Article Figures & Data
Figures
- Figure 1.
Expression and responses to capsaicin of TRPV1 mutants. A, Top, Expression levels of TRPV1 alanine mutations in the range of 736-747 in HEK293 cells detected with anti-V5 in Western blots. Similar results obtained with all mutants in at least three experiments. B, TRPV1 mutant channel function assessed from calcium increase in response to capsaicin (200 nm, 30 s, black bars). Responses normalized to calcium increases evoked by ionomycin (open bars). Traces are averages ± SEM of calcium signals from n = 37–129 individual cells. C, Δ[Ca]i response to capsaicin (200 nm, 30 s) relative to response to ionomycin for mutants shown in B and others. TRPV1 mutants D738A, Q and S, and R740A did not respond to capsaicin up to 5 μm, but R740Q was capsaicin responsive.
- Figure 2.
Sensitization of TRPV1 is disrupted by specific mutations in the AKAP79 binding site. A, Sensitization of responses to capsaicin (200 nm, 1 s) by PMA (1 μm) in HEK293 cells transfected with WT TRPV1 (both with and without PMA), two mutants in which sensitization was ablated (C742A and V745A), and two mutants that had no significant effect on sensitization (D746A and E747A). Traces are averages ± SEM of calcium signals from n = 52–230 capsaicin-responsive cells per trace. B, Ratio of calcium increase after PMA (6th to 4th capsaicin application, black bars) compared with control (ratio of 4th to 3rd response). Bars show average ± SEM. n.s., Not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 3.
Trafficking of TRPV1 to the surface membrane and interaction with AKAP79 is reduced by specific mutations. A, Images show HEK293 cell transfected with WT TRPV1. The image from a brief exposure to WGA (first panel) was used to create a juxtamembrane ROI (2nd panel) that was superimposed on the image of TRPV1 fluorescence (3rd panel) to measure TRPV1 levels at the membrane (4th panel, TRPV1 image plus ROI). A separate mask obtained from the thresholded fluorescence within the cell defined the cytoplasm excluding the nucleus (see 4th panel). B, Ratio of mean fluorescence at the plasma membrane to cytoplasm for TRPV1 WT and alanine mutants, n = 12–30. C, Coimmunoprecipitation of AKAP79 by TRPV1 mutants. Blot stripped and reprobed for TRPV1 (bottom blot). D, Intensity of AKAP79 band after immunoprecipitation, normalized to TRPV1 expression level and expressed relative to WT. Dotted line represents the nonspecific signal in the absence of TRPV1. Bars are average of four repetitions. IB, Immunoblot; IP, immunoprecipitation. *p < 0.05; ***p < 0.001.
- Figure 4.
Intracellular peptides inhibit the PMA-induced sensitization of currents evoked by capsaicin in DRG neurons. A, Representative voltage-clamp experiment showing that inward current evoked by capsaicin (Caps.; 200 nm, application shown by black bars) is sensitized by bradykinin (BK; 1 μm, white bar). B, Comparison of sensitization by PMA or bradykinin (30 s) compared with control. Note that PMA generated a more robust and less variable sensitization; the SD/mean was 0.41 for PMA compared with 0.60 for bradykinin (n = 9–13). C, Sample traces showing effect of peptides corresponding to subsets of the TRPV1 AKAP79 binding site on sensitization of capsaicin by PMA. Peptides (200 μm) were added to pipette solution and the cell was held in whole-cell configuration for ∼13 min without negative pressure before application of PMA to allow diffusion of peptide into cytoplasm. D, Peak current evoked by capsaicin (100 nm, 5 s), normalized to mean of last two stimuli before PMA. Location of peptide shown by colored bars. Mean sensitization over responses 8–10 shown in the right panel. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control, n = 8–16. E, Sensitization of heat-evoked TRPV1 currents by PMA (1 μm) was reduced with 736-745 peptide in the recording pipette. F, Specimen of heat-evoked current and temperature ramp measured at the application tip. Heat ramps lasted 5 s and were applied every 45 s. Bottom, Mean of peak responses to three heat stimulations after PMA (see E), normalized to response before. *p < 0.05 compared to control, n = 9 each.
- Figure 5.
TAT-conjugated 736-745 peptide inhibits TRPV1 sensitization. A, Example trace showing PMA-induced sensitization of TRPV1 in DRG neurons with intracellular application of 736-745-TAT or scramble-TAT (200 μm) in patch pipette; protocol as in Figure 4. Time course (B) and average of TRPV1 sensitization (C) in DRG neurons with intracellular addition of TAT-conjugated and unconjugated peptides, n = 6–16. *,#p < 0.05 compared to control and scramble-TAT, respectively. Data for control and 736–745 taken from Figure 4. D, HEK293 cells transfected with TRPV1 and loaded with fura-2 AM for 30 min, exposed to extracellular TAT-conjugated peptide (5 μm) for 20 min, and subsequently exposed to capsaicin pulses as in Figure 2, n = 65–128. E, Average sensitization ratio after application of PMA (filled bars) compared with control (open bars). Significant sensitization was observed in control experiments without peptide and in experiments with scramble-TAT (**p < 0.01) but not with 736-745-TAT. n.s., Not significant.
- Figure 6.
736-745-TAT suppresses pain-related behavior in mice. A, Formalin test of inflammatory hyperalgesia. TAT-conjugated peptides injected in plantar hindpaw, followed by Formalin (4%, 30 min later) at the same site. No difference was found in phase I (0–10 min), but 736-745-TAT (10 μm, 20 μl) suppressed phase II (10–60 min) compared with both saline and scramble-TAT (100 μm, 20 μl); p < 0.05 compared with control (*) or scramble-TAT (#), n = 9 per group. B, PMA test of inflammatory hyperalgesia. TAT-conjugated peptides coinjected with PMA (50 pmol/hindpaw). At 10–25 min after injection, licking of paw was attenuated by 736-745-TAT (1, 10, and 100 μm as indicated). p < 0.05 compared with controls (*) and scramble-TAT (#), n = 8–12 per group. C, 736-745-TAT blocks inflammatory thermal hyperalgesia without affecting acute thresholds. 736-745-TAT, scramble-TAT (10 μm in saline), or saline were injected 30 min before λ-carrageenan (Carr.; 2%, 20 μl) or saline, n = 12 per group. Ordinate shows latency for paw withdrawal to radiant heat after λ-carrageenan injection, baseline measured before injection (0 min). Right panel shows difference of area under the curve (AUC) between test samples and saline–carrageenan inflammation control (30–180 min). ***,###p < 0.001 versus control or scramble-TAT.
- Figure 7.
Key residues for the TRPV1–AKAP79 interaction are unique to TRPV1. Within the human genome, more than five consecutive amino acids of the TRPV1 736-749 range are only shared by TRPV4 and TRPV2 (http://blast.ncbi.nlm.nih.gov). Important residues for the inhibition of sensitization as determined by site-directed mutagenesis (indicated by *) and by antagonist peptides (see Fig. 4), are only found in TRPV1. Shaded background indicates identical amino acids. The human 736-749 TRPV1 sequence range is highly conserved and is identical for Macaca fascicularis, Crotalus atrox, Carollia brevicauda, Canis lupus familiaris, Bos taurus, Scapanus orarius, Rattus norvegicus, and Cavia porcellus; one amino acid difference is found in Mus musculus, Oryctolagus cuniculus, Xenopus laevis, and Gallus gallus.
