Sensing molecular carbon dioxide: a translational focus for respiratory disease

Abstract

The last two decades of research on carbon dioxide have demonstrated that CO₂ is far more than a waste product of aerobic metabolism leading to acidosis and that it elicits biological responses directly via non-pH-dependent molecular interactions. New specialized methodologies have mapped CO₂ incorporation into specific regions of CO₂-sensitive proteins and linked these events to altered cellular function. CO₂ affects a host of biological responses related to respiratory disease, including control of respiration, protein maturation, alveolar fluid homeostasis, wound repair, innate immunity, host defense, and airway contractility. Elevated CO₂ (hypercapnia) appears to be primarily deleterious in pulmonary diseases, leading to a heightened interest in strategies to reduce excess CO₂ in patients with hypercapnic respiratory failure. Here, we summarize recently generated knowledge on molecular CO₂ sensing and signaling and the potential translational relevance of these processes in the context of respiratory disease. We need to grow this field further by encouraging experts in basic and translational science to contribute to more fully elucidating CO₂ sensing, signaling, and downstream effects. Understanding the biology and clinical consequences of perturbations in CO₂ homeostasis should no longer be considered secondary to studying oxygen sensing and signaling in respiratory medicine.

Keywords: CO2 sensing; CO2 signaling; carbon dioxide; hypercapnia; respiratory failure.

Figure 1.

Schematic representation of the mechanisms that may lead to the deleterious effects of hypercapnia in various tissues and organs. Created with BioRender.com.

Figure 2.

Mechanisms of CO₂ signaling and sensing in mammals. This schematic illustrates some or the mechanisms by which CO₂ is sensed and signals in mammalian systems, with selected examples. CO₂ in aqueous solution forms carbonic acid (H₂CO₃), which can dissociate to hydrogen ion (H⁺) and bicarbonate ion (HCO₃⁻). Thus, signaling can potentially be achieved through changes in CO₂ itself, pH or HCO₃⁻. Molecular CO₂ may be sensed through carbamylation of key residues in multiple proteins. CO₂-mediated carbamylation of connexin 26 (32), hemoglobin (–35) and ubiquitin (36) is well-documented. These direct CO₂-dependent modifications may mediate the chemosensing of CO₂ in the brain, alter the affinity of hemoglobin for oxygen, and activate gene transcription by triggering nuclear translocation of NF-κB). CO₂-dependent activation of transmembrane adenylyl cyclases (tmACs) and cAMP-dependent signaling have also been demonstrated (37). pH changes elicited by alterations in CO₂ contribute to changes in the rate and depth of breathing via pH-sensitive brainstem ion channels, including TASK-2 (20, 38) and the G-protein coupled receptor GPR4 (21). Fear associated with CO₂ exposure is elicited via pH-sensitive ASIC ion channels in the amygdala (26). HCO₃⁻ is sensed by soluble adenylyl cyclase (sAC) enzymes whose activity is required for sperm activation (39, 40). HCO3⁻ also mediates olfaction via guanylyl cyclase D and subsequent activation of cyclic nucleotide-gated channels in the mouse (41). Some mechanisms for detecting changes in CO₂ are proposed to be mediated by both molecular CO₂ and HCO₃⁻, such as activity of the receptor tyrosine kinase gamma (RPTPγ) in the proximal renal tubule (42).

Figure 3.

Detection of reversible protein carboxylation. A) CO₂-dependent formation of protein carbamate. B) Schematic of the TEO trapping method. TEO transfers an ethyl group (red) to the anionic carbamate derived from CO₂ (blue) and protein primary amine (green). C) Schematic of LysCarComp-MS. Lys-CO₂ is detected indirectly by OCNH/CO₂ competition. Homocitrulline (hCit), lysine carbamate (Lys-CO₂), tandem mass tags (TMT), and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Figure 4.

Chemical technologies for CO₂ sensing. A) Sensors based on the reactivity of amines. B) Sensors based on the reactivity of N-heterocyclic carbenes. C) Sensors based on the reactivity of iminophosphorane.

Figure 5.

Connexin 26 in CO₂ sensing. A) Model of human connexin 26 (2ZW3, (103)) indicating, with a dashed red line, the carbamate bridge proposed to form in the presence of CO₂ between Lysine 125 in subunit A (K125(a)) and Arginine 104 in subunit B (R104(b)). B) Ribbon cartoon of a cross-section of connexin 26 hemichannel, with membrane indicated with dotted blue lines (I: intracellular face, E: extracellular face). The four transmembrane helices are indicated 1–4, the N termini with N. The cytoplasmic loop is shown in cerise, and residues R104 and K125 are shown with yellow- and cerise-space-filling atoms, respectively. C) Cryo-EM coulomb shells for human connexin 26 vitrified at pH 7.4 in the presence of 2.5% CO₂ (blue) or 15% CO₂ (pink) viewed from the cytoplasmic side of the molecule. D) Schematic representation of the same view as (C) indicating the changes to the protein structure between 2.5% and 15% CO₂. (1) Anti-clockwise twist of the transmembrane helices, TM2 in particular (royal blue). E) Position of 4 KIDS mutations on a cross-section of human Cx26, indicating their proximity to regions of flexion in the structure. (Models for (B) and (E) were generated using Alphafold2 (105)).

Figure 6.

Schematic representation of signaling pathways involved in the downregulation of Na,K-ATPase during hypercapnia. Elevated CO₂ levels initiate specific signaling events that drive internalization of the Na,K-ATPase from the plasma membrane and its subsequent degradation. This response is mediated by both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms. Additionally, activation of endoplasmic reticulum-associated degradation (ERAD) occurs due to a hypercapnia-induced reduction of the endoplasmic reticulum (ER) Ca²⁺ pool and subsequent activation of inositol-requiring enzyme 1α (IRE1α). Moreover, hypercapnia enhances protein oxidation within the ER, resulting in oxidative modification of the Na,K-ATPase β-subunit and its ER retention, thereby further reducing Na,K-ATPase plasma membrane abundance and impairing alveolar fluid clearance. sAC, soluble adenylyl cyclase; cAMP, cyclic adenosine monophosphate; ERK1/2, extracellular signal-regulated kinase 1/2; CAMKK-β, calmodulin-dependent kinase (CAMKK)-β; PKC-ζ, protein kinase C-ζ; JNK, c-Jun N-terminal kinase; PM, plasma membrane; Ub, ubiquitin. Created with BioRender.com.

Figure 7.

Schematic depiction of hypercapnia-induced changes in the endoplasmic reticulum. Elevated CO₂ levels lead to a reduction in ATP levels, mitochondrial dysfunction and increased endoplasmic reticulum (ER) oxidation. Furthermore, activation of Ca²⁺ signaling, in part by ER membrane-localized inositol trisphosphate receptor (InsP3R), and impairment in the function of ATP-dependent Ca²⁺ transporters, such as sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), may result in the depletion of ER Ca²⁺ stores, phosphorylation of inositol-requiring enzyme 1 α (IRE1α), and promotion of ER-associated degradation (ERAD). Additionally, elevated CO₂ levels activate several key components of the unfolded protein response (UPR) cascade, including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), AMP-activated protein kinase (AMPK), B-cell lymphoma 2 protein (Bcl-2), B-cell lymphoma-extra large protein (Bcl-xL) heat shock protein 70 (HSP70) and caspase-7. Created with BioRender.com.

Figure 8.

Model of hypercapnia-mediated inhibition of β-catenin (βcat) signaling in AT2 cells. In normocapnia, AT2 progenitors are spatially proximal to PDGFRα expressing fibroblasts, which secret canonical Wnts, such as Wnt2, and maintain βcat signaling and AT2 proliferative capacity. PDGFRα /Wnt5a-expressing fibroblasts are spatially farther from the AT2 cell. Hypercapnia leads to reduced βcat signaling in AT2 cells, impairing cell renewal and differentiation by changing Wnt expression in PDGFRα stromal cells toward Wnt5a. The narrowness of the AT2 progenitor niche raises the possibility that elevated Wnt5a secretion in close proximity to the Wnt2 signal antagonizes βcat signaling in AT2 cells, inhibiting proliferative capacity.

Figure 9.

Schematic of the effects of hypercapnia on innate immunity and host defense and emerging information on mechanisms by which elevated CO₂ produces these effects.

Figure 10.

Proposed model of hypercapnia-mediated airway contractility. Lung airway cells sense and respond to changes in CO₂ levels, which modulates the airway tone, leading to airway contraction or relaxation via either vagal reflexes or specific molecular CO₂ signaling. Elevated CO₂ conditions, particularly those associated with acute respiratory acidosis, can have a relaxing effect on contracted airways due to pH effects. Airway smooth muscle (ASM) cell response to CO₂ and other gaseous molecules, Oxygen (O₂) and nitric oxide (NO), modulates intracellular Ca²⁺ signals to alter ASM cell contractility. The intracellular Ca²⁺ response may play a key role in the systems-level understanding of gaseous molecular signaling in lung airways. sGC, soluble guanylate cyclase; SOCE, store-operated Ca²⁺ entry. Created with BioRender.com.