(Supplementary information) TRPV1 EXHIBITS DYNAMIC IONIC SELECTIVITY DURING AGONIST STIMULATION. Man-Kyo Chung, Ali D. Güler, and #Michael J.
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1 (Supplementary information) TRPV1 EXHIBITS DYNAMIC IONIC SELECTIVITY DURING AGONIST STIMULATION Man-Kyo Chung, Ali D. Güler, and #Michael J. Caterina Title Supplementary Figure 1. Capsaicin evokes changes in TRPV1 permeability to small alkali cations but not anion permeability. Supplementary Figure 2. Estimation of pore diameter in TRPV1 M644C. Supplementary Figure 3. MTS reagent effects on capsaicin-evoked currents in wild-type TRPV1 and TRPV1 M644C Supplementary Figure 4. Effects of MTSET and MTSEA on capsaicin evoked currents in TRPV1 M644C in the presence of extracellular TRIS. Comparison of the effects of MTSET on the capsaicin evoked Supplementary Figure 5. currents in TRPV1 M644C expressing cells showing differential changes in P MAE /P Na. Supplementary Figure 6. Differential changes in current amplitude and P NMDG /P Na evoked by different TRPV1 agonists. Supplementary Figure 7. Effects of extracellular Ca 2+ on agonist-evoked changes in TRPV1 P NMDG /P Na. Supplementary Figure 8. External Ca 2+ and capsaicin concentration-dependent changes in TRPV1 P Ca /P Na. Supplementary Figure 9. Agonist-evoked changes in TRPV1 P Ca /P Na in the presence of 10 mm Ca 2+ and 150 mm Na + in TRPV1-HEK293 cells. Supplementary Table I. Capsaicin (1 µm)-evoked changes in TRPV1 relative permeabilities of organic cations to Na +. Supplementary Table II. Capsaicin (0.1 µm)-evoked changes in TRPV1 relative permeabilities of alkali cations to Na +. Supplementary Table III. Extracellular Ca 2+ -dependent changes in TRPV1 P Ca /P Na. Supplementary Methods References
2 Supplementary Figure 1. Capsaicin evokes changes in TRPV1 permeability to small alkali cations but not anion permeability. (a) Capsaicin evokes changes in relative TRPV1 permeability to small alkali cations. E rev was repeatedly monitored in external 150 mm K +, Cs +, Na +, or Li + and internal Na + (int A) during stimulation with capsaicin (100 nm) in TRPV1-HEK293 cells (n=5). At the earliest times after development of the capsaicin-evoked current, TRPV1 exhibited only a slight degree of discrimination among these cations, with the rank order Cs + K + >Li + Na +. Over time, however, relative permeability to the larger ions (Cs + and K + ) underwent a small but statistically significant increase (P Cs /P Na from 1.10±0.001 to 1.14±0.007, n=5 p<0.005; P K /P Na from 1.10±0.014 to 1.12±0.010, n=5 p<0.05, paired t- test) whereas relative permeability to the smaller ion, Li +, decreased with time (from 1.03±0.009 to 0.95±0.005, n=5, p<10 4, paired t-test). Thus, capsaicin evokes changes in TRPV1 permeability even among small monovalent cations. See Supplementary Table II
3 for summary of capsaicin-evoked E rev and P X /P Na changes. Similar results were obtained from 4 separate experiments. (b) TRPV1 remains cation selective during prolonged capsaicin stimulation. Traces show E rev change (mean+sem, n=6) evoked by capsaicin (1 µm) in the presence of different concentrations of external NMDG-Cl (150 mm NMDG and 138 mm Cl versus 57 mm NMDG and 38 mm Cl ). Dotted line represents predicted E rev value when [NMDG] o was 57 mm, based on the assumption of zero anionic conductance. Open circle represents E rev (mean±sem, n=6) measured in a separate experiment, in the presence of 150 mm external NMDG, after 1 min 1 µm capsaicin stimulation. E rev values were then measured in the same cell upon changing [NMDG] o to 57 mm (filled circle). Again, the measured value was close to the predicted value, based on preserved exclusion of anions. A similar correspondence to the predicted shift was observed when [NMDG] o was initially 57 mm and increased abruptly to 150 mm after one minute of capsaicin stimulation (data not shown). Internal solution in these experiments was composed of 150 NaOH, 135 HCl, 1 MgCl 2, 10 HEPES, 5 EGTA (in mm, ph 7.4). Bath solution was composed of 150 NMDG, 118 HCl, 10 HEPES, 10 EDTA (in mm, ph 7.4) or 57 NMDG, 38 HCl, 10 HEPES, 10 EDTA (in mm, ph 7.4).
4 Supplementary Figure 2. Estimation of pore diameter in TRPV1 M644C. Estimation of initial (open circles) and final (filled circles, dashed lines) pore diameter of TRPV1 WT (black) and M644C (red), transiently transfected into HEK293 cells, using the excluded volume equation (Equation (5)) as in Fig. 2. Estimated minimum diameters at initial and steady-state were 10.2 and 12.7 Å in WT and 10.0 and 12.3 Å in M644C. n=6 for each point.
5 Supplementary Figure 3. MTS reagent effects on capsaicin-evoked currents in wildtype TRPV1 and TRPV1 M644C. (a) Effects of MTSET (1 mm) on amplitudes of capsaicin-evoked (1 µm) currents measured at +40 (upper trace) and 40 mv (lower trace), in the presence of external Na + (ext A/int A) in HEK 293 cells transiently transfected with TRPV1 wild-type (WT, left) or M644C (right). Horizontal dotted lines represent zero current level. (b) Normalized capsaicin-evoked current amplitudes at +40 mv in the presence of external Na + in TRPV1 WT (left panels) or TRPV1 M644C (right panels) following the application of 1 mm MTS reagents (cyan : MTSES, blue : MTSEA, red : MTSET) or no MTS reagents (black). Amplitudes are normalized to that measured just prior to MTS addition. n=5 6. Data are presented as mean + or SEM. (c-e) Current-voltage relationships recorded in cells expressing TRPV1 WT (left panels) or M644C (right panels) before capsaicin application (trace a), initially following capsaicin application (trace b), just prior to MTSET application (trace c), during MTSET application (trace d), and after MTSET removal (trace e) in the presence of external Na + (Top), 2-MAE (Middle), or TRIS (Bottom).
6 Supplementary Figure 4. Effects of MTSET and MTSEA on capsaicin evoked currents in TRPV1 M644C in the presence of extracellular TRIS. Comparison of the effects of 1 mm MTSEA (a) and MTSET (b) on 1 µm capsaicinevoked currents in TRPV1 M644C. Current amplitude measured at +30 mv (I +30mV ) and P TRIS /P Na were normalized to the values just prior to the inclusion of MTS reagent in the bath.
7 Supplementary Figure 5. Comparison of the effects of MTSET on the capsaicin evoked currents in TRPV1 M644C expressing cells showing differential changes in P MAE /P Na. (a) Comparison of the effects of MTSET on TRPV1 M644C P MAE /P Na (left panels), outward current amplitudes at +50 mv (middle panels), and inward current amplitudes at 60 mv (right panels) in the presence of extracellular 2-MAE in two groups of cells showing either low P MAE /P Na change (black, 0.10±0.02, n=5) or high P MAE /P Na change (red, 0.31±0.01, n=6) evoked by application of 1 µm capsaicin. (b) Comparison of low (black) and high (gray) P MAE /P Na change groups with respect to 20 80% decay time (left; P, P MAE /P Na ; I, currents at +50 mv) and inhibition of P MAE /P Na, currents at +50 mv (I +50mV ), and currents at 60 mv (I 60mV ). *, p<0.05; **, p<0.005; ***, p<
8 Supplementary Figure 6. Differential changes in current amplitude and P NMDG /P Na evoked by different TRPV1 agonists. (a) (Top) Maximum current amplitudes measured at +20 mv in the experiment shown in Fig. 4. (Middle) Maximum evoked current amplitude measured at 100 mv within 10 s of current response onset (gray bars) or during the second current increase (black bars). CAP, 10 µm capsaicin; NADA, 10 µm N-arachidonoyl dopamine; PIP, 100 µm piperine; RTX, 10 nm resiniferatoxin; HT, heat 25 to 45 C (τ=4.5s); CMP, 10 mm camphor. Recordings were performed using solutions int A/ext C. (b) Mean initial (open bars) and maximal (filled bars) P NMDG /P Na measured during the current responses shown in Fig. 4a. In all panels, *, p < 0.05; **, p < 0.005; ***, p < 0.001;, p < 10 4 ;, p < 10 6, compared with corresponding capsaicin value, unpaired student s t-test.
9 Supplementary Figure 7. Effects of extracellular Ca 2+ on agonist-evoked changes in TRPV1 P NMDG /P Na. (a) TRPV1 currents ( 100 mv) evoked by 10 µm capsaicin (Top) or 10 nm RTX (Bottom) in the presence of 150 mm NMDG and x mm Ca 2+ (x = [Ca 2+ ] o indicated next to each trace). Periods of agonist stimulation and Ca 2+ quenching (performed as in Fig. 5d) are indicated by bars above the traces. Internal solution int A was used in every experiment. (b) I-V relations measured at the times designated in panel a. (c) P NMDG /P Na calculated from the E rev measured immediately upon Ca 2+ quenching as in panel a. Dashed lines represent mean initial P NMDG /P Na measured during stimulation with each agonist in Supplementary Fig. 6B. n=6. *, p<10 3 ; **, p<10 4 ; +, p<10 5 ;, p<10 6. The P NMDG /P Na following the application of capsaicin in the presence of 1 mm Ca 2+ was significantly smaller than that measured following activation at 0.1 mm or nominally absent Ca 2+. As with capsaicin, the P NMDG /P Na evaluated after TRPV1 activation by 10 nm RTX was significantly reduced by the presence of 1 mm Ca 2+ during agonist stimulation. However, the value measured at the time of Ca 2+ quenching was still ~2.4- fold greater than that measured at the beginning of RTX stimulation in the absence of Ca 2+.
10 Supplementary Figure 8. External Ca 2+ and capsaicin concentration-dependent changes in TRPV1 P Ca /P Na. (a e) Changes in TRPV1 P Ca /P Na in the presence of various concentrations of external Ca 2+ and capsaicin. (Top) P Ca /P Na measured during the application of capsaicin (purple : 3 nm, orange : 10 nm, blue: 30 nm, red: 0.1 µm, black : 0.3 µm, cyan: 1 µm) under different ionic conditions, as indicated. E rev was measured until either the current desensitized to 20% of peak or 20 s after capsaicin application, whichever came first. P Ca /P Na was calculated using Equations (2) under int A/ext G. In the experiment using int A and external solutions containing 150 NMDG, 3, 5 or 10 mm Ca 2+, 10 HEPES (ph 7.4 with HCl), we assumed α=0.05 and used Equation of P Ca /P Na =(([Na + ] i exp(e rev F/RT)) α [NMDG] o ) (exp(e rev F/RT)+1)/4[Ca 2+ ] o, where F is the Faraday constant, R is the gas constant, T is absolute temperature and [X + ] is the activity of ion X +. When P Ca /P Na was calculated using the E rev derived from the experiments using int A/ext D and E, this equation and Equations (3) in Methods yielded virtually identical results. Mean+sem or sem. n=3 8 cells for each point. (Bottom) Capsaicin concentration-dependence of
11 P Ca /P Na measured at three time points in upper panels (circles, initial; squares, maximum; triangles, minimum after peak). n = 5 8 for each point. (f) Relationship of P Ca /P Na to ionic activity of extracellular Ca 2+. P Ca /P Na values were measured at three points: initial (circles), maximum (squares), and minimum-followingmaximal (triangles), at the capsaicin concentration yielding the maximum P Ca /P Na at each Ca 2+ concentration (1 µm at 1 mm Ca 2+ ; 0.3 µm at 3 mm Ca 2+ ; 30 nm at 5 mm Ca 2+ ; 10 nm at 10 mm Ca 2+ ; 3 nm at mm Ca 2+ ). Data for initial and minimum-followingmaximal P Ca /P Na were fitted separately using logistic functions, with an imposed lower limit of 5. The resulting EC 50 values for initial and minimum-after-peak Ca 2+ ionic activities were 0.9 mm and 3.9 mm, respectively (corresponding to Ca 2+ concentrations of 2.4 mm and 11.4 mm, with Hill slopes of 2.2 and 1.3, and maxima of 21.1 and 23.8, respectively). Maximum P Ca /P Na values were fit by linear regression. Data used to derive panel f are provided in Supplementary Table III.
12 Supplementary Figure 9. Agonist-evoked changes in TRPV1 P Ca /P Na in the presence of 10 mm Ca 2+ and 150 mm Na + in TRPV1-HEK293 cells. (a) (Top) Representative currents (at 80 mv) evoked in TRPV1-HEK293 cells by 0.1 µm (black), 0.3 µm (blue), and 1 µm (red) capsaicin in an external solution containing 10 mm Ca 2+ and 150 mm Na + (int A/ext F). (Bottom) P Ca /P Na (calculated using Equation (4) in Methods) measured during the responses shown at Top. (b) Mean+SEM initial (open bars), maximum (black bars) and minimum following the maximum (gray bars) P Ca /P Na at the indicated capsaicin concentrations (n=5 6). * p<0.05, ** p<0.01, paired Student s t-test
13 Supplementary Table I. Capsaicin (1 µm)-evoked changes in TRPV1 relative permeabilities of organic cations to Na +. E rev (mv) P X /P Na a. Paired Student s t-test, n=6 Na + 2-MAE TRIS NMDG initial 1.3± ± ± ±1.5 Final 1.5± ± ± ±3.6 p a <0.01 <10-5 <10-3 <10-5 initial ± ± ±0.002 Final ± ± ±0.03 p a - <10-5 <10-3 <10-3
14 Supplementary Table II. Capsaicin (0.1 µm)-evoked changes in TRPV1 relative permeabilities of alkali cations to Na +. Na + Li + K + Cs + Initial 0.39± ± ± ±0.16 E rev (mv) P X /P Na a. Paired Student s t-test, n=5 Final 0.30± ± ± ±0.15 p a >0.6 <10-4 <0.05 <10-3 Initial ± ± ±0.001 Final ± ± ±0.007 p a - <10-4 <0.05 <0.005
15 Supplementary Table III. Extracellular Ca 2+ -dependent changes in TRPV1 P Ca /P Na Extracellular [Ca 2+ ] (mm) initial 52.7 ± ± ± ± ± 0.29 E rev (mv) P Ca /P Na maximum 31.4 ± ± ± ± ± 0.29 minimum 32.9 ± ± ± ± ± 2.48 p a <10-4 <0.001 <0.005 >0.1 - p b >0.1 >0.05 <0.05 >0.1 <0.001 initial 6.60 ± ± ± ± ± 0.45 maximum 24.2 ± ± ± ± ± 0.45 minimum 22.3 ± ± ± ± ± 0.83 p a <10-4 <0.001 <0.001 >0.1 - p b >0.3 >0.05 <0.05 >0.05 <10-5 n a. Paired Student s t-test between initial and maximum b. Paired Student s t-test between maximum and minimum-following-maximum Capsaicin concentrations : 1 mm Ca 2+, 1 µm; 3 mm Ca 2+, 0.3 µm; 5 mm Ca 2+, 30 nm; 10 mm Ca 2+, 10 nm; mm Ca 2+, 3 nm.
16 Supplemental Methods DNA constructs, cell culture, and transfections HEK293T cells were transiently transfected with pcdna3-based plasmids using Lipofectamine 2000 (Invitrogen). Green fluorescent protein (GFP) or ds-red was cotransfected with cdnas encoding rat TRPV1, mouse TRPV1 (gift of Michael Xhu, Ohio State University), or 5HT 3 (gift of David Julius, UCSF). Stable transfectants of rat TRPV1 and pcdna3 have been described 1. Site-directed mutagenesis of rat TRPV1 was performed by PCR using complementary synthetic oligonucleotides encoding the mutation of interest and Proofstart high fidelity polymerase (Qiagen). TG dissected from euthanized Sprague-Dawley rats (P1 P14, Charles River) were washed once in Puck s saline (171 NaCl, 6.7 KCl, 1.4 Na 2 HPO 4, 0.5 KH 2 PO 4, 6.0 glucose, ph 7.3 with NaOH), incubated in Puck s saline containing 0.125% collagenase (Roche) for min at 37 C then treated with 0.05 % trypsin-1mm EDTA in phosphate-buffered saline ( NaCl, 2.97 Na 2 HPO 4, 1.06 KH 2 PO 4, ph 7.4) for 2 minutes. After washing with culture medium (MEM containing 10% horse serum, 1% vitamins, and 1% penicillin/streptomycin/glutamine, all from Invitrogen), ganglia were triturated with flame-polished Pasteur pipettes. Dissociated cells were centrifuged through 25% Percoll, resuspended in culture medium containing 100 ng/ml nerve growth factor (NGF, 2.5S, Invitrogen), and plated on polyornithine- and laminin-coated coverslips. The cells were incubated at 32 C in 5% CO 2, then assayed 16 to 24 hours later. All experiments were in accordance with protocols approved by the Johns Hopkins Animal Care and Use Committee. Chemicals Unless otherwise indicated, chemical reagents were obtained from Sigma-Aldrich. Capsaicin, NADA, PMA, and RTX were dissolved in ethanol, CBX and RR in water, and other drugs in DMSO. Stocks were diluted in the aqueous medium on the day of experiment. Vehicle did not exceed 0.1 % DMSO or 0.01% ethanol, except in the case of
17 camphor, which was applied in 0.5 % DMSO. The corresponding amounts of vehicle were included in controls. Patch Clamp Electrophysiology In int A, we noticed a slowly developing endogenous current whose properties resemble the Mg 2+ -inhibited current 2. The endogenous current recorded from naïve or pcdna3- transfected HEK293 cell showed no difference from that in HEK293 cells stably expressing TRPV1, and was not affected by capsaicin. In some experiments, we used int B, containing 1 mm Mg 2+, to suppress the endogenous current. This maneuver did not appear to alter our monovalent cationic permeability measurements. Bath temperature was controlled using an in-line heater, alone or together with an in-line heater/cooler (Warner Instruments) and monitored continuously with a thermistor (Physitemp). The bath (Warner Instruments, <200 µl in volume) was perfused throughout the experiment at a rate of ~3 ml min 1 and drug and temperature stimulus application by gravity-driven flow was performed using valve manifolds which allowed a solution exchange at τ of ~1.2 s (Warner Instruments ). Data Analysis In all cases, P Ca /P Na calculation was discontinued once the agonist-evoked current had desensitized to 20% of its maximum value. Some cells exhibited a large current evoked by exposure to external 112 mm Ca 2+ alone, consistent with the reported agonist effect of Ca 2+ on TRPV1 3. These cells were excluded from analysis. In establishing the relationship between P Ca /P Na and [Ca 2+ ] or capsaicin concentration (Fig. 5h, Supplementary Fig. 4), we did not consider the likely contribution of Ca 2+ as a TRPV1 agonist 3. Time courses of inhibition were compared using 20% 80% decay time in the presence of extracellular 2-MAE or TRIS due to the complex kinetics. In the calculation of P X /P Na, we did not consider the permeation of MTS reagents.
18 The concentration-response curves were fitted using a logistic function, y=max/(1+10^((logec 50 -x) h)), where y is response, x is the logarithm of concentration, h is hill slope. We used Prism for curve-fitting and correlation analysis. YO-PRO uptake At the indicated time, YO-PRO1 (1 µm, Molecular Probes) was added to the perfusate. After an additional 40 s, cells were stimulated with chemical agonist or heat (25 C to 45 C within 30s), still in the presence of YO-PRO1. Fluorescence intensity (470 nm excitation, 525 nm emission) was monitored in isolated cells at 2 s intervals using an upright fluorescence microscope (Nikon), excitation filter changer (Ludl), and CCD camera (Coolsnap ES, Roper) and Ratiotool software (Isee Imaging). For comparisons among agonists, only cells exhibiting responses >85 fluorescence units were analyzed. To correct for variations in TRPV1 expression levels, fluorescence intensity data for all agonists were normalized to the peak fluorescence change evoked by 10 µm capsaicin on that same day. References for Supplementary Methods 1. Guler, A.D., et al. Heat-evoked activation of the ion channel, TRPV4. J Neurosci 22, (2002). 2. Runnels, L.W., Yue, L. & Clapham, D.E. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 291, (2001). 3. Ahern, G.P., Brooks, I.M., Miyares, R.L. & Wang, X.B. Extracellular cations sensitize and gate capsaicin receptor TRPV1 modulating pain signaling. J Neurosci 25, (2005).
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