*2.2. Elevated CO2 Levels Decrease Na,K-ATPase Plasma Membrane Abundance and Function*

It has been previously demonstrated that NKA-α cannot leave the ER before being assembled with the regulatory NKA-β subunit and that only a functional NKA α:β complex exported from the ER can reach the cellular surface [19,20]. Thus, to determine whether the hypercapnia-induced increase in the amount of ER-resident NKA-β influenced PM expression of the NKA, we performed a cell-surface protein biotinylation and the streptavidin pull-down assay (Figure 2A).

**Figure 2.** Exposure to elevated CO2 levels for up to 12 h decreases Na,K-ATPase plasma membrane abundance and function. (**A**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) for different time-points. Plasma membrane (PM) abundance of NKA-α and NKA-β was determined by biotin-streptavidin pull-down and immunoblotting. Representative immunoblots of NKA-α, NKA-β, and transferrin receptor (TfR) at the PM are shown. Bars represent the NKA-α or NKA-β/TfR ratio. Values are expressed as mean ± SD (*n* = 3, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). (**B**) Primary rat ATII and (**C**) human A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with extracellular pH = 7.4 for 12 h. The PM fraction was isolated by ultracentrifugation and ouabain-sensitive NKA activity was measured by using a colorimetric ATP bioluminescence assay kit. Values are expressed as mean ± SD (*n* = 5, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

Exposure of AEC to elevated CO2 concentrations decreased the cell surface abundance of the NKA-α and NKA-β subunits in a time-dependent manner. These findings correlated with the above-mentioned increase in the levels of ER-resident NKA-β (Figure 1D), which suggests that the ER retention of NKA-β contributed to the decreased cell-surface expression of the enzyme. Next, NKA function was assessed by measuring ouabain-sensitive ATPase activity in isolated PM fractions from primary rat alveolar epithelial type II (ATII) and human A549 cells (Figure 2B,C). Consistent with the results of our biotinylation assays, in both cellular cultures, elevated CO2 levels markedly decreased ouabain-sensitive ATPase activity, which indicates reduced NKA function.

#### *2.3. Hypercapnia Induces Endoplasmic Reticulum Retention of the Na,K-ATPase* β*-Subunit*

The increased abundance of ER-resident NKA-β (that was not efficiently delivered to the plasma membrane) might be explained by retention of NKA-β in the ER [20]. To test this hypothesis, we performed isolation of ER and Golgi subcellular fractions from total cellular lysates and determined the amount of high mannose and complex N-glycan type NKA-β (Figure 3A). We observed increased amounts of NKA-β in the ER upon hypercapnia, which was associated with decreased Golgi-resident complex forms of the enzyme. These results suggested that newly synthesized forms of NKA were not processed to further maturation steps but were retained in the ER, which leads to decreased cell-surface abundance of the transporter.

Recent reports showed that the ER chaperones, binding immunoglobulin protein (BiP), and calnexin are required for the folding and retention of the NKA-β subunit upon normal and stress conditions [20]. To further assess if those chaperones were involved in the hypercapnia-induced ER retention of the NKA-β, AEC were exposed to normal or elevated CO2 levels and localization of NKA-β, calnexin, and BiP were detected by immunofluorescent microscopy (Figure 3B,C). Enhanced co-localization of NKA-β with calnexin and BiP was observed upon hypercapnia, which indicates that both chaperons might be involved in the process of ER retention.

**Figure 3.** Elevated CO2 levels promote endoplasmic reticulum (ER) retention of Na,K-ATPase-β. (**A**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. Subcellular fractions were isolated by ultracentrifugation and protein levels of the NKA-β, protein disulfide isomerase (PDI; an ER marker), and Golgin subfamily A member 2 (GM130;a Golgi marker) were analyzed by immunoblotting. Representative Western blots are shown. Bars represent ER NKA-β/Coomassie ratio or complex type NKA-β/Coomassie ratio. Values are expressed as mean ± SD (*n* = 4, \*\* *p* < 0.01, \*\*\* *p* < 0.001). (**B**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. Cellular localization of NKA-β and calnexin were determined by immunofluorescence. Immunofluorescence staining of NKA-β (red), calnexin (green), and nuclei (blue) are shown. Scale bar—20 μM. (**C**) A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. Cellular localization of NKA-β and BiP were determined by immunofluorescence. Representative immunofluorescence staining of NKA-β (red), BiP (green), and nuclei (blue) are shown. Scale bar—20 μM.

#### *2.4. Hypercapnia Attenuates Na,K-ATPase* α*:*β *Complex Formation*

Previous studies have reported that ER quality control allows only export of assembled NKA α:β complexes at a 1:1 stoichiometric ratio to the Golgi, which sustains an equimolar ratio of NKA α-subunits and β-subunits at the cellular surface [19,21]. Having demonstrated that elevated CO2 levels caused ER retention of the NKA-β, we further analyzed the formation of the NKA α:β complex upon hypercapnia treatment. To this end, we employed A549-α1-green fluorescent protein (A549-α1-GFP) cells in which GFP was fused to the NKA-α subunit and exposed those cells to normal or elevated CO2 levels. Afterward, we immunoprecipitated NKA-β from either total cell lysates or ER fractions by using specific antibodies and the amount of co-immunoprecipitated NKA-α was detected by immunoblotting (Figure 4A,B). Our results showed that hypercapnia decreased the amount of precipitated NKA α-subunit, which suggests that the ER-retained NKA-β was only partially assembled with NKA-α. Similar results were obtained when reverse co-immunoprecipitation with an antibody against GFP was performed (Figure 4C).

**Figure 4.** Elevated CO2 levels decrease formation of the Na,K-ATPase-α:β complex. (**A**) A549-α1-GFP expressing cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. NKA-β was immunoprecipitated from the whole cell lysate by using an NKA-β-specific antibody and levels of co-immunoprecipitated NKA-α were analyzed by immunoblotting. Representative Western blots are shown (*n* = 4). (**B**) A549-α1-GFP expressing cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. The ER fraction was isolated and NKA-β was immunoprecipitated and the levels of co-immunoprecipitated NKA-α were analyzed by immunoblotting. Representative Western blots are shown (*n* = 3). (**C**) A549-α1-GFP expressing cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. NKA-α1-GFP (GFP-α) was immunoprecipitated from the whole cell lysate by using a GFP-specific antibody and the levels of co-immunoprecipitated NKA-β were analyzed by immunoblotting. Representative Western blots are shown (*n* = 4).

### *2.5. Elevated CO2 Levels Alter the Oxidizing Environment of the Endoplasmic Reticulum and Promote Oxidation of the Na,K-ATPase* β*-Subunit*

It is well documented that a fully functional ER is dependent on physiological levels of Ca2<sup>+</sup> and ATP and requires an oxidizing environment [22,23]. However, it has been shown that sustained hypercapnia and hypercapnic acidosis may decrease ATP production and perturb mitochondrial function [24,25]. Thus, we speculated that elevated CO2 levels may prevent NKA-β maturation in the ER by altering the metabolic status of the cell. In line with this notion, we observed a significant decrease in intracellular ATP levels in hypercapnia-exposed AEC (Figure 5A). Of note, cell viability was not affected by elevated CO2 levels even when AEC were exposed to hypercapnia for up to 72 h (Figure 5B).

**Figure 5.** Hypercapnia decreases intracellular ATP production but does not affect cell viability. (**A**) A549 were treated with normal (Ctrl, 40 mmHg, pHe = 7.4) or elevated CO2 levels (CO2, 120 mmHg, pHe = 7.4) for different time-points. Intracellular ATP levels were measured by using an ATP bioluminescence assay kit. Bars represent total ATP/total protein ratio. Values are expressed as mean ± SD (*n* = 3, \*\* *p* < 0.01, \*\*\* *p* < 0.001). (**B**) A549 cells were exposed to normal (Ctrl, 40 mmHg, pHe = 7.4) or elevated CO2 levels (CO2, 120 mmHg, pHe = 7.4) for different time-points. Cell viability was measured by assessing the plasma membrane integrity. Graph bars represent the percentage of viable cells. Values are expressed as mean ± SD (*n* = 3).

It has been previously reported that decreased cellular ATP levels can promote protein oxidation by activating redox reactions [26,27]. Therefore, we next hypothesized that hypercapnia may alter the oxidizing environment of cells. To determine whether increased CO2 levels affect protein oxidation in hypercapnia-treated AEC, we isolated total, cytosolic, and ER fractions from whole-cell homogenates by ultracentrifugation and, subsequently, assessed protein oxidation in these fractions. Importantly, in contrast to the total protein and cytosolic fractions (Figure 6A,B), hypercapnia treatment augmented the levels of protein oxidation in the ER (Figure 6C).

In addition, to determine whether NKA was a substrate of protein oxidation, AEC were exposed to normal or elevated CO2 concentrations and the levels of protein oxidation in immunoprecipitated NKA-β were detected by immunoblotting (Figure 6C). Of note, we detected that the levels of NKA-β oxidation were increased when exposed to elevated CO2 levels, which suggests that hypercapnia dysregulated the oxidizing status of the ER environment. Therefore, it induces the oxidation of NKA-β and potentially disrupts normal maturation of the enzyme.

**Figure 6.** Hypercapnia increases ER protein and Na,K-ATPase-β oxidation levels. A549 cells were exposed to 40 (Ctrl) or 120 mmHg CO2 (CO2) with an extracellular pH = 7.4 for 12 h. Next, cellular fractions were isolated by ultracentrifugation and protein oxidation, which was determined in (**A**) total protein, (**B**) cytosolic, and (**C**) ER fraction by immunoblotting against 2,4-dinitrophenylhydrazone (2,4-DNPH). Bars represent a 2,4-DNPH/Coomassie ratio. Values are expressed as mean ± SD (*n* = 5, \* *p* < 0.05). (**D**) A549 cells were exposed to normal (Ctrl, 40 mmHg, pHe = 7.4) or elevated CO2 levels (CO2, 120 mmHg, pHe = 7.4) for 12 h. NKA-β was then immunoprecipitated and protein oxidation was determined as described above. Representative Western blots are shown.
