Fig. 9

Essential role of TRPV1 SUMOylation in inflammatory thermal hyperalgesia. a Schematic diagram for the design of AAV-Control, AAV-TRPV1, and AAV-TRPV1-K822R. b Diagram for AAV viral infection into L3–L4 DRG of TRPV1^−/− mice. The viruses (2 μl) were directly injected into the DRG. c The expression of TRPV1 in the left L3-L4 DRG of TRPV1^−/− mice 4 weeks after the injection of AAV-Control, AAV-TRPV1, and AAV-TRPV1-K822R. DRG sections were stained with antibodies against TRPV1 (red) and NeuN (green). d Whole-cell currents at −60 mV evoked by capsaicin (1 μM) in neurons isolated from left L3-L4 DRG of TRPV1^−/− mice that received the injection of AAV-Control, AAV-TRPV1, or AAV-TRPV1-K822R, as indicated, 4 weeks prior to DRG dissection. Representative traces (right) and quantification of current densities (left) are shown. The current densities are: AAV-Control, 0.58 ± 0.09 pA/pF (n = 10); AAV-TRPV1, 187.5 ± 18.5 pA/pF (n = 11); AAV-TRPV1-K822R, 194.3 ± 33.4 pA/pF (n = 13). e Capsaicin-evoked Ca²⁺ transients in neurons as prepared in d. Cells were loaded with Fluo-4 and then images taken while capsaicin (1 μM) was applied at 60 sec. Time courses (left) and peak responses to capsaicin (right) are shown as ΔF/F₀, where F is fluorescence at a given time point, F₀ is the baseline fluorescence, and ΔF = F−F₀. The peak values are: AAV-Control (gray), 0.06 ± 0.02 (n = 21); AAV-TRPV1 (black), 2.99 ± 0.52 (n = 14); AAV-TRPV1-K822R (wine) = 3.35 ± 0.67 (n = 9). f AAV-TRPV1-K822R failed to rescue inflammatory thermal hyperalgesia in TRPV1^−/− mice. At 4 weeks after the injection of AAV-Control, AAV-TRPV1, and AAV-TRPV1-K822R in the left L3-L4 DRG, the TRPV1^−/− mice were subject to the carrageenan edema model. The paw withdrawal latency (PWL) was measured as in Fig. 3e. Only AAV-TRPV1, but not AAV-Control or AAV-TRPV1-K822R, rescued thermal hyperalgesia induced by carrageenan administration to the hindpaws (n = 5 per group). ***P < 0.0001, AAV-TRPV1 vs. AAV-TRPV1-K822R. g AAV-TRPV1-K822R exhibited weak dominant negative effect on inflammatory thermal hyperalgesia in wild-type mice. Similar to f but AAV-TRPV1 and AAV-TRPV1-K822R were injected to the left L3–L4 DRG of wild type mice. At 4 weeks after the injection, mice were subject to the carrageenan edema model and PWL was measured by Hargreaves test (n = 8 per group). ***P < 0.0001, AAV-TRPV1 vs. AAV-TRPV1-K822R. Data are means ± s.e.m. Two-way ANOVA for f and g