High CO₂levels impair Na, K-ATPase activity independently of pH.

<p>(A) ATII cells were exposed to 40, 60, 80 and 120 mmHg CO₂with extracellular pH (pH_e): 7.4 or to 40 mmHg CO₂with pH_e: 7.2 for 30 min, and Na,K-ATPase activity was measured as [γ-³²P]ATP hydrolysis. Graph represents the mean±SEM, (n = 5). (B) ATII cells were treated as described in (A) and the Na,K-ATPase protein abundance at the plasma membrane (PM) was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α₁-subunit at the PM and total protein abundance are shown. (C) Live cell imaging of GFPα₁-A549. Cells were exposed to 40 mmHg CO₂(pH_e: 7.4) for 10 min (left panels) and then switched to 120 mmHg CO₂(pH_e: 7.4) (right, upper panel) or to 40 mmHg CO₂(pH_e: 7.2) (right, lower panel) for 30 min. White arrows indicate the plasma membrane Na,K-ATPase. (D) Live cell imaging of GFPα₁-A549. Cells were exposed to 40 mmHg CO₂(pH_e: 7.4) for 10 min (left panel), switched to 120 mmHg CO₂(pH_e: 7.4) (middle panel) for 30 min and switched back to 40 mmHg CO₂(pH_e: 7.4) for 60 min (left panel). White arrows indicate the plasma membrane Na,K-ATPase * p<0.05, ** p<0.01.