esters have been found to directly displace resinifera- some other PKC-independent mechanism. Second, our data toxin binding to TRPV1 (22), further obfuscating the issue of argue that PKC-mediated phosphorylation of TRPV1 enhances whether phorbol ester-mediated activation of TRPV1 involves currents activated by PMA by means of an overlapping mech- phosphorylation by PKC or an agonist-like action. Fourth, anism distinct from that involving phosphorylation-dependent phorbol ester potentiation of capsaicin-evoked currents is vari- enhancement of capsaicin-evoked currents. We found that the able. We and others report a relatively ephemeral 2-fold poten- enhancement of currents gated by either PMA or capsaicin was tiation (18), whereas others obtain extremely robust 5- to 10-fold eliminated when S800 was mutated, whereas a mutation at S502 potentiation of capsaicin-evoked currents (16, 20, 21, 23). Many reduced PKC potentiation of capsaicin-evoked currents but not of the disparities may simply reflect different expression systems, PMA-evoked currents. Similarly, a mutation at T704 dramati- culture conditions, and drug application paradigms. For in- cally reduces the PKC-dependent enhancement of PMA- ␣ stance, it has been reported recently that expression of the mediated activation of TRPV1 but has no effect on PKC- isoform of PKC correlates with the ability of phorbol esters to dependent enhancement of capsaicin-evoked currents. The directly activate TRPV1 (27). Other studies clearly implicate an reason for this distinction is not clear, but likely involves different important role for PKC␧ in modulation of TRPV1 (21). Thus, it ␣ ␧ mechanisms of channel-gating induced by phorbol esters and is possible that expression systems vary in PKC and PKC capsaicin. In general, the results suggest a model in which expression, leading to variable phorbol ester activation of phosphorylation of S800 by PKC is a necessary step for modu- TRPV1. Ultimately a more important question is whether PKC␣ ␧ lation of TRPV1 function by PKC, and phosphorylation of S502 and PKC play different roles in modulating sensory transduc- or T704 is additionally required, depending on the mechanism of tion by TRPV1. channel activation. Whereas this model is the best fit for our However, at the crux of the matter lies the issue of whether

data, it is important to keep in mind that TRPV1 is a polymodal NEUROSCIENCE phorbol esters activate and͞or potentiate TRPV1 through direct receptor, and, although under our conditions we saw no evidence binding or by activation of PKC or both. Whereas phorbol ester for direct gating of the channel by PKC-mediated phosphory- binding to TRPV1 appears to support an agonist or allosteric lation, it is possible that at higher temperatures or under slightly modulator model, experiments involving PKC catalytic inhibi- different recording conditions phosphorylation of TRPV1 by tors that compete with ATP or substrate, rather than diacyl- PKC could directly lead to channel gating. glycerol, point to phosphorylation. The data here show that phosphorylation contributes to phorbol ester potentiation of 32 We thank Dr. Richard Cook and the Baylor College of Medicine Protein TRPV1 currents because phorbol esters increase P incorpo- Core Facility for assistance with Edman sequencing and mass spectros- ration into TRPV1, and mutations at S502 and S800 eliminate copy, and Dr. David Julius for kindly providing rat TRPV1 cDNA. This phorbol ester potentiation of capsaicin-evoked currents. The work was supported by grants from the National Institutes of Health findings with the mutants agree with previous reports (21). (R01MH60230 and R01NS42595 to R.W.G., F32MH65766 to K.S.G., However, the previous findings do not address phorbol ester and R01NS18788 and P01NS39420 to G.S.O.). G.B. is a McNair Scholar activation of TRPV1, nor do they eliminate the possibility that of the Baylor College of Medicine Medical Scientist Training Program.

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