The angiotensin II type 1 receptor (AT1R) closely interacts with large conductance voltage- and Ca2+-activated K+ (BK) channels and inhibits their activity independent of G-protein activation

Abstract

Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 μM) abolished the ANG-II (1 μM)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 μM GDPβS). In BK-expressing HEK293T cells, ANG-II (1 μM) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.

Keywords: Angiotensin II; Angiotensin II Type 1A Receptor; Cell Surface Receptor; Large Conductance Voltage- and Ca2+-activated K+ Channel; Patch Clamp; Potassium Channel; Protein Complex; Vascular Smooth Muscle Cells.

FIGURE 1.

ANG-II inhibits whole cell BK currents in freshly dissociated rat renal arterial smooth muscle cells via losartan-sensitive AT1R and independent of G-protein activation. A and B, whole cell BK currents at baseline (control) and after 1 μm ANG-II treatment in the same arterial myocyte. Whole cell currents were elicited by 20-ms pulses from −90 to 160 mV from a holding potential of 0 mV. C, mean percentage values normalized to control. The currents were reduced to 62 ± 7% (n = 5 cells, four independent experiments) of the original current after ANG-II treatment. D and E, ANG-II failed to inhibit BK whole cell currents when the same myocyte was pretreated with 10 μm losartan (20 min), an AT1R inhibitor. F, mean percentage BK current values were the same in cells pretreated with losartan and after stimulation with ANG-II (n = 9 cells, three independent experiments). G and H, superimposed traces with test pulses of 100 mV before and after ANG-II (1 μm) treatment in a control cell and in a cell intracellularly perfused with 500 μm GDPβS, an inhibitor of G-protein activation. I, mean values of paired experiments as in H showing that in the presence of GDPβS, ANG-II reduced currents to 68 ± 5% (n = 5 cells, two independent cell isolations) of its original value. Measurements were at the end of test pulses to 100 mV. In this and the following figures, the current traces are just prior to drug application and after the effect of drug treatment reached steady state. Error bars indicate S.D. values. *, p < 0.05.

FIGURE 2.

AT1R expression is essential for ANG-II inhibitory effect on BK channels. A and C, representative traces before and after application of 1 μm ANG-II in BK and AT1R-IRES-BK transfected cells, respectively. B and D, corresponding mean % values ± S.D. (n = 6 cells, four independent transfections for BK alone, and n = 3 cells, one transfection for AT1R + BK). Only when AT1R is expressed, ANG-II reduced BK currents to 66 ± 10% of its original value. E, example of time course of ANG-II action on BK currents. A similar time course was collected for other drugs. F, current traces before and after 1 μm ANG-II in AT1R-IRES-BK transfected cells pretreated with 10 μm losartan. G, corresponding mean % values (n = 5 cells, one transfection). H, example of immunoblot analysis of pERK1/2 in lysates from cells expressing AT1R and BK that were untreated (lane 1), treated with ANG-II alone (lane 2), treated with losartan alone (lane 3), and pretreated with losartan and stimulated with ANG-II (lane 4). pERK1/2 levels were higher in ANG-II treated cells (lane 2) and returned to near baseline in cells preincubated with losartan (lane 4) (n = 3 experiments). Loading and expression controls were ERK1/2, AT1R, and BK blots. All lanes were loaded with 30 μg of protein. Antibody concentrations were 186 ng/ml pERK1/2 pAb, 2.85 ng/ml ERK1/2 pAb, 250 ng/ml c-Myc pAb, and 525 ng/ml BK mAb. Error bars indicate S.D. values. *, p < 0.05. Ctrl, control; Los, losartan.

FIGURE 3.

BK and AT1R co-localization in HEK293T cells and in arterial myocytes. A–C, live labeling of BK (green) and AT1R (red) in transiently expressing HEK293T cells displaying co-localization at the plasma membrane. HEK293T cells were co-transfected with N-terminally tagged FLAG-AT1R and c-Myc-BK. Anti-FLAG mAb and anti-c-Myc pAb were used for labeling. D–F, labeling of BK (red) and AT1R (green) after permeabilization showing total expression and subplasmalemma co-localization in rat renal arterial smooth muscle cells. Anti-BK mAb and anti-AT1R pAb were used for labeling. G, a representative three-dimensional cross-correlation plot of BK → AT1R as a function of pixel shift in the x and y axes (arrows in A and B indicate the cell used for the plot). The cross-correlation surface has a peak at zero pixel shift that decays abruptly by shifting the image few pixels indicative of specific co-localization. H, quantification of co-localization in HEK293T cells and smooth muscle cells (SMCs) by PPI analysis (see “Experimental Procedures”). In HEK293T cells, PPI is 0.68 ± 0.08 for BK → AT1R and 0.87 ± 0.08 for AT1R → BK (n = 16 cells, three independent transfections). In rat renal arterial SMCs, PPI is 0.62 ± 0.08 for AT1R → BK and 0.71 ± 0.08 for BK → AT1R (n = 18 cells, two independent cell isolations). Error bars indicate S.D. values.

FIGURE 4.

Constitutive inhibition of BK channel activity by AT1R expression. A, voltage stimulation protocol. B and C, representative inside-out patch recordings of type 1 and 3 currents in HEK293T cells co-transfected with BK and AT1R. Fitted V_½ values were 0.4 and 36.8 mV, respectively. V_½ values were calculated using instantaneous tail currents, I, to obtain FP_o (G/G_max = I/I_max) as a function of the preceding test pulse voltage and fitting the data to a Boltzmann distribution as in F (see “Experimental Procedures”). D, examples of normalized BK tail currents (I/I_max) in cells expressing BK alone or BK + AT1R. The scheme (top trace) shows the pulse protocol. Current traces demonstrate that peak I/I_max (FP_o) magnitude followed the trend BK ≈ type 1 AT1R + BK > type 2 AT1R + BK ≈ type 3 AT1R + BK. E, BK activation kinetics was slowed down by AT1R co-expression in the order type 3 > type 2 > type 1 (test pulse = 60 mV) consistent with a larger decrease in P_o in type 3 AT1R + BK channels. F, average voltage activation curves (FP_o versus voltage) of BK expressed alone or in combination with AT1R. The error bars are within symbols and indicate S.E. Continuous lines are the means of the fitted curves of each experiment. Average V_½ values were −0.4 ± 3.2 for BK (n = 24 cells), −0.3 ± 2 for AT1R + BK type 1 (n = 22 cells), 13 ± 2 for AT1R + BK type 2 (n = 17 cells), and AT1R + BK type 3 = 35 ± 2 (n = 12 cells). Type 3 channels displayed the highest inhibition by AT1R co-expression (FP_o is lower at a given potential when compared with BK expressed alone). G, V_½ distribution in all patches (n = 79) from cells co-expressing BK and AT1R. Ca²⁺ concentration facing the intracellular side of the channels was 6.7 μm.

FIGURE 5.

AT1R forms a complex with BK in HEK293T cells. A, cartoons of seven-transmembrane domain BK α-subunit (left) and AT1R (right). B, AT1R pulls down BK (n = 3 independent experiments). Lanes 1–4, IP using anti-c-Myc pAb recognizing AT1R-c-Myc in lysates from transfected (+) or untransfected (−) cells with AT1R-c-Myc and/or BK; lanes 5 and 6, IP with rabbit IgG or protein G beads using lysates from cells co-transfected with AT1R-c-Myc and BK; lane 7, IP using a mixture of lysates from cells independently expressing AT1R-c-Myc or BK. Loading was equal in all lanes (25 μl). Lower panel, control immunoblot of BK expression in input cell lysates (30 μg of protein/lane). Immunoblots were with 525 ng/ml anti-BK mAb. C, control showing effective IP of AT1R in same cell lysates or mixture of lysates. Loading was equal in all lanes (5 μl). Lower panel, AT1R expression in input cell lysates (30 μg of protein/lane) probed with 250 ng/ml anti-c-Myc pAb. D, BK also pulls down AT1R (n = 2 independent experiments). Lanes 1–3, IP of lysates from cells expressing only AT1R, expressing only BK or co-expressing both proteins. IP was with anti-c-Myc pAb recognizing c-Myc-BK, and immunoblot was with 495 ng/ml anti-FLAG mAb recognizing AT1R. Lane 4, negative control using IgG to IP lysates from cells expressing both AT1R and BK. Lower panel, immunoblot of input lysates. E, control of effective IP of BK. Lower panel, expression of BK in corresponding input cell lysates. Immunoblot was with 525 ng/ml anti-BK mAb. IB, immunoblot; IE, independently expressing.

FIGURE 6.

Molecular analysis of BK-AT1R association. A, BK topology (left) and truncated BK constructs that were used for immunocytochemistry (right). RCK, regulator of conductance for K⁺. B, overlaid confocal images of cells co-expressing AT1R (red) and full-length or truncated BK (green) molecules: BK, BK_1–711, BK_1–441, BK_1–343, and BK_323–1113. Note that BK_323–1113 appears intracellular. Larger yellow squares show 5× magnification of the regions in the smaller yellow squares. C, specific co-localization analysis by PPI (25) demonstrating that the first 1–343 amino acids of BK are sufficient for its association with AT1R. PPI values were 0.68 ± 0.1 for BK to AT1R and 0.87 ± 0.08 for AT1R to BK (n = 16 cells, four independent experiments); 0.63 ± 0.06 for BK_1–711 to AT1R and 0.83 ± 0.12 for AT1R to BK_1–711 (n = 9 cells, two independent experiments); 0.75 ± 0.1 for BK_1–441 to AT1R and 0.88 ± 0.12 for AT1R to BK_1–441 (n = 13 cells, two independent experiments); 0.83 ± 0.11 for BK_1–343 to AT1R and 0.91 ± 0.07 for AT1R to BK_1–343 (n = 15 cells, three independent experiments); and 0.19 ± 0.08 for BK_322–1113 to AT1R and 0.26 ± 0.09 for AT1R to BK_322–1113 (n = 17 cells, three independent experiments). Error bars indicate S.D. values. *, p < 0.05.

FIGURE 7.

AT1R and BK channels co-localized FRET. A, co-localized FRET in cells co-expressing AT1R-YFP and BK-CFP fusion proteins, expressing CFP-linker-YFP (positive control), and co-expressing CFP and YFP independently (negative control). Bleed-through was calculated using cells individually expressing each molecule (data not shown). B, correlation of CFP-YFP co-localization and FRET signal. Red dots indicate high correlation, and blue dots indicate no correlation. C, mean values of normalized co-localized FRET were as follows for cells expressing: (i) CFP-linker-YFP, 46 ± 10.3 (n = 14); (ii) CFP + YFP 0.5 ± 0.8 (n = 16, two independent experiments); (iii) BK-CFP + AT1R-YFP, 28 ± 7.5 (n = 20); (iv) CFP + AT1R-YFP, 2.4 ± 3 (n = 10); and (v) BK-CFP + YFP, 1.1 ± 1.6 (n = 11). Co-localized FRET was analyzed with a pixel by pixel method using a FRET analyzer from ImageJ. *, p < 0.05 with respect to controls. coFRET, co-localized FRET.