Anchored PKA synchronizes adrenergic phosphoregulation of cardiac Cav1.2 channels

Figure 1. PKA anchoring contributes to adrenergic modulation of Cav1.2.A, schematic of anchored PKA modulation of Cav 1.2 channels. AKAP79 orients PKA toward phosphorylation sites in the cytoplasmic tail of the channel and the small GTPase Rad. B, PKA exists of two type, type I and type II, which are distinguished by their different regulatory subunits RI and RII, respectively. Type II regulatory subunit (RII) is localized via interaction with specific AKAPs. An RII overlay assay was performed to investigate the ability of AKAP to bind RIIα of PKA. Characterization of anchoring disruptor peptides were conducted by RII overlay assay of tsA201 cell extracts immobilized on polyvinylidene fluoride membranes. Individual membranes were incubated with RII and vehicle (1), scrambled AKAP-IS peptide control (2), or AKAP-IS peptide (3). Immunoblot detection of immobilized RII revealed AKAP’s. Green arrows present RII binding proteins. C–K, AKAP-IS eliminates upregulation of Cav1.2 activity by PKA activation in the presence of RAD. Whole-cell patch-clamp recording techniques were used to measure the effects of vehicle (C, F, and I) and 1 μM AKAP-IS (D, G, and J) perfused in intracellular solution.…

Figure 2. Phosphosites on Cav1.2 and Rad are necessary for cAMP stimulation of currents.A–C, basal electrophysiological properties of truncated (A) Cav1.2 and (B) the STAA nonphosphorylatable mutant were assessed. Recordings were conducted in the presence of Rad (open circles) or the mRad (gray circles) mutant that cannot be phosphorylated by PKA. C, summarized data on peak current induced by depolarizing potential 10 mV in (A) and (B). D–F, I/V curves of Cav1.2Δ1800 + dCT coexpressed with RAD WT (D) and m RAD (E) in presence (red) or absence (white) of forskolin. F, summarized data on peak current induced by depolarizing potential 10 mV in (D) and (E). G–I, I/V curves of (G) Cav1.2Δ1800/STAA + dCT coexpressed with RAD WT and (H) mRAD (H) with phosphoregulatory in presence (red) or absence (white) of forskolin. I, summarized data on peak current induced by depolarizing potential 10 mV in (G) and (H). J and K, I/V curves of vehicle (J) and AKAP-IS (K) in presence of RAD. L, summarized data on peak current induced by depolarizing potential 10 mV in presence (red) and absence (white) of forskolin (J) and (K). Cav1.2Δ1800/STAA + vehicle, N = 19; Cav1.2Δ1800/STAA + scramble AKAP-IS N = 18;…

Figure 3. Altered basal and β-adrenergic stimulation of L-type Ca2+currents in adult RAD deficiency ventricular cardiomyocytes.A, immunoblot analysis of RAD expression in (top) cardiac and (middle) spleen lysates from control (RAD+/+, black, lanes 1-3) and cardiac selective (RAD−/−, red, lanes 4-6) KO mice. Immunoblot detection of actinin and Cav1.2 (4 weeks posttamoxifen treatment) serve as loading controls Quantification by densitometry of (B) RAD levels and Cav1.2 a subunit (C) in the heart tissues. Statistical significance determined via unpaired t test, ∗∗∗∗p < 0.0001 versus RAD+/+ mice. D, I/V curves of basal L-type Ca2+ current in RAD+/+ (n = 23) and RAD−/− ventricular cardiomyocytes (n = 12). E–G, I/V curves of L-type Ca2+ current in absence and presence of Iso at 3 nM (E), 10 nM (F), and 100 nM (G). H, peak current density at 0, 3, 10, and 100 nM Iso in RAD+/+ and RAD−/− ventricular cardiomyocytes. I, concentration–response curve of Iso-elicited increase in current (baseline subtracted). Statistical significance determined via Holm–Sidak test following the two-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 versus groups without Iso. Iso, isoproterenol; Rad, Ras…

Figure 4. Inotropic dose response to β-adrenergic stimulation in RAD−/−mice. Isoproterenol dose response assessed (A) ejection fraction, (B) fractional shortening, and (C) heart rate in RAD+/+ (black; n = 24) and RAD−/− mice (red; n = 18) mice. Isoproterenol administration was by intraperitoneal injection. D and E, isoproterenol dose responsive changes (D) ejection fraction, (E) fractional shortening and (F) heart rate are indicated for RAD+/+ (black; n = 24) and RAD−/−(red; n = 18) mice. Summarized data of (G) the ratio of heart weight to body weight and in RAD+/+ (white, n = 15) and RAD−/− (red; n = 6) mice. H, summarized data on left ventricular posterior wall in RAD+/+ (white; n = 24) and RAD−/− mice (red; n = 18) mice. Statistical significance determined via Holm–Sidak test following the two-way ANOVA. ∗∗p < 0.01 and ∗∗∗p < 0.001 versus RAD+/+ mice; #p < 0.05 and ##p < 0.01 versus saline injection. Rad, Ras associated with diabetes.