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. 2010 Nov 17;286(3):1828–1835. doi: 10.1074/jbc.M110.160390

FIGURE 3.

FIGURE 3.

G protein signaling and Kir4.1 cell surface expression and activity. cDNAs coding for Kir4.1 and the CaRWT or GTPase-deficient G protein α subunit mutants (Gαi2Q204L, Gα13Q226L, GαqQ209L, or GαsQ227L) were expressed transiently in HEK-293 cells. A, cell surface expression was measured using cell surface biotinylation. The top panel shows summary data (n = 4, mean ± S.E.). The bands corresponding to biotinylated Kir4.1 were quantitated using densitometry (Scion Image), and the values in each experiment were normalized for the level of expression of Kir4.1 alone. The middle panel Kir4.1 (Bio) shows a representative blot for biotinylated Kir4.1, and the bottom panel shows a representative cell extract blotted for Kir4.1 (Extr). All cells expressed Kir4.1 and the CaR or G protein α subunit constructs as shown. *, p < 0.05 compared with Kir4.1 alone, CaRWT and Gαq were not statistically different from each other (ANOVA). B, current density in HEK-293 cells expressing Kir4.1 and vector or the G protein α subunit constructs are shown (n = 8, ± S.E.). *, p < 0.05 compared with Kir4.1 (Vector) alone (ANOVA). C, the effect of RGS4 on CaR-mediated reduction in Kir4.1 whole cell current density is shown. cDNAs coding for Kir4.1 the CaRWT and RGS4 were expressed transiently in HEK-293 cells as indicated. Summary data for whole cell current density ± S.E. (n = 10). All cells expressed Kir4.1 and vector, and the CaRWT is as shown, and 0 to 1.5 μg of RGS4 cDNA as shown. *, p < 0.05 compared with CaR without RGS4 (ANOVA). D, cell surface expression was measured using cell surface biotinylation. The top panel shows summary data (n = 5, mean ± S.E.). The bands corresponding to biotinylated Kir4.1 were quantitated using densitometry (Scion Image), and the values in each experiment were normalized for the level of expression of Kir4.1 alone. The middle panel Kir4.1 (Bio) shows a representative blot for biotinylated Kir4.1, and the bottom panel shows a representative cell lysate blotted for Kir4.1 (Lys). *, p < 0.05 compared with Kir4.1 and CaRWT (ANOVA).