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

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.

Figure 1

Characterization of M-1 cell line by immunoblotting. Lysates from M-1 cells were immunoblotted with antibodies against anti-α-epithelial sodium channel (ENaC; A)and anti-renal outer medullary potassium (ROMK; B) for cell line characterization. Arrows indicate the predicted bands that represent each protein, including full-length and the cleaved form of α-ENaC.

Figure 2

Angiotensin II (Ang II) inhibits ROMK-mediated K⁺ conductance in M-1 cells. (A) M-1 cells were treated with different Ang II concentrations or vehicle for 30 min (n = 3). ^**p < 0.01 vs. vehicle-treated M-1 cells. The p value was calculated using ANOVA followed by the Bonferroni post hoc test. (B) M-1 cells were pretreated with telmisartan for 60 min and then treated with vehicle or Ang II (10⁻¹⁰ M) for 40 min. (C) M-1 cells were treated with Ang II (10⁻¹⁰ M) or vehicle in the presence or absence of the selective ROMK inhibitor VU591 (10 μM) for 40 min (n = 15). ^*p<0.05; ^***p<0.001; ^****p<0.0001 vs. vehicle-treated M-1 cells. The p value was calculated by using the Kruskal-Wallis test, followed by Dunn’s post hoc test.

Figure 3

Angiotensin II type 1 receptor-associated protein (ATRAP)-overexpression attenuates Ang II-mediated ROMK inhibition in M-1 cells. (A) Representative immunoblot of M-1 cells transfected with galactosidase (LacZ-V5) and ATRAP-V5 vectors probed with an anti-V5 antibody. (B) Evaluation of lipotransfection protocol efficiency in M-1 cells that were transfected with a GFP-encoding vector. Green fluorescence emission in M-1 cells was detected using a 505–550 nm filter in a confocal microscope after exciting GFP-transfected and mock-transfected M-1 cells at 488 nm laser light. Images were captured at 10x magnification. Mock-transfected M-1 cells are displayed in the inset. (C) K⁺ conductance was determined as the increase of fluorescence emission after thallium incubation in M-1 cells overexpressing LacZ or ATRAP treated with 10⁻¹⁰ M Ang II or vehicle for 40 min (n = 4). ^*p < 0.05 vs. vehicle-treated LacZ M-1 cells. The p value was calculated by the Kruskal-Wallis test, followed by Dunn’s post hoc test. (D) K⁺ conductance inhibition by Ang II treatment in LacZ‐ and ATRAP-overexpressing M-1 cells. ^*p < 0.05 vs. LacZ-overexpressing M-1 cells, using unpaired t-test.

Figure 4

AT1R-associated protein-overexpression blunts Ang II-mediated decrease of plasma membrane ROMK expression. M-1 cells overexpressing LacZ-V5 or ATRAP-V5 were treated with vehicle or 10⁻¹⁰ M Ang II for 30–40 min before assessing plasma membrane ROMK expression by biotinylation protocol. (A) Representative immunoblot image of biotinylated ROMK, total ROMK, and GAPDH. GAPDH was also used as a control to assess purity in biotinylated samples (Supplementary Material). (B) Densitometry analysis of bands from biotinylated ROMK (~45-kda) normalized to total ROMK expression (n = 4). As biotinylation protocol was performed in six-well plates, band intensities were normalized to each plate’s respective control (vehicle-treated LacZ sample). ^*p < 0.05 vs. vehicle-treated LacZ-overexpressing M-1 cells, using Kruskal-Wallis test followed by Dunn’s post hoc test. (C) Ang II-mediated reduction of ROMK expression at the plasma membrane in LacZ‐ and ATRAP-overexpressing M1-cells. ^*p < 0.05 vs. LacZ-overexpressing M-1 cells, using unpaired t-test. (D) Densitometry analysis of bands from total ROMK normalized to GAPDH expression (n = 4).

Figure 5

The effect of Ang II on the levels of phospho-c-Src in radioimmunoprecipitation assay (RIPA)-soluble lysates from M-1 cells overexpressing LacZ and ATRAP protein. M-1 cells overexpressing LacZ-V5 and ATRAP-V5 were acutely treated with vehicle or 10⁻¹⁰ M Ang II for 15 min and assessed for c-Src phosphorylation status. (A) Representative immunoblot of phospho-c-Src (Supplementary Material). (B) Graphical representation of the ratio of phosphorylated Src to GAPDH (n = 8). ^*p < 0.05; ^****p < 0.0001 vs. vehicle-treated LacZ-M-1 cells. ^††††p < 0.0001 vs. Ang II-treated-LacZ-M-1 cells. All statistical analyses were performed using ANOVA, followed by Bonferroni post hoc test.

Figure 6

Schematic representation of ATRAP-mediated actions on c-Src phosphorylation status, ROMK expression at the plasma membrane, and ROMK activity. M-1 cells overexpressing control LacZ protein has a decreased K⁺ conductance after Ang II treatment, an effect that was associated with lower expression of ROMK at the plasma membrane and induction of c-Src phosphorylation, the classical signaling pathway for ROMK inhibition by Ang II. Conversely, M-1 cells overexpressing the ATRAP do not show significant decreases in K⁺ conductance after Ang II treatment and plasma membrane ROMK expression, which was also associated with a blunted response of c-Src phosphorylation.