Journal: Journal of Biological Chemistry

Article Title: 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

doi: 10.1074/jbc.m114.595603

Figure Lengend Snippet: 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 M 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 M ANG-II in AT1R-IRES-BK transfected cells pretreated with 10 M 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 g 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.

Article Snippet: For biochemistry and immunocytochemistry we used C-terminal c-Myc-tagged AT1R (AT1R-c-Myc) in pCMV6Entry (Origene), N-terminal FLAG-tagged AT1R (FLAGAT1R) in pcDNA3 (that includes the signal peptide, KTIIALSYIFCLVFA, to promote membrane expression), and BK -subunit without tag (BK) or with an N-terminal c-Myc tag (c-Myc-BK) (21) in pcDNA3.

Techniques: Expressing, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: 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

doi: 10.1074/jbc.m114.595603

Figure Lengend Snippet: 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 3 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, quantifica- tion 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 3 AT1R and 0.87 0.08 for AT1R 3 BK (n 16 cells, three independent transfections). In rat renal arterial SMCs, PPI is 0.62 0.08 for AT1R 3 BK and 0.71 0.08 for BK 3 AT1R (n 18 cells, two independent cell isolations). Error bars indicate S.D. values.

Article Snippet: For biochemistry and immunocytochemistry we used C-terminal c-Myc-tagged AT1R (AT1R-c-Myc) in pCMV6Entry (Origene), N-terminal FLAG-tagged AT1R (FLAGAT1R) in pcDNA3 (that includes the signal peptide, KTIIALSYIFCLVFA, to promote membrane expression), and BK -subunit without tag (BK) or with an N-terminal c-Myc tag (c-Myc-BK) (21) in pcDNA3.

Techniques: Labeling, Expressing, Clinical Proteomics, Membrane, Transfection

Journal: Journal of Biological Chemistry

Article Title: 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

doi: 10.1074/jbc.m114.595603

Figure Lengend Snippet: 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 V1⁄2 values were 0.4 and 36.8 mV, respectively. V1⁄2 values were calculated using instantaneous tail currents, I, to obtain FPo (G/Gmax I/Imax) 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/Imax) in cells expressing BK alone or BK AT1R. The scheme (top trace) shows the pulse protocol. Current traces demonstrate that peak I/Imax (FPo) 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 Po in type 3 AT1R BK channels. F, average voltage activation curves (FPo 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 V1⁄2 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 (FPo is lower at a given potential when compared with BK expressed alone). G, V1⁄2 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 M.

Article Snippet: For biochemistry and immunocytochemistry we used C-terminal c-Myc-tagged AT1R (AT1R-c-Myc) in pCMV6Entry (Origene), N-terminal FLAG-tagged AT1R (FLAGAT1R) in pcDNA3 (that includes the signal peptide, KTIIALSYIFCLVFA, to promote membrane expression), and BK -subunit without tag (BK) or with an N-terminal c-Myc tag (c-Myc-BK) (21) in pcDNA3.

Techniques: Inhibition, Activity Assay, Expressing, Transfection, Activation Assay, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: 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

doi: 10.1074/jbc.m114.595603

Figure Lengend Snippet: FIGURE 5. AT1R forms a complex with BK in HEK293T cells. A, cartoons of seven-transmembrane domain BK -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 l). Lower panel, control immunoblot of BK expression in input cell lysates (30 g 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 l). Lower panel, AT1R expression in input cell lysates (30 g 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.

Article Snippet: For biochemistry and immunocytochemistry we used C-terminal c-Myc-tagged AT1R (AT1R-c-Myc) in pCMV6Entry (Origene), N-terminal FLAG-tagged AT1R (FLAGAT1R) in pcDNA3 (that includes the signal peptide, KTIIALSYIFCLVFA, to promote membrane expression), and BK -subunit without tag (BK) or with an N-terminal c-Myc tag (c-Myc-BK) (21) in pcDNA3.

Techniques: Transfection, Expressing, Control, Western Blot, Negative Control

Journal: Journal of Biological Chemistry

Article Title: 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

doi: 10.1074/jbc.m114.595603

Figure Lengend Snippet: 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, BK1–711, BK1–441, BK1–343, and BK323–1113. Note that BK323–1113 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 BK1–711 to AT1R and 0.83 0.12 for AT1R to BK1–711 (n 9 cells, two independent experiments); 0.75 0.1 for BK1–441 to AT1R and 0.88 0.12 for AT1R to BK1–441 (n 13 cells, two independent experiments); 0.83 0.11 for BK1–343 to AT1R and 0.91 0.07 for AT1R to BK1–343 (n 15 cells, three independent experiments); and 0.19 0.08 for BK322–1113 to AT1R and 0.26 0.09 for AT1R to BK322–1113 (n 17 cells, three independent experiments). Error bars indicate S.D. values. *, p 0.05.

Article Snippet: For biochemistry and immunocytochemistry we used C-terminal c-Myc-tagged AT1R (AT1R-c-Myc) in pCMV6Entry (Origene), N-terminal FLAG-tagged AT1R (FLAGAT1R) in pcDNA3 (that includes the signal peptide, KTIIALSYIFCLVFA, to promote membrane expression), and BK -subunit without tag (BK) or with an N-terminal c-Myc tag (c-Myc-BK) (21) in pcDNA3.

Techniques: Construct, Immunocytochemistry, Expressing