Fig. 6: Disrupting DOCK4/Nav1.7 interaction enhances heat nociception, and heat stimulation maintains the membrane localization of Nav1.7.

a Schematic diagram of the full-length C-terminus of Nav1.7 (Nav1.7CT) and various truncated fragment variants. b A GST pull-down assay with two purified proteins, GST-flag-Nav1.7CT and DOCK4, shows the direct interaction between DOCK4 and Nav1.7. The major interaction site is F2 (1825-1899 aa) of Nav1.7CT. This experiment was repeated three times. c Schematic diagram of the DOCK4 full-length and various truncated fragment variants. d A GST pull-down assay reveals the interaction between DOCK4 and Nav1.7, with the interaction site identified as the DC (1311-1966 aa) region of DOCK4. This experiment was repeated three times. e Nav1.7CT-F2 fragment (1825-1899 aa) AAV overexpression vector construction schematic. f The expression of GFP in the DRG neurons of AAV-injected mice indicates successful Nav1.7CT-F2 overexpression. Scale bar, 100 μm. g, h The effect of Nav1.7CT-F2 overexpression on the interaction between DOCK4 and Nav1.7. n = 3 repeats per group. t₄ = 5.18, P = 0.0066. i–k The impact of Nav1.7CT-F2 overexpression on behavioral responses to Hargreaves, tail-flick, and hot plate tests. n = 6 mice per group. t₁₀ = 3.296, P = 0.0081 in (i). t₁₀ = 3.155, P = 0.0103 in 48 °C; t₁₀ = 4.859, P = 0.0007 in 50 °C; t₁₀ = 4.03, P = 0.0024 in 52°C in (j). t₁₀ = 4.551, P = 0.0011 in 50 °C; t₁₀ = 2.611, P = 0.026 in 52 °C; t₁₀ = 4.679, P = 0.0009 in 55°C in k. l The CPA scores induced by Hargreaves training was assessed in control and Nav1.7CT-F2 overexpressed mice. n = 6 mice per group. t₁₀ = 5.667, P = 0.0002. m The impact of Nav1.7CT-F2 overexpression on behavioral responses to intense mechanical pinprick tests. n = 6 mice per group. n The influence of Nav1.7CT-F2 overexpression on the distribution of Nav1.7 in the membrane and cytoplasm of cultured DRG neurons. Profile plots were utilized to delineate membrane staining. Scale bar, 10 μm. o The impact of Nav1.7CT-F2 overexpression on the peak Nav1.7 currents in cultured DRG neurons. n = 7 neurons in control group, n = 6 neurons in Nav1.7CT-F2 group. F_{(1, 407)} = 74.63, P = 1.31 × 10⁻¹⁶. p The diagram illustrates the experimental procedure. The hindpaws of the mice were immersed in a 43 °C water bath for 30 seconds, after which the mice were placed at RT for 5, 10, 20, or 40 minutes. Behavioral and biochemical experiments were then conducted (Created in BioRender. https://BioRender.com/xs51blc). q The impact of heat stimulation on the behaviors of mice response to the Hargreaves. n = 8 mice in RT group, n = 10 mice in 43 °C group. F_{(1, 80)} = 10.88, P = 0.0072 in 5 min, P = 0.0004 in 10 min. r The impact of heat stimulation on the interaction between DOCK4 and Nav1.7 in DRG lysates of mice. s The impact of heat stimulation on the membrane expression of Nav1.7 in DRGs of mice. n = 3 samples per group. F_{(2, 6)} = 23.14, P = 0.0021 in RT vs. 10 min, P = 0.0032 in 10 min vs. 40 min. t, u The impact of heat stimulation on the total expression of Nav1.7 and DOCK4 in DRGs of mice. n = 3 samples per group. v The diagram outlines the experimental procedure. Isolated DRG neurons were immersed in a 43 °C water bath for 30 seconds and then placed at RT for either 10 or 40 minutes. Subsequently, the neurons were lysed to assess the membrane abundance of Nav1.7 (Created in BioRender. https://BioRender.com/xs51blc). w Nav1.7 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 4 repeats. F_{(2, 9)} = 25.61, P = 0.0002 in RT vs. 10 min, P = 0.0021 in 10 min vs. 40 min. x, y The effect of DOCK4 deleted in DRG neurons on the behaviors of mice response to the Hargreaves after heat stimulation. n = 8 mice in AAV-GFP group, n = 11 mice in AAV-Cre group, n = 9 mice in Dock4^WT group, n = 10 mice in AvCreERT2; Dock4^CKO group. F_{(4, 68)} = 19.05, P = 0.0115 in 5 min, P = 0.00000002 in 10 min, P = 0.0007 in 20 min in x; F_{(4, 68)} = 13.79, P = 0.0002 in 5 min, P = 0.00000005 in 10 min, P = 0.0085 in 20 min in y. h–m, t, u, Two-tailed Independent Student’s t test; s, w, One-way ANOVA followed by Tukey’s multiple comparisons test; o, q, x, y, Two-way ANOVA followed by Bonferroni’s multiple comparisons test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s. means not significant. Data are presented as mean ± SEM. Complete sample size and sex is provided in the Supplementary Data. Source data are provided as a Source Data file.