Hypercapnia Induces Inositol-Requiring Enzyme 1α--Driven Endoplasmic Reticulum--associated Degradation of the Na,K-ATPase β-Subunit.

Acute respiratory distress syndrome is often associated with elevated levels of CO₂ (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA β-subunit (NKA-β) in the ER. Exposing murine precisioncut lung slices and human alveolar epithelial A549 cells to elevated CO₂ levels led to a rapid decrease of NKA-β abundance in the ER and at the cell surface. Knockdown of ER mannosidase a class 1B member 1 and ER degradationenhancing a-mannosidase like protein 1 by siRNA or treatment with the mannosidase a class 1B member 1 inhibitor kifunensine rescued loss of NKA-β in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1a (IRE1α), and treatment with an siRNA against IRE1a prevented the decrease of NKA-β in the ER. Of note, the hypercapniainduced phosphorylation of IRE1a was triggered by a Ca²⁺-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1a in precision-cut lung slices and A549 cells, suggesting that Ca²⁺ efflux from the ER might be responsible for IRE1a activation and ERAD of NKA-b. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia. [ABSTRACT FROM AUTHOR]