Title: Biphasic Currents Evoked by Chemical or Thermal Activation of the Heat-gated Ion Channel, TRPV3
Abstract: 2-Aminoethyl diphenylborinate was recently identified as a chemical activator of TRPV1, TRPV2, and TRPV3, three heat-gated members of the transient receptor potential vanilloid (TRPV) ion channel subfamily. Here we demonstrated that two structurally related compounds, diphenylboronic anhydride (DPBA) and diphenyltetrahydrofuran (DPTHF), can also modulate the activity of these channels. DPBA acted as a TRPV3 agonist, whereas DPTHF exhibited prominent antagonistic activity. However, all three diphenyl-containing compounds promoted some degree of channel activation or potentiation, followed by channel block. Strong TRPV3 activation by DPBA often leads to the appearance of a secondary, enhanced, current phase. A similar biphasic response was observed during TRPV3 heat stimulation; an initial, gradually sensitizing phase (I1) was followed by an abrupt transition to a secondary phase (I2). I2 was characterized by larger current amplitude, loss of outward rectification, and alterations in the following properties: permeability among cations; ruthenium red and DPTHF sensitivity; temperature dependence; and voltage-dependent gating. The I1 to I2 transition depended strongly on TRPV3 current density. Removal of extracellular divalent cations resulted in heat-evoked currents resembling I2, whereas mutation of a putative Ca2+-binding residue in the pore loop domain, aspartate 641, facilitated detection of the I1 to I2 transition, suggesting that the conversion to I2 resulted from the agonist- and time-dependent loss of divalent cationic inhibition. Primary keratinocytes overexpressing exogenous TRPV3 also exhibited biphasic agonist-evoked currents. Thus, strong activation by either chemical or thermal stimuli led to biphasic TRPV3 signaling behavior that may be associated with changes in the channel pore. 2-Aminoethyl diphenylborinate was recently identified as a chemical activator of TRPV1, TRPV2, and TRPV3, three heat-gated members of the transient receptor potential vanilloid (TRPV) ion channel subfamily. Here we demonstrated that two structurally related compounds, diphenylboronic anhydride (DPBA) and diphenyltetrahydrofuran (DPTHF), can also modulate the activity of these channels. DPBA acted as a TRPV3 agonist, whereas DPTHF exhibited prominent antagonistic activity. However, all three diphenyl-containing compounds promoted some degree of channel activation or potentiation, followed by channel block. Strong TRPV3 activation by DPBA often leads to the appearance of a secondary, enhanced, current phase. A similar biphasic response was observed during TRPV3 heat stimulation; an initial, gradually sensitizing phase (I1) was followed by an abrupt transition to a secondary phase (I2). I2 was characterized by larger current amplitude, loss of outward rectification, and alterations in the following properties: permeability among cations; ruthenium red and DPTHF sensitivity; temperature dependence; and voltage-dependent gating. The I1 to I2 transition depended strongly on TRPV3 current density. Removal of extracellular divalent cations resulted in heat-evoked currents resembling I2, whereas mutation of a putative Ca2+-binding residue in the pore loop domain, aspartate 641, facilitated detection of the I1 to I2 transition, suggesting that the conversion to I2 resulted from the agonist- and time-dependent loss of divalent cationic inhibition. Primary keratinocytes overexpressing exogenous TRPV3 also exhibited biphasic agonist-evoked currents. Thus, strong activation by either chemical or thermal stimuli led to biphasic TRPV3 signaling behavior that may be associated with changes in the channel pore. Four members of the transient receptor potential vanilloid (TRPV) 1The abbreviations used are: TRPV, transient receptor potential ion channel vanilloid; 2-APB, 2-aminoethyl diphenylborinate; TRPM; transient receptor potential ion channel melastatin; DPBA, diphenylboronic anhydride; DPTHF, 2,2-diphenyltetrahydrofuran; HEK, human embryonic kidney; [Ca2+]i, free intracellular Ca2+ concentration; NMDG, N-methyl-d-glucamine; GFP, green fluorescent protein; YFP, yellow fluorescent protein; RR, ruthenium red; DVC, divalent cation; DVF, divalent cation free; pF, picofarad; CI, confidence interval. protein subfamily have been identified as heat-gated nonselective cation channels. TRPV1 and TRPV2 are activated by noxious heat at temperatures above ∼42 and 52 °C, respectively (1Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. 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TRPV4, in contrast, can be activated by the synthetic phorbol ester, 4α-phorbol didecanoate, or by 5′,6′-epoxyeicosatrienoic acid, a cytochrome P450 metabolite of arachidonic acid (11Nilius B. Vriens J. Prenen J. Droogmans G. Voets T. Am. J. Physiol. 2004; 286: C195-C205Crossref PubMed Scopus (370) Google Scholar). Recently, another small organic molecule, 2-aminoethyl diphenylborinate (2-APB), was shown to activate TRPV3, both in heterologous expression systems and in keratinocytes expressing this channel endogenously (12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Neurosci. 2004; 24: 5177-5182Crossref PubMed Scopus (251) Google Scholar, 13Hu H.Z. Gu Q. Wang C. Colton C.K. Tang J. Kinoshita-Kawada M. Lee L.Y. Wood J.D. Zhu M.X. J. Biol. Chem. 2004; 279: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). At higher concentrations, this same compound can activate TRPV1 and TRPV2 but not TRPV4 (13Hu H.Z. Gu Q. Wang C. Colton C.K. Tang J. Kinoshita-Kawada M. Lee L.Y. Wood J.D. Zhu M.X. J. Biol. Chem. 2004; 279: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). 2-APB was initially demonstrated to act as an inhibitor of inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular stores (14Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (774) Google Scholar). Subsequent studies revealed that this compound could also block store-operated calcium entry, which is believed by some investigators to be mediated by TRP channels (15Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar), independent of inositol 1,4,5-trisphosphate receptors, and that under certain circumstances, it could paradoxically potentiate both of these activities (16Braun F.J. Broad L.M. Armstrong D.L. Putney Jr., J.W. J. Biol. 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In addition, 2-APB has been reported to activate an unidentified nonselective cation channel in RBL-2H3 m1 rat basophilic leukemia cells (23Braun F.J. Aziz O. Putney Jr., J.W. Mol. Pharmacol. 2003; 63: 1304-1311Crossref PubMed Scopus (47) Google Scholar) and to inhibit volume-regulated anion channels (24Lemonnier L. Prevarskaya N. Mazurier J. Shuba Y. Skryma R. FEBS Lett. 2004; 556: 121-126Crossref PubMed Scopus (28) Google Scholar). Two structural analogs of 2-APB, diphenylboronic anhydride (DPBA) and 2,2-diphenyltetrahydrofuran (DPTHF), have also been reported to inhibit store-operated calcium entry (18Dobrydneva Y. Blackmore P. Mol. Pharmacol. 2001; 60: 541-552PubMed Google Scholar). However, the activity profiles of these compounds are not identical. For example, although 2-APB inhibits volume-regulated anion channels, DPTHF apparently does not (24Lemonnier L. Prevarskaya N. Mazurier J. Shuba Y. Skryma R. FEBS Lett. 2004; 556: 121-126Crossref PubMed Scopus (28) Google Scholar). Given these findings, we sought to determine whether and how DPBA and DPTHF might modulate the TRPV3 activity. Here we report that, like 2-APB, DPBA activates recombinant TRPV3. In contrast, DPTHF inhibits TRPV3 activation by either of these compounds. We also find that repetitive or prolonged activation of TRPV3 with either DPBA or heat evokes not only an initial phase, consisting of gradually sensitizing, outwardly rectifying currents, but also a subsequent phase, characterized by a much higher current amplitude and absence of outward rectification. Further analysis reveals that changes in both voltage-dependent gating and divalent cation block may contribute to these time-dependent changes in heat-evoked TRPV3 currents. DNA Constructs, Cell Culture, and Transfections—Unless otherwise noted, molecular biology and cell culture reagents were obtained from Invitrogen or New England Biolabs (Beverly, MA), and chemicals were from Sigma. Cell culture and transfection with Lipofectamine 2000 (Invitrogen) were performed as described previously (6Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar). A TRPV3-YFP cDNA encoding mouse TRPV3 (25Chung M.K. Lee H. Caterina M.J. J. Biol. Chem. 2003; 278: 32037-32046Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) with yellow fluorescent protein (YFP) fused onto its C terminus was subcloned into pcDNA3 (Invitrogen) and transfected into human embryonic kidney (HEK) 293 cells (gift of J. Nathans, The Johns Hopkins University). A single G418 (500 μg/ml)-resistant clone exhibiting a high level of YFP fluorescence and TRPV3 immunoreactivity was used for Ca2+ imaging studies. Stable transfectants of rat TRPV1, rat TRPV4, and pcDNA3 alone have been described previously (6Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar). HEK 293 cells expressing SV40 large T-antigen were transiently co-transfected with cDNA encoding mouse TRPV2 (25Chung M.K. Lee H. Caterina M.J. J. Biol. Chem. 2003; 278: 32037-32046Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) or pcDNA3 and green fluorescent protein (GFP). For whole-cell voltage clamp experiments, HEK 293/T-antigen cells were transiently transfected with the mammalian expression vector, pTracer (Invitrogen), or pcDNA3 containing mouse TRPV3 cDNA. In TRPV3-pTracer, TRPV3 expression is driven by a cytomegalovirus promoter and GFP-Zeocin expression by an EM-7 promoter. pTracer without TRPV3 was used for vector control experiments. Site-directed mutagenesis of TRPV3 was performed by PCR using complementary synthetic oligonucleotides encoding the mutation of interest and Proofstart high fidelity polymerase (Qiagen, Valencia, CA). The resulting cDNA, subcloned into pcDNA3, was co-transfected with GFP. Cells were replated 15–18 h post-transfection on polyornithine-coated glass coverslips and subjected to patch clamp recording 2 h later. Transiently transfected cells were identified by GFP fluorescence. For most experiments, primary keratinocytes were cultured from newborn mice (postnatal day 1–3) as described previously (12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Neurosci. 2004; 24: 5177-5182Crossref PubMed Scopus (251) Google Scholar, 26Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M. J. Biol. Chem. 2004; 279: 21569-21575Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), plated at 105/cm2 on uncoated glass coverslips, and assayed after 40–80 h at 37 °C, 5% CO2. In several cases, we used an alternative protocol incorporating 2.5% dispase instead of trypsin in an effort to increase the expression level of native TRPV3. In this protocol, cells collected after proteolysis were centrifuged through a bovine serum albumin column (3 g/20 ml of media) and then resuspended and cultured in Serum-free Keratinocyte Medium (Invitrogen) supplemented with 50 μg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor, and 0.05 mm CaCl2. The cells were plated on polyornithine/laminin/collagen IV-coated coverslips and recorded after 40–60 h. Keratinocyte transfection was performed 24 h after cell plating using Lipofectamine 2000 (Invitrogen), and voltage clamp was performed after an additional 18 h. Ca2+ Imaging—Ca2+ imaging bath solution contained (in mm) 130 NaCl, 3 KCl, 2.5 CaCl2, 0.6 MgCl2, 10 HEPES, 1.2 NaHCO3, and 10 glucose, adjusted to pH 7.45 with NaOH. Cells were loaded with fura 2-AM as described previously (6Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar). Ratiometric Ca2+ imaging was performed using an upright fluorescence microscope (Nikon, Melville, NY), excitation filter changer (Ludl, Hawthorne, NY), and charge-coupled device camera (Coolsnap ES, Roper, Tucson, AZ). Pairwise images (340 and 380 nm excitation, 510 nm emission) were collected at 2–10-s intervals, and the data were analyzed using Ratio Tool software (Isee Imaging, Raleigh, NC) as described previously (6Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar). For 50 cells from each coverslip, fura ratios (emission at 340 nm excitation/emission at 380 nm excitation) were calculated, base line-subtracted, and averaged. Data from ≥4 independent coverslips were used to calculate mean ± S.E. for each experimental group. Patch Clamp Electrophysiology—Unless otherwise indicated, in whole-cell voltage clamp experiments using 2-APB, DPBA or DPTHF, recording pipettes were filled with “low Cl– pipette solution” containing (in mm) 120 cesium aspartate, 10 CsCl, 1 MgCl2, 5 EGTA, and 10 HEPES (pH 7.4 with CsOH). Cells were superfused with “low Cl– bath solution” containing (in mm) 130 sodium aspartate, 6 NaCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES (pH 7.4 with NaOH). Standard bath solution for recording heat-evoked currents contained (in mm) 140 NaCl, 10 glucose, 1 CaCl2, 0.5 MgCl2, 10 HEPES (pH 7.4 with NaOH). Standard pipette solution contained (in mm) 140 NaCl, 10 HEPES, 5 EGTA (pH 7.4 with NaOH). In the experiment evaluating the voltage dependence of heat-evoked currents, NaCl in the bath and pipette solutions was reduced from 140 to 40 mm with equimolar substitution of N-methyl-d-glucamine (NMDG) to minimize the series resistance error derived from the large current amplitudes. To evaluate the relative permeabilities of NMDG and Ca2+ to Na+, we first measured the reversal potential (Erev) using a bath solution containing 140 NaCl, 10 HEPES (pH 7.4 with NaOH) combined with the standard pipette solution. Then we measured the Erev change using a solution containing 140 NMDG-Cl, 10 HEPES (pH 7.4 with HCl) for NMDG permeability, or 140 NaCl, 5 CaCl2, 10 HEPES (pH 7.4 with NaOH) for Ca2+ permeability. Unless otherwise indicated, keratinocytes were recorded in whole-cell voltage clamp mode using pipette solution containing (in mm) 140 CsCl, 1 MgCl2, 10 HEPES, 5 EGTA (pH 7.4 with CsOH) and the standard bath solution. An Axopatch 200B or Multiclamp 700A amplifier was used with pClamp 9 software (Axon Instruments, Union City, CA). Borosilicate glass electrodes had tip resistances of 2–4 megohms. The series resistance was usually in the range of 4–7 megohms, which was not compensated. Recording was aborted if this value exceeded 10 megohms. In the experiment evaluating voltage dependence, series resistance compensation was performed to ∼75%. A 3 m KCl agar salt bridge was used throughout the experiments. Bath temperature was controlled by using an in-line heater, alone or together with an in-line heater/cooler (Warner Instruments, Hamden, CT) and monitored continuously with a thermistor (Physitemp, Clifton, NJ). The bath was perfused throughout the experiment and drug and temperature stimulus application was performed using an automated valve manifold (Warner Instruments, Hamden, CT, or Automate Scientific Inc, San Francisco). Data Analysis—Data are expressed as mean ± S.E. Unless otherwise indicated, statistical comparisons were made using unpaired Student's t test. Concentration-response relations were fitted using Prism software (Graphpad, San Diego) with the following equations: y = Emax/(1 + (EC50/x)H) for 2-APB and DPBA or y = Imax/(1 + (IC50/x)H) for DPTHF, where Emax or Imax is maximal agonistic or inhibitory effect, respectively; x is agonist or antagonist concentration; H is the Hill coefficient; and EC50 or IC50 is half-maximal effective concentration for activation or inhibition, respectively. In the case of DPTHF, separate fits were conducted from 1 to 32 μm and from 32 μm to 1 mm. Linear regression fits (y = mx + b, where m is the slope and b is the intercept) were achieved with Clampfit 9 (Axon Instruments) or Prism software. Curves plotting the relationship between voltage and tail current amplitude were fitted using the Boltzmann equation: I = Imin + (Imax – Imin)/(1 + exp((V½ – V)/k)), where Imin is the minimum current amplitude; Imax is the maximum current amplitude; V½ is the potential at which I is halfway between Imin and Imax; and k is the slope. To calculate cationic permeabilities relative to Na+, we used the following equations: PNMDG/PNa = exp((F/RT)(ENMDG – ENa)), and PCa/PNa = {[Na+]/4[Ca2+]} {exp((F/RT)(ECa – ENa))–1} {1 + exp(ECa(F/RT))}, where P is the permeability of the indicated ion; F is Faraday's constant; R is the gas constant; T is absolute temperature; and E is the reversal potential for the ion indicated by the subscript, corrected for liquid junction potential calculated in pClamp 9. To explore the effects of 2-APB structural analogs on TRPV3, we performed fura 2 microscopic fluorescent Ca2+ imaging on HEK 293 cells stably expressing mouse TRPV3 bearing a C-terminal yellow fluorescent protein tag (TRPV3-YFP). DPBA (100 μm, Fig. 1, A and C), like 2-APB (100 μm, Fig. 1, A and B), evoked robust free intracellular Ca2+ concentration ([Ca2+]i) increases in these cells at room temperature that reversed upon washout (mean change in fura 340 nm/380 nm excitation ratio, 0.73 ± 0.07 units, n = 7 coverslips). No such responses were observed in cells stably transfected with the control vector pcDNA3 (0.02 ± 0.01 fura ratio units, n = 4 coverslips, p < 10–4 versus TRPV3-YFP, unpaired Student's t test) or in TRPV3-YFP cells treated with vehicle alone (0.1% Me2SO, not shown). In contrast to the other two compounds, DPTHF (100 μm) failed to evoke a detectable increase in [Ca2+]i in cells expressing TRPV3-YFP. However, in the presence of DPTHF, responses to 100 μm 2-APB were reduced by 73.2% (0.18 ± 0.01 ratio units, n = 4, versus 0.65 ± 0.13 ratio units, n = 8, in the absence of DPTHF, p < 0.01, Fig. 1, A and B). DPTHF also produced a robust (93.2%) inhibition of 100 μm DPBA-induced responses (0.05 ± 0.02 ratio units, n = 6, p < 10–5 versus DPBA alone, Fig. 1, A and C). Reportedly, TRPV1 and TRPV2 can also be activated by higher concentrations of 2-APB, whereas TRPV4 cannot (12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Neurosci. 2004; 24: 5177-5182Crossref PubMed Scopus (251) Google Scholar, 13Hu H.Z. Gu Q. Wang C. Colton C.K. Tang J. Kinoshita-Kawada M. Lee L.Y. Wood J.D. Zhu M.X. J. Biol. Chem. 2004; 279: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Confirming these results, upon stimulation with 320 μm of 2-APB, we observed a strong rise in [Ca2+]i in HEK 293 cells stably expressing rat TRPV1 (1.36 ± 0.10 ratio units, p < 0.001 versus pcDNA3 control, n = 4) or transiently expressing rat TRPV2 (0.58 ± 0.04 ratio units, p < 0.01 versus pcDNA3 control, n = 4) (Fig. 1B). Therefore, we tested whether DPBA or DPTHF could activate or inhibit these other channels. DPBA (100 μm) evoked a robust rise in [Ca2+]i in TRPV1-expressing cells (0.77 ± 0.06 ratio units, n = 8, p < 10–6 versus pcDNA3 control, n = 4) or TRPV2-expressing cells (0.99 ± 0.03, n = 4, p < 10–4 versus pcDNA3 control, n = 4) (Fig. 1, A and C). In HEK 293 cells stably expressing rat TRPV4, even at 500 μm, DPBA failed to evoke [Ca2+]i increases distinguishable from the small background responses of stable pcDNA3 transfectants (Fig. 1, A and C). When DPTHF (100 μm) was included in the bath solution during DPBA stimulation of TRPV1- or TRPV2-expressing cells, the sizes of the responses were not altered significantly (data not shown). However, at a higher concentration (500 μm) DPTHF produced a modest inhibition of these channels (TRPV1, 25.2% inhibition, n = 4, p < 0.05; TRPV2, 33.2% inhibition, n = 4, p < 0.001), (Fig. 1, A and C). Given the relatively limited DPTHF activity at these two channels, we focused our subsequent analyses on TRPV3. In order to confirm that TRPV3 can be activated by DPBA and that this activity is inhibited by DPTHF, we performed whole-cell voltage clamp experiments on wild-type mouse TRPV3 transiently transfected into HEK 293 cells. In these cells, both 2-APB and DPBA evoked robust currents with Erev near 0 mV in current-voltage (I-V) traces (Fig. 2, A, B, F, and G). In both cases, response amplitudes increased, and outwardly rectifying initial responses became dually rectifying with successive drug applications. No such current responses were observed in cells transiently transfected with the control vector, pTracer (–3.7 ± 1.4 pA/pF at +80 mV and 0.2 ± 0.2 pA/pF at –80 mV, n = 5 versus TRPV3-expressing cells 247.2 ± 33.7 pA/pF at +80 mV and –59.1 ± 8.9 pA/pF at –80 mV, n = 23, p < 10–5). Likewise, inclusion of DPBA (100 μm) in the pipette resulted in no detectable current increase in cells expressing TRPV3 (data not shown), suggesting an extracellular site of action for this drug, as reported previously for 2-APB (13Hu H.Z. Gu Q. Wang C. Colton C.K. Tang J. Kinoshita-Kawada M. Lee L.Y. Wood J.D. Zhu M.X. J. Biol. Chem. 2004; 279: 35741-35748Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). As in the Ca2+-imaging assay, both 2-APB- and DPBA-evoked currents were reversibly inhibited by DPTHF (2-APB, 50.7 and 81.4% inhibition; DPBA, 58.9 and 90.8% inhibition at +80 and –80 mV, respectively, n = 6 to 11) (Fig. 2, C, D, F, and G). The DPBA-evoked inward current response was also robustly inhibited by 10 μm ruthenium red (RR; 99% less than control at –80 mV, p < 0.0001, n = 6), an organic cation known to block influx through many cation channels, including most TRPV subtypes (Fig. 2, E and G). However, as reported previously (12Chung M.K. Lee H. Mizuno A. Suzuki M. Caterina M.J. J. Neurosci. 2004; 24: 5177-5182Crossref PubMed Scopus (251) Google Scholar), RR alone evoked outward currents in TRPV3-expressing cells. Moreover, DPBA-evoked outward currents were inhibited only slightly by the presence of this compound (24.5% less than control at +80 mV, p < 0.05, n = 6). To characterize DPBA-evoked TRPV3 responses more quantitatively, we applied varying concentrations of 2-APB or DPBA for 2 min to cells transiently expressing TRPV3, and we plotted the maximal current amplitude during the stimulus at ±80 mV (Fig. 3A). Responses to each compound were evident at concentrations as low as 10 μm. With increasing concentration, the rising phase of these responses became steeper and the plateau higher. Careful inspection of the resulting concentration-response profiles (Fig. 3B) revealed that, at low concentrations, currents evoked by 2-APB and DPBA were similar in amplitude. However, at 100 μm, responses to DPBA (336.1 ± 29.1 pA/pF at +80 mV and –188.9 ± 24.7 pA/pF at –80 mV) were significantly larger than those to 2-APB (166.0 ± 58.7 pA/pF at +80 mV and –67.5 ± 35.7 pA/pF at –80 mV, n = 9, p < 0.05). Above 100 μm, DPBA-evoked responses tended to reach an apex and then decline in mid-response (Fig. 3A). This effect became more evident with increasing concentrations of DPBA and led to an apparent decline in maximal current amplitude at 1 mm. This decline in response amplitude was also observed in 2-APB-treated cells, but only at 1 mm. To minimize the impact of this phenomenon on our estimate of drug potency, we excluded the 1 mm DPBA data point during curve-fitting. The resulting concentration-response profiles yielded EC50 values for DPBA (64.1 μm at +80 mV, 95% confidence interval (95% CI) 43.7 to 94.1 μm; and 85.1 μm at –80 mV, 95% CI 61.1 to 118.6 μm) that were comparable with those for 2-APB (90.6 μm at +80 mV, 95% CI 52.0 to 157.9 μm; and 165.8 μm at –80 mV, 95% CI 71.3 to 386.0 μm), with similar Hill coefficients (DPBA, 2.46 and 2.75 at +80 and –80 mV, respectively; 2-APB, 1.69 and 1.68). Whereas the calculated values for maximal DPBA responses (Emax, 448 pA/pF at +80 mV, –310 pA/pF at –80 mV) were slightly greater than those for responses evoked by 2-APB (321 pA/pF at +80 mV, –232 pA/pF at –80 mV), 95% CI for these two values overlapped. To investigate further the inhibition of TRPV3 by DPTHF, we applied 100 μm DPBA to cells expressing TRPV3, allowed responses to reach steady state, then superimposed varying concentrations of DPTHF (Fig. 3C). Under these conditions, DPTHF concentration-dependently inhibited DPBA-evoked currents. However, this concentration dependence extended over several orders of magnitude, with two apparent kinetic components (Fig. 3D). Accordingly, we fitted the response profiles at +80 mV and –80mV with two separate curves each, using 32 μm (10–4.5 in Fig. 3D) as the boundary between the two components. At –80 mV, this fit yielded IC50 values of 6.0 μm for the initial component and 151.5 μm for the secondary component, with Hill coefficients of 2.40 and 2.26, respectively. At +80 mV, IC50 values were 10.0 μm for the initial component and 226.7 μm for the secondary component with Hill coefficients of 1.17 and 1.34, respectively (Fig. 3D). This kinetic pattern suggests that DPTHF has two or more sites and/or mechanisms of action on TRPV3. To analyze further DPBA-evoked TRPV3 responses, we applied sequentially increasing concentrations of DPBA to a given TRPV3-expressing cell (Fig. 4, A–C). DPBA evoked currents in a concentration-dependent manner (EC50 = 38.8 μm at +80 mV, 95% CI 18.4 to 82.0 μm; and EC50 = 38.7 μm at –80 mV, 95% CI 24.7 to 60.6 μm; Hill coefficients, 2.13 at +80 mV and 1.93 at –80 mV). These values are similar to those obtained in the single concentration protocol, with a slight difference in EC50 only at –80 mV. Using the increasing concentration protocol, a mid-stimulus current decline again became apparent at around 320 μm (20 ± 4% inhibition at +80 mV and 42 ± 6% inhibition at –80 mV, n = 8 cells, Fig. 4D). After the current reached a plateau, a further increase in DPBA concentration to 1 mm caused a further reduction in current amplitude. Upon drug washout, current a