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.
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.
authors: From the Departments of Anesthesiology.
authors: Zhu
authors: Z
authors: Zhang
authors: From the Departments of Anesthesiology.
authors: Min
authors: M
authors: Li
authors: From the Departments of Anesthesiology.
authors: Rong
authors: R
authors: Lu
authors: From the Departments of Anesthesiology.
authors: Abderrahmane
authors: A
authors: Alioua
authors: From the Departments of Anesthesiology, Physiology, the Brain Research Institute, and the Cardiovascular Research Laboratory, University of California, Los Angeles, California 90095.
authors: Enrico
authors: E
authors: Stefani
authors: From the Departments of Anesthesiology, the Brain Research Institute, and the Cardiovascular Research Laboratory, University of California, Los Angeles, California 90095 Molecular and Medical Pharmacology, and ltoro@ucla.edu.
authors: Ligia
authors: L
authors: Toro
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D047228:Angiotensin II Type 1 Receptor Blockers / Q000008:administration & dosage*; D000818:Animals; D020013:Calcium Signaling; D031541:Fluorescence Resonance Energy Transfer; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D019808:Losartan / Q000008:administration & dosage; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D012077:Renal Artery / Q000187:drug effects / Q000378:metabolism
chemical_list: D047228:Angiotensin II Type 1 Receptor Blockers; D051036:Large-Conductance Calcium-Activated Potassium Channels; D044140:Receptor, Angiotensin, Type 1; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D019808:Losartan
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