Fig. 4. ZPT and Zn²⁺ restore GTPase activity and cellular interactions of pathologic Gα_o mutants.

(A to C) ZPT (A) restores GTPase activity of Gα_o[E246K] (B), not affecting GTP binding and hydrolysis of WT Gα_o (C). Representative curves of BODIPY-labeled GTP binding and hydrolysis are provided. (D and E) Quantification of k_hydr of Gα_o WT, Gα_o[G203R], Gα_o[R209C], and Gα_o[E246K] treated with ZPT (D) and ZnCl₂ (E). Note the log scale in y axes. (F and G) N2a cells were cotransfected with His₆-RGS19 and Gα_o-GFP (C-terminally tagged) variants. The next day, cells were treated with dimethyl sulfoxide (DMSO) or 1 μM ZPT for 3 hours before IP of Gα_o with nanobody against GFP; coprecipitation of RGS19 was analyzed by SDS-PAGE and Western blot. Abs against GFP and His₆-tag were used to detect Gα_o and RGS19, respectively (F). Quantification of co-IP of His₆-RGS19 by Gα_o variants, normalized to the binding levels of WT Gα_o (G). (H and I) Cotransfection with AGS3-GFP and Gα_o-GST-HA (C-terminally tagged) variants was similarly followed by treatment with DMSO or 1 μM ZPT before Gα_o pull-down with glutathione beads. Ab against HA was used to detect Gα_o and against GFP to detect AGS3 (H). Quantification of the coprecipitation of AGS3-GFP by Gα_o-GST-HA variants normalized to the binding levels of WT Gα_o (I). Data in (B) to (E), (G), and (I) are means of ≥3 biological replicates ± SEM; the means in (B) and (C) are shown as dots, and SEM deviations are shown as the error bars (black for no drug; red for ZPT). (D and E) ****P < 0.0005; ns, not significant by one-way ANOVA to verify the significance of the concentration dependence (as significance of the mean change upon changing concentrations of ZPT/ZnCl₂). (G and I) **P < 0.01; ****P < 0.0001 on log-transformed data by two-way ANOVA with Sidak corrections for multiple comparisons.