The Angiotensin II Type 1 Receptor-Associated Protein Attenuates Angiotensin II-Mediated Inhibition of the Renal Outer Medullary Potassium Channel in Collecting Duct Cells

Juliano Zequini Polidoro; Nancy Amaral Rebouças; Adriana Castello Costa Girardi

doi:10.3389/fphys.2021.642409

The Angiotensin II Type 1 Receptor-Associated Protein Attenuates Angiotensin II-Mediated Inhibition of the Renal Outer Medullary Potassium Channel in Collecting Duct Cells

Front Physiol. 2021 May 14:12:642409. doi: 10.3389/fphys.2021.642409. eCollection 2021.

Authors

Juliano Zequini Polidoro¹, Nancy Amaral Rebouças², Adriana Castello Costa Girardi¹

Affiliations

¹ Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil.
² Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Abstract

Adjustments in renal K⁺ excretion constitute a central mechanism for K⁺ homeostasis. The renal outer medullary potassium (ROMK) channel accounts for the major K⁺ secretory route in collecting ducts during basal conditions. Activation of the angiotensin II (Ang II) type 1 receptor (AT1R) by Ang II is known to inhibit ROMK activity under the setting of K⁺ dietary restriction, underscoring the role of the AT1R in K⁺ conservation. The present study aimed to investigate whether an AT1R binding partner, the AT1R-associated protein (ATRAP), impacts Ang II-mediated ROMK regulation in collecting duct cells and, if so, to gain insight into the potential underlying mechanisms. To this end, we overexpressed either ATRAP or β-galactosidase (LacZ; used as a control), in M-1 cells, a model line of cortical collecting duct cells. We then assessed ROMK channel activity by employing a novel fluorescence-based microplate assay. Experiments were performed in the presence of 10^-10 M Ang II or vehicle for 40 min. We observed that Ang II-induced a significant inhibition of ROMK in LacZ, but not in ATRAP-overexpressed M-1 cells. Inhibition of ROMK-mediated K⁺ secretion by Ang II was accompanied by lower ROMK cell surface expression. Conversely, Ang II did not affect the ROMK-cell surface abundance in M-1 cells transfected with ATRAP. Additionally, diminished response to Ang II in M-1 cells overexpressing ATRAP was accompanied by decreased c-Src phosphorylation at the tyrosine 416. Unexpectedly, reduced phospho-c-Src levels were also found in M-1 cells, overexpressing ATRAP treated with vehicle, suggesting that ATRAP can also downregulate this kinase independently of Ang II-AT1R activation. Collectively, our data support that ATRAP attenuates inhibition of ROMK by Ang II in collecting duct cells, presumably by reducing c-Src activation and blocking ROMK internalization. The potential role of ATRAP in K⁺ homeostasis and/or disorders awaits further investigation.

Keywords: K+ channels; K+ homeostasis; angiotensin II type 1 receptor; angiotensin II type 1 receptor-associated protein; c-Src; kidney.