The Angiotensin II Type 1 Receptor (AT1R) Closely Interacts with BK Channels and Inhibits their Activity Independent of G-protein Activation [Membrane Biology]

July 28th, 2014 by Zhang, Z., Li, M., Lu, R., Alioua, A., Stefani, E., Toro, L.

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 support AT1R role. 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 Forster 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.
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