Fig. 4: Biochemical basis underlying CXCR2-mediated TRPA1 activation. | Nature Communications

Fig. 4: Biochemical basis underlying CXCR2-mediated TRPA1 activation.

From: CXCL5 activates CXCR2 in nociceptive sensory neurons to drive joint pain and inflammation in experimental gouty arthritis

Fig. 4

A Ca2+ imaging of HEK293T cells upon recombinant mouse CXCL5 (rmCXCL5, 100 nM), AITC (100 μM), and ionomycin (10 μM) application. Cells were co-transfected with plasmids containing mouse Cxcr2 (mCxcr2) and mouse Trpa1 (mTrpa1) genes. Scale bar = 50 µm. B–E Ca2+ traces evoked by rmCXCL5 in HEK293T cells transfected with vector alone (B), mCxcr2 (C), mTrpa1 (D), or mCxcr2+mTrpa1 (E). F Summary of rmCXCL5 evoked Ca2+ responses (normalized with ionomycin) in HEK293T cells. G Summary of rmCXCL5 evoked Ca2+ responses in HEK293T cells transfected with mCXCR2 + mTRPA1. Cells were simultaneously transfected with empty vectors or genes encoding Gαt, GRK2ct, or siRNAs against Gnb1 and Gnb2 or treated with U73122 (5 μM), wortmannin (10 nM), U0126 (10 μM), and H89 (10 μM). H The same as panel G, except AITC was used. I and J Co-IP showing coupling of Gβ with TRPA1 in HEK293T cells in vehicle- and CXCL5-treated conditions. K–M Docking model of mTRPA1 (yellow) with Gβγ (blue and purple). Proteins are shown as surfaces in panel K, and proteins in L and M are shown as secondary structures. Blue arrow indicates the K992-M1018 segment of mTRPA1 for direct binding. N Electrostatic surface potential of mTRPA1 and Gβγ proteins. ESP: electrostatic surface potential. O Five key residues in K992-M1018 segment of WT channel. Point mutations were illustrated in red. P rmCXCL5 or AITC evoked Ca2+ responses in HEK293T cells transfected with mCXCR2 and WT mTRPA1 or mutations. Q Co-IP showing Gβ coupling with WT or TRPA1 with 5 mutations under CXCL5-treated condition. R Proposed working model for Gβγ binding with mTRPA1 and channel opening, Gray oval: Cross-section of the largest part of mTRPA1 radius. One-way ANOVA with Bonferroni’s post hoc test was used for panels (F, G, H, and P). Student’s unpaired t-test (two-tailed) was used for panels (J and Q). *p < 0.05, **p < 0.01. Data are shown as mean ± SEM. The n number, exact p-value, and statistical results are provided in Source Data file.

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