Endoplasmic Reticulum Calcium Levels Research Articles

Calcium Efflux From the Endoplasmic Reticulum Leads to β-Cell Death

It has been established that intracellular calcium homeostasis is critical for survival and function of pancreatic β-cells. However, the role of endoplasmic reticulum (ER) calcium homeostasis in β-cell survival and death is not clear. Here we show that ER calcium depletion plays a critical role in β-cell death. Various pathological conditions associated with β-cell death, including ER stress, oxidative stress, palmitate, and chronic high glucose, decreased ER calcium levels and sarcoendoplasmic reticulum Ca(2+)-ATPase 2b expression, leading to β-cell death. Ectopic expression of mutant insulin and genetic ablation of WFS1, a causative gene for Wolfram syndrome, also decreased ER calcium levels and induced β-cell death. Hyperactivation of calpain-2, a calcium-dependent proapoptotic protease, was detected in β-cells undergoing ER calcium depletion. Ectopic expression of sarcoendoplasmic reticulum Ca(2+)-ATPase 2b, as well as pioglitazone and rapamycin treatment, could prevent calcium efflux from the ER and mitigate β-cell death under various stress conditions. Our results reveal a critical role of ER calcium depletion in β-cell death and indicate that identification of pathways and chemical compounds restoring ER calcium levels will lead to novel therapeutic modalities and pharmacological interventions for type 1 and type 2 diabetes and other ER-related diseases including Wolfram syndrome.

Abstract LB-80: Role of ER calcium in Bcl-2 resistance to dosage and duration-related cytotoxic efficacy of Taxol

Abstract Background and Objectives: Calcium is one of the key regulators in apoptosis. Among various calcium sources, our previous study shows that the endoplasmic reticulum (ER), the main intracellular calcium storage site, provides a specific target for the chemotherapy agent Taxol and thus contributes to apoptosis. Although the expression of the anti-apoptotic protein Bcl-2 has been shown to confer resistance to Taxol, the exact mechanism of how it inhibits apoptosis and the role of ER calcium in this process remains unclear. This study investigated whether ER-associated calcium changes were generated and related to resistance by Bcl-2 to Taxol-induced apoptosis. Materials and methods: Fluo4-AM and Cameleon D1ER was used to monitor cytosolic and ER calcium dynamics by advanced time-lapsed imaging. Annexin V-FITC and propidium iodide were used to detect various phases of apoptosis induced by Taxol at different dosages from 10−12 to 10−5 M and at different durations from 5 min to 12 hours. Breast cancer cell lines MDA-MB-M468 and MDA-MB-231 were used and Bcl-2 transfectant cell lines were constructed by stable transfection. Results and Significance: Our previous data shows that Taxol (above 10−6 M) induces a rapid ER calcium release (peaking at 5 minutes) that promotes apoptosis. In this study, upon the expression of Bcl-2, basal ER calcium levels are decreased and Taxol-induced ER calcium release is inhibited, playing a major role in Bcl-2 resistance to Taxol. Depending upon dose and exposure time, long-term (hours) Taxol treatment partially overcame the Bcl-2 inhibition of ER calcium release by continually stimulating the ER calcium release, ultimately inducing significant, although smaller apoptosis in the presence of Bcl-2 expression. In addition to the effect on ER calcium, our data shows that the extracellular calcium pool is involved in Bcl-2 resistance through increased capacitative calcium entry to refill the ER calcium store, preventing ER calcium depletion. Taxol does not have a significant effect on the enhanced calcium influx from the extracellular medium. Taken together, out results indicate that the ER calcium store is the focus of the Taxol attack and plays a key role in Bcl-2 resistance to cytotoxic efficacy of Taxol in breast cancer cells. This finding provides insight to better understand the complex relationship between Bcl-2, calcium and Taxol towards improving breast cancer chemotherapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-80. doi:1538-7445.AM2012-LB-80

Regulation of Cathelicidin Antimicrobial Peptide Expression by an Endoplasmic Reticulum (ER) Stress Signaling, Vitamin D Receptor-independent Pathway

Vitamin D receptor (VDR)-dependent mechanisms regulate human cathelicidin antimicrobial peptide (CAMP)/LL-37 in various cell types, but CAMP expression also increases after external perturbations (such as infection, injuries, UV irradiation, and permeability barrier disruption) in parallel with induction of endoplasmic reticulum (ER) stress. We demonstrate that CAMP mRNA and protein expression increase in epithelial cells (human primary keratinocytes, HaCaT keratinocytes, and HeLa cells), but not in myeloid (U937 and HL-60) cells, following ER stress generated by two mechanistically different, pharmacological stressors, thapsigargin or tunicamycin. The mechanism for increased CAMP following exposure to ER stress involves NF-κB activation leading to CCAAT/enhancer-binding protein α (C/EBPα) activation via MAP kinase-mediated phosphorylation. Furthermore, both increased CAMP secretion and its proteolytic processing to LL-37 are required for antimicrobial activities occur following ER stress. In addition, topical thapsigargin also increases production of the murine homologue of CAMP in mouse epidermis. Finally and paradoxically, ER stress instead suppresses the 1,25(OH)(2) vitamin D(3)-induced activation of VDR, but blockade of VDR activity does not alter ER stress-induced CAMP up-regulation. Hence, ER stress increases CAMP expression via NF-κB-C/EBPα activation, independent of VDR, illuminating a novel VDR-independent role for ER stress in stimulating innate immunity.

Dendritic EGFP‐STIM1 activation after type I metabotropic glutamate and muscarinic acetylcholine receptor stimulation in hippocampal neuron

Several signaling pathways in neurons engage the endoplasmic reticulum (ER) calcium store by triggering calcium release. After release, ER calcium levels must be restored. In many non-neuronal cell types, this is mediated by store-operated calcium entry (SOCE), a cellular homeostatic mechanism that activates specialized store-operated calcium channels (SOC). Although much evidence supports the existence of SOCE in neurons, its importance has been difficult to determine because of the abundance of calcium channels expressed and the lack of SOC-specific pharmacological agents. We have explored the function of the SOCE-inducing protein STIM1 in neurons. In EGFP-STIM1-expressing hippocampal neurons, the sarco- and endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin caused rapid aggregation (i.e., activation) of STIM1 in soma and dendrites. Upon STIM1 activation by thapsigargin, a dramatic reduction in STIM1 mobility was detected by fluorescence recovery after photobleaching (FRAP). By triggering release of ER calcium with 3,5-dihydroxyphenylglycine (DHPG) or carbachol (Cch), agonists of type I metabotropic glutamate receptors (mGluR) and muscarinic acetylcholine receptors (mAChR), respectively, STIM1 was activated, and calcium entry (likely to represent SOCE) occurred in dendrites. It is therefore possible that neuronal SOCE is activated by physiological stimuli, some of which may alter the postsynaptic calcium signaling properties.

Endoplasmic reticulum Ca2+ increases enhance mutant glucocerebrosidase proteostasis

SummaryAltering intracellular calcium levels is known to partially restore mutant enzyme homeostasis in several lysosomal storage diseases, but why? We hypothesize that endoplasmic reticulum (ER) calcium level increases enhance the folding, trafficking and function of these mutant misfolding/degradation-prone lysosomal enzymes by increasing chaperone function. Herein, we report that increasing ER calcium levels by reducing ER calcium efflux through the ryanodine receptor (antagonists or RNAi) or by promoting ER calcium influx by SERCA2b overexpression enhances mutant glucocerebrosidase (GC) homeostasis in Gaucher’s disease patient-derived cells. Post-translational regulation of the calnexin folding pathway by increasing the ER calcium concentration appears to enhance the capacity of this chaperone system to fold mutant misfolding-prone enzymes, increasing the folded mutant GC population that can engage the trafficking receptor at the expense of ER-associated degradation, increasing the lysosomal GC concentration.

Calreticulin: non‐endoplasmic reticulum functions in physiology and disease

Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.

NMDA Receptor Stimulation Induces Reversible Fission of the Neuronal Endoplasmic Reticulum

With few exceptions the endoplasmic reticulum (ER) is considered a continuous system of endomembranes within which proteins and ions can move. We have studied dynamic structural changes of the ER in hippocampal neurons in primary culture and organotypic slices. Fluorescence recovery after photobleaching (FRAP) was used to quantify and model ER structural dynamics. Ultrastructure was assessed by electron microscopy. In live cell imaging experiments we found that, under basal conditions, the ER of neuronal soma and dendrites was continuous. The smooth and uninterrupted appearance of the ER changed dramatically after glutamate stimulation. The ER fragmented into isolated vesicles in a rapid fission reaction that occurred prior to overt signs of neuronal damage. ER fission was found to be independent of ER calcium levels. Apart from glutamate, the calcium ionophore ionomycin was able to induce ER fission. The N-methyl, D-aspartate (NMDA) receptor antagonist MK-801 inhibited ER fission induced by glutamate as well as by ionomycin. Fission was not blocked by either ifenprodil or kinase inhibitors. Interestingly, sub-lethal NMDA receptor stimulation caused rapid ER fission followed by fusion. Hence, ER fission is not strictly associated with cellular damage or death. Our results thus demonstrate that neuronal ER structure is dynamically regulated with important consequences for protein mobility and ER luminal calcium tunneling.

Mitochondrial apoptosis induced by BH3-only molecules in the exclusive presence of endoplasmic reticular Bak

Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3-only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3-only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER-stress surveillance machinery IRE1alpha/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3-only mediators can rely on reticular Bak to activate an ER-to-mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax-dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.

Endoplasmic reticulum generates calcium signalling microdomains around the nucleus and spindle in syncytial Drosophila embryos

Cell cycle calcium signals are generated by inositol trisphosphate-mediated release of calcium from internal stores [Ciapa, Pesando, Wilding and Whitaker (1994) Nature (London) 368, 875-878; Groigno and Whitaker (1998) Cell 92, 193-204]. The major internal calcium store is the ER (endoplasmic reticulum): the spatial organization of the ER during mitosis is important in defining a microdomain around the nucleus and mitotic spindle in early Drosophila embryos [Parry, McDougall and Whitaker (2005) J. Cell Biol. 171, 47-59]. Nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Mitosis is prevented by the InsP(3) antagonists Xestospongin C and heparin. Nuclear-localized transients and cortical transients rely on extraembryonic calcium, suggesting that ER calcium levels are maintained by calcium influx.

Maintaining Low Ca2+ Level in the Endoplasmic Reticulum Restores Abnormal Endogenous F508del‐CFTR Trafficking in Airway Epithelial Cells

The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del-CFTR/calnexin interaction and to restore the cAMP-dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5-di(t-butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del-CFTR transport activity. In particular, cyclopiazonic acid restores a 2-aminoethyoxydiphenyl borate-sensitive F508del-CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5-trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del-CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del-CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del-CFTR in human airway epithelial cells.

Rescue of Vasopressin V2 Receptor Mutants by Chemical Chaperones: Specificity and Mechanism

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.

Store-operated calcium entry in physiology and pathology of mammalian cells.

One of the numerous calcium-involving processes in mammalian cells is store-operated calcium entry (SOCE) -- the process in which depletion of calcium stores in the endoplasmic reticulum (ER) induces calcium influx from the extracellular space. Previously supposed to function only in non-excitable cells, SOCE is now known to play a role also in such excitable cells as neurons, muscles and neuroendocrine cells and is found in many different cell types. SOCE participates not only in processes dependent on ER calcium level but also specifically regulates some important processes such as cAMP production, T lymphocyte activation or induction of long-term potentiation. Impairment of SOCE can be an element of numerous disorders such as acute pancreatitis, primary immunodeficiency and, since it can take part in apoptosis or cell cycle regulation, SOCE may also be partially responsible for such serious disorders as Alzheimer disease and many types of cancer. Even disturbances in the 'servant' role of maintaining ER calcium level may cause serious effects because they can lead to ER homeostasis disturbance, influencing gene expression, protein synthesis and processing, and the cell cycle.

Anti-apoptotic protein Bcl-2 interacts with and destabilizes the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis.

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.

Presenilin-1 deficiency impairs glutamate-evoked intracellular calcium responses in neurons

Presenilin 1 (PS1) plays a critical role in cleaving amyloid precursor protein (APP) to produce amyloid-β (Aβ), the primary proteinaceous component of the senile plaques associated with Alzheimer's disease. In addition to mediating the cleavage of APP and a number of other proteins, a growing body of evidence suggests that PS1 also regulates intracellular endoplasmic reticulum calcium levels. Such findings suggest that PS1 activity may modulate neuronal excitability, as well. To address this issue we examined cytosolic intracellular calcium responses in PS1-deficient neurons stimulated by the excitatory amino acid neurotransmitter, glutamate. We found that glutamate-induced intracellular calcium levels were markedly reduced in neurons lacking PS1 (−/−) compared with heterozygous (+/−) and wild-type (+/+) neurons. To prove that PS1 was sufficient to mediate normal glutamate-induced calcium responses, we used a Semliki-forest virus (SFV) vector to express wild-type PS1 in PS1 knock-out neurons. We found that heterologous PS1 expression restored glutamate-evoked calcium responses in PS1-deficient neurons to levels matching non-infected wild-type cells. PS1-deficient neurons infected with SFV directing expression of β-galactosidase failed to rescue the wild-type phenotype. These results support the idea that normal PS1 activity regulates neuronal responses to neurotransmitter stimulation.

ER Calcium Critical for Necrosis

Necrosis is cell death that results from injury or cell stress and is biochemically and morphologically distinct from apoptosis, or programmed cell death. In the worm Caenorhabditis elegans , activated mutants of degenerin channels, such as the Na + channel, can cause necrotic cell death in the cells expressing the mutant channels. Xu et al. identified calreticulin in a screen for mutations that suppress necrosis in a degenerin Na + channel mutant background. Calreticulin mutants suppressed necrosis in neurons and muscles and in response to two different necrosis-inducing stresses: activated Na + channels and ectopic expression of activated Gα s . Calreticulin mutants could not suppress necrosis induced by activated Ca 2+ channels, suggesting that calreticulin was affecting calcium homeostasis not protein synthesis. Loss of function mutants of the inositol triphosphate receptor or the ryanodine receptor [two calcium release channels in the endoplasmic reticulum (ER)] or suppression of expression of calnexin were all also able to suppress necrosis in the activated Na + channel mutants. Pharmacological manipulations that decreased ER calcium levels or ER calcium release also inhibited necrosis. Thus, ER calcium release and buffering capacity controlled by the calcium binding proteins calreticulin and calnexin appear essential for stimulating necrosis by stresses not involving calcium influx across the plasma membrane. K. Xu, N. Tavernarakis, M. Driscoll, Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca 2+ release from the endoplasmic reticulum. Neuron 31 , 957-971 (2001). [Online Journal]

Reduced Capacitative Calcium Entry Correlates with Vesicle Accumulation and Apoptosis

A preneoplastic variant of Syrian hamster embryo cells, sup(+), exhibits decreased endoplasmic reticulum calcium levels and subsequently undergoes apoptosis in low serum conditions (Preston, G. A., Barrett, J. C., Biermann, J. A., and Murphy, E. (1997) Cancer Res. 57, 537-542). This decrease in endoplasmic reticulum calcium appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30-100 microM) or cell-impermeant 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N', N'-tetraacetic acid (5-10 mM) is able to induce apoptosis of cells in conditions where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis of cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of cells with bafilomycin (30-100 nM), an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles, decreased capacitative entry, and induced apoptosis. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.

Systolic blood pressure measurement in newborn with transcutaneous Doppler.

Elevated carbon dioxide levels (hypercapnia) are frequently observed in patients with acute and chronic lung diseases. Na,K-ATPase is a sodium transporter and junctional molecule that is critically required for the maintenance of optimal alveolar fluid balance and thus gas exchange. Here, we explored how hypercapnia affects maturation of the Na,K-ATPase in the endoplasmic reticulum (ER). Murine precision-cut lung slices and human alveolar epithelial cells exposed to elevated CO₂ levels showed a rapid and dose-dependent reduction of the Na,K-ATPase β-subunit in the ER and decreased Na,K-ATPase plasma membrane abundance. By combining chemical and genetic approaches, we identified that hypercapnia activated inositol-requiring enzyme 1α (IRE1α) and initiated a specific signaling pathway that resulted in ER-associated degradation (ERAD) of the Na,K-ATPase β-subunit. ERAD of the β-subunit prevented assembly with the α-subunit of the enzyme and thus downregulated formation of functional transporter molecules as well as delivery of the complex to the cell surface. Moreover, we observed that IRE1α activation was triggered by changes in ER and cytosolic calcium levels, and were dependent on the activity of the inositol trisphosphate ER receptor (IP3R). Treatment with the IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB) prevented calcium dysregulation and increased plasma membrane abundance of the Na,K-ATPase α/β-complex. Accordingly, our results suggest that elevated CO₂ levels decrease Na,K-ATPase plasma membrane abundance by altering ER calcium homeostasis and enhancing degradation of the Na,K-ATPase β-subunit in the ER.