1. Rad[R208A/L235A] does not bind CaVβ and selectively inhibits CaV1
Rad[R208A/L235A] does not bind CaVβ and selectively inhibits CaV1.2 and CaV2.2 channels.
Rad[R208A/L235A] does not bind CaVβ and selectively inhibits CaV1.2 and CaV2.2 channels. (A) Crystal structure of Rad G domain with residues R208 and L235 highlighted. (B) FRET efficiency measurements in HEK293 cells coexpressing CFP-FRB + β3-YFP (control), CFP-Rad + β3-YFP, or CFP-Rad[R208A/L235A] + β3-YFP. Data are means ± SEM. *P < 0.01, one-way ANOVA. (C) Binding-curve analyses of FRET experiments. (D) Population Ipeak–V plots for cells expressing α1C + β2a (black squares; n = 11), α1C + β2a + Rad[R208A/L235A] (red squares; n = 10); α1D + β2a (black circles; n = 10), α1D + β2a + Rad[R208A/L235A] (red circles; n = 13); α1A + β2a (black triangles; n = 10), α1A + β2a + Rad[R208A/L235A] (red triangles; n = 11); and α1B + β2a (black diamonds; n = 10), α1B + β2a + Rad[R208A/L235A] (red diamonds; n = 14). P < 0.05, Student’s t test. (E) Exemplar Ba2+ currents from cultured adult guinea pig ventricular cardiomyocytes expressing Rad (Top) and Rad[R208A/L235A] (Bottom). (F) Population Ipeak–V for cardiomyocytes expressing either CFP-Rad (red squares; n = 4) or CFP-Rad[R208A/L235A] (blue squares; n = 8). Dotted line is mean current density for control cardiomyocytes expressing GFP, reproduced from Fig. 2G. (G) Exemplar currents (Top) and diary plot (Bottom) showing the impact of 1 μM forskolin on IBa in cardiomyocytes expressing CFP-Rad[R208A/L235A]. (H) Bar chart showing differential impact of forskolin in up-regulating CaV1.2 IBa in cardiomyocytes expressing CFP-Rad or CFP-Rad[R208A/L235A]. Data are means ± SEM.
Akil A. Puckerin et al. PNAS 2018;115:47:
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