Figure 4.
Protein arginine methyltransferase 7 modulates NaV1.9 currents by binding and methylating hLoop1. (A) Schematic representation of one deletion mutant and 4 single-alanine–scanning constructs spanning the region between residues 563 and 572 used in the Y2H assay. The percentage of yeast transformant growth on QDO medium was analysed correspondingly by one-way ANOVA (n = 3). ***P < 0.001, n.s., no significance. (B) Immunoblotting analysis showing the enhanced monomethylation of hLoop1 in hPRMT7-overexpressing cells with the indicated antibodies. (C) Immunoblotting with the indicated antibodies showed decreased MMA levels of hLoop1 in hPRMT7-knockdown cells and increased MMA levels of hLoop1 in hPRMT7-overexpressing cells. (D) Immunoprecipitation of FLAG-tagged wild-type (WT) R519A, R521A, or R519A/R521A hLoop1 proteins expressed in HEK293T cells using anti-FLAG antibodies. The methylation signals and target bands were immunoblotted with the indicated antibodies. (E) Representative whole-cell hNaV1.9 currents evoked by voltage from Scn11a −/− mouse small DRG neurons electroporated with the indicated groups. (F) Current–voltage relationships of the indicated experimental groups. The peak current density (normalized by membrane capacitance) was statistically analysed (hNaV1.9-4A, n = 14; hNaV1.9-4A+hPRMT7, n = 12; hNaV1.9-R519A, n = 10; hNaV1.9-R519A+hPRMT7, n = 9), and significant differences were tested by two-way ANOVA. ANOVA, analysis of variance; DRG, dorsal root ganglion; hNaV1.9, human NaV1.9; MMA, monomethylarginine; PRMT7, protein arginine methyltransferase 7; QDO, quadruple dropout; WCLs, whole-cell lysates; Y2H, yeast two-hybrid.