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.
Highlights
Appropriate regulation of K+ content in the extracellular fluid is critical for cell function, especially for excitable tissues, since the K+ gradient largely determines resting membrane potential (Unwin et al, 2011)
In order to assess whether the angiotensin II (Ang II)-mediated effect on K+ conductance was induced by Ang II type 1 receptor (AT1R) activation, M-1 cells were pretreated with telmisartan (10−8 M) before exposing cells to vehicle or Ang II (10−10 M)
It is well established that AT1R upregulation in collecting duct cells with consequent ROMK inhibition constitutes an essential mechanism for K+ conservation during low-K+ diets
Summary
Appropriate regulation of K+ content in the extracellular fluid is critical for cell function, especially for excitable tissues, since the K+ gradient largely determines resting membrane potential (Unwin et al, 2011). Seminal studies by Giebisch, Malnic, and Klose during the 1960s have shown the crucial role of distal nephron to the regulated kaliuresis under different experimental conditions (Malnic et al, 1964). They have found that the proximal tubule and the Henle of Loop segments reabsorb ~90% of all filtered K+, regardless of the amount of K+ contained in the diet. The appropriate regulation of renal losses occurs in the downstream segments by further K+ reabsorption or secretion from the peritubular space (Malnic et al, 1964)
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