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
| Autor: | Rong Lu, Abderrahmane Alioua, Ligia Toro, Enrico Stefani, Zhu Zhang, Min Li |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
BK channel
Patch-Clamp Techniques G protein Green Fluorescent Proteins Biochemistry Losartan Receptor Angiotensin Type 1 Renal Artery GTP-binding protein regulators GTP-Binding Proteins Membrane Biology Fluorescence Resonance Energy Transfer Animals Humans Calcium Signaling Large-Conductance Calcium-Activated Potassium Channels Patch clamp Receptor Molecular Biology Calcium signaling Muscle Cells biology Chemistry Angiotensin II Cell Biology Potassium channel Rats HEK293 Cells biology.protein Biophysics Angiotensin II Type 1 Receptor Blockers |
| Zdroj: | Journal of Biological Chemistry. 289:25678-25689 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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