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A3796 - Hypercapnia Decreases Na,K-ATPase β-Subunit Cell Surface Abundance by Inducing ER Retention of the Protein in Alveolar Epithelial Cells
Author Block: V. Kryvenko, W. Seeger, I. Vadász; Department of Internal Medicine, Justus Liebig University, Universities of Giessen and Marburg Lung Center, Member of the German Center for Lung Research, Giessen, Germany.
Rationale: High levels of CO2 (hypercapnia) and alveolar edema are often observed in patients with acute respiratory distress syndrome (ARDS) due to the dysfunction of the alveolo-capillary barrier. Na,K-ATPase is a heterodimeric glycoprotein, which plays a key role in alveolar fluid clearance and thus edema resolution that are critically required for survival. A disturbance of Na,K-ATPase protein folding in the endoplasmic reticulum (ER) may decrease protein abundance at the plasma membrane and impair fluid clearance. The aim of this study was to analyze how hypercapnia affects ER folding of the Na,K‑ATPase β‑subunit. Methods and Results: Exposing human alveolar epithelial A549 cells to elevated CO2 concentrations (up to 120 mmHg, pH 7.4) promoted significant retention of the Na,K‑ATPase β‑subunit in the ER in a time- and dose-dependent manner followed by a significant reduction of the enzyme abundance at the plasma membrane. Additionally, hypercapnia was found to increase the amount of deglycosylated Na,K‑ATPase β‑subunit. The ER-retained Na,K‑ATPase β‑subunit was not assembled with the Na,K-ATPase α‑subunit, suggesting that protein misfolding in ER prevents the formation of α:β complexes. Furthermore, treatment with α-ketoglutaric acid, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) or 4-phenylbutyric acid decreased the amount of ER-retained Na,K‑ATPase β‑subunit, suggesting that metabolic and Ca2+-dependent mechanisms were potentially involved in the retention of the enzyme. Moreover, hypercapnia-induced accumulation of misfolded proteins activated the unfolded protein response (UPR) by increasing phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by PKR-like endoplasmic reticulum kinase (PERK). Of note, knockdown of PERK increased the ER amount of the Na,K‑ATPase β‑subunit, suggesting an adaptive UPR. Conclusions: Taken together, our results show that hypercapnia disrupts normal folding of the Na,K‑ATPase β‑subunit by promoting ER retention of the enzyme and decreases protein abundance at the plasma membrane in alveolar epithelial cells. Elevated CO2 concentrations cause ER stress and activate the PERK branch of UPR by promoting phosphorylation of eIF2α. Given that alveolar fluid clearance is driven by the Na,K-ATPase located at the plasma membrane, revealing the mechanisms which interfere with the turnover and subcellular localization of the enzyme may provide a novel therapeutic approach for patients with ARDS.