Fig 2: Heat stimulation stabilises TRPM3 in the DRG membrane.a The diagram shows the experimental procedure. The hindpaws of mice were soaked in a 43 °C water bath for 30 s, and the mice were placed at RT for 2, 5, 10, 20 or 40 min. Then, behavioural, co-IP and membrane protein immunoblotting experiments were performed. b, c ATF4/TRPM3 interactions (b) and TRPM3 membrane expression (c) in mice were evaluated at different time points after heat stimulation. The experiment was repeated four times in b. n = 6 mice per group in c. F(5,18) = 20.17, P = 0.0007 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0044 in 20 min in b. F(5,30) = 10.59, P = 0.0277 in 2 min, P = 0.0022 in 5 min, P < 0.0001 in 10 min, P = 0.0027 in 20 min in c. *P < 0.05, **P < 0.01 versus the RT group. d The diagram shows the experimental procedure. Isolated DRG neurons were placed in a 43 °C water bath for 30 s and placed at RT for 2, 5, 10, 20 or 40 min. The neurons were then lysed to detect the membrane abundance of TRPM3. e TRPM3 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 3. F(5,12) = 12.3, P = 0.0358 in 2 min, P = 0.0100 in 5 min, P < 0.0001 in 10 min, P = 0.0085 in 20 min. *P < 0.05, **P < 0.01 versus the RT group. f–h Naïve (f), ATF4 siRNA-injected (g) and Atf4+/- (h) mice were subjected to the Hargreaves test at different time points after heat stimulation. n = 12 mice per group in f, g. n = 6 mice per group in h. F(5,66) = 14.36, P = 0.0004 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0057 in 20 min. **P < 0.01 versus RT group. One-way ANOVA followed by Tukey’s multiple comparisons test. The error bars indicate the SEMs.

Fig 3: ATF4 interacted with KIF17 in the DRG tissues of mice.a–f Co-IP showing the ATF4/KIF interaction in the DRG. DRG lysates were immunoprecipitated with an ATF4 antibody and immunoblotted with a KIF17, KIF3A, KIF3B, KIF5A, KIF5B, KIFC2 or ATF4 antibody as indicated. This experiment was repeated three times. g SIM images show that the colocalization between ATF4 and KIF17 in DRG neurons. Scale bar, 5 µm. h The GST pull-down assay with two purified proteins, KIF17-GST-Flag and ATF4, showed a direct interaction between ATF4 and KIF17. This experiment was repeated three times. i Interaction between KIF17 and ATF4 mutants. The ATF4 mutants were transiently co-expressed with KIF17, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. j The interaction level between TRPM3 and KIF17 was examined by co-IP in scrambled and ATF4 siRNA-treated mice DRG lysates. DRG lysates were immunoprecipitated with a TRPM3 antibody and immunoblotted with a KIF17 or TRPM3 antibody as indicated. This experiment was repeated three times. t4 = 12.88, P = 0.0002. k SIM images showed the colocalization between TRPM3 and KIF17 in DRG neurons from naïve, ATF4 siRNA and ATF4-overexpresing mice. Quantification data showed the colocalization rates of KIF17 with TRPM3 (colocalized yellow spots/total KIF17 positive spots) and those of TRPM3 with KIF17 (colocalized yellow spots/total TRPM3 positive spots) in DRG neurons. n = 3 mice per group. F(2,6) = 27.17, P = 0.0224 in naïve vs. ATF4-siRNA, P = 0.0255 in naïve vs. ATF4-over in KIF17 with TRPM3. F(2,6) = 33.5, P = 0.0069 in naïve vs. ATF4-siRNA, P = 0.0375 in naïve vs. ATF4-over in TRPM3 with KIF17. Scale bar, 10 µm. j Two-tailed Independent Student’s t test. k One-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01. The error bars indicate the SEMs.

Fig 4: ATF4 mediates TRPM3 trafficking in DRG neurons.a–c TRPM3 expression in the DRG membrane fraction (a), cytoplasmic fraction (b) and total lysate (c) from WT and Atf4+/- mice. n = 3 mice per group. t4 = 10.3, P = 0.0005 in a. t4 = 6.043, P = 0.0038 in b. d TRPM3 surface levels were measured in cultured DRG neurons prepared from WT and Atf4+/- mice using a surface biotinylation assay. n = 3 cultures per group. t4 = 6.28, P = 0.0033. e Co-IP showing the ATF4/TRPM3 interaction in DRGs. DRG lysates were immunoprecipitated with an ATF4 (top) or TRPM3 (bottom) antibody and immunoblotted with a TRPM3 or ATF4 antibody as indicated. This experiment was repeated three times. f The SIM images show that the colocalization between ATF4 and TRPM3 in DRG neurons. Scale bar, 5 µm. g The GST pull-down assay with two purified proteins, TRPM3-GST-Flag and ATF4, showed a direct interaction between ATF4 and TRPM3. This experiment was repeated three times. h Interaction between TRPM3 and ATF4 mutants. The ATF4 mutants was transiently co-expressed with TRPM3, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. i–k TRPM3 expression in the DRG membrane fraction (i), cytoplasmic fraction (j) and total lysate (k) after ATF4 overexpression. n = 6 mice per group. F(2,15) = 14.31, P = 0.0007 in i. F(2,15) = 30.15, P < 0.0001 in j. l, n, p Time course of a whole-cell patch-clamp recording showing the effect of CIM0216 (3 µM) and isosakuranetin (Iso; 10 µM) on the TRPM3 current in DRG neurons from WT (l), Atf4+/- (n) and rescued (p) mice. The open circle indicates the outward current recorded at + 75 mV, and the closed circle indicates the inward current recorded at -75 mV. m, o, q I–V relationship of the TRPM3 current at the time points indicated in I, n and p. r Histogram of the TRPM3 current density (outward and inward, current before CIM0216 was subtracted) of DRG neurons from WT, Atf4+/- and ATF4 rescued mice after CIM0216 treatment. n = 6 neurons per group. F(2,15) = 8.735, P = 0.0049 in WT vs. Atf4+/-, P = 0.0097 in Atf4+/- vs. rescue in +75 mV; F(2,15) = 9.158, P = 0.0038 in WT vs. Atf4+/-, P = 0.0089 in Atf4+/- vs. rescue in -75 mV. a–d Two-tailed Independent Student’s t test. i, j, k, r, One-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01, n.s. means not significant. The error bars indicate the SEMs.

Fig 5: Distribution of ATF4 in primary sensory neurons.a Left, image of immunostaining showing broad ATF4 expression in mouse DRG neurons. Middle, ATF4 expression was decreased in Atf4+/- mice. Right, absence of ATF4 immunostaining upon treatment with a blocking peptide. Scale bar, 200 µm. b Size frequency distribution of ATF4-positive and total neurons in the mouse DRG. A total of 1489 neurons from n = 4 mice were analysed. c, d Colocalization of ATF4 and cell markers (IB4, CGRP and NF200) in the DRG (c) and sciatic nerve (d). Scale bar, 200 µm.