A number of gaseous molecules, including nitric oxide and carbon monoxide, play important roles in many cellular processes by acting as signalling molecules. Surprisingly, however, it has long been assumed that carbon dioxide – a gaseous molecule that is produced during cellular metabolism – is not a signalling molecule.

Controlling the concentration of carbon dioxide (CO₂) in a biological system is essential to sustain life, and it was thought that the body used pH – which is the concentration of hydrogen ions – as a proxy for the level of CO₂. The concentration of CO₂ is related to pH because CO₂ reacts with water to form carbonic acid, which quickly breaks down to form hydrogen ions and bicarbonate ions. This close relationship has led many researchers to equate pH-sensing with CO₂-sensing, and to suggest that a physiological receptor for CO₂ does not exist.

Recent research into structures called connexin hemichannels has challenged this view. Researchers found that when pH levels were held constant, increasing the level of CO₂ caused the structures to open up, suggesting that CO₂ could be directly detected by the hemichannels. Each hemichannel contains six connexin subunits, but the details of how the CO₂ molecules interact with the individual connexin subunits to open up the hemichannels remained mysterious.

Now Meigh et al. show that CO₂ molecules bind to a specific amino acid (lysine) at a particular place (residue 125) in one of the connexin subunits to form a carbamate group. This group then interacts with the amino acid (arginine) at residue 104 in a neighbouring connexin subunit to form a carbamate bridge between the two subunits. This leads to structural changes that cause the gap junction hemichannels to open and release signals that can activate other cells. Since connexin hemichannels are found throughout the human body, these results suggest that CO₂ might act as a signalling molecule in processes as diverse as the control of blood flow, breathing, hearing and reproduction.

CO₂ is the unavoidable by-product of cellular metabolism. Humans produce approximately 20 moles of CO₂ per day (Marshall and Bangert, 2008). The dissolved CO₂ can readily combine with water, aided by carbonic anhydrase, to form H₂CO₃, which dissociates rapidly to H⁺ and HCO₃⁻. In any physiological solution therefore, the partial pressure of CO₂ (PCO₂) will be in equilibrium with, and inescapably related to, the pH and the concentration of HCO₃⁻ of that solution. Regulation of PCO₂ is thus a vital homeostatic function that is linked to acid-base balance.

As might be expected, chemosensory reflexes regulate the frequency and depth of breathing to ensure homeostatic control of blood gases. The field of respiratory chemosensitivity has been dominated by ‘reaction theory’ which posits that pH is a sufficient signal for detection of changes in PCO₂ (Loeschcke, 1982). Many investigators therefore equate pH-sensing with CO₂-sensing. There are several areas of the medulla oblongata which contain neurons that respond to changes in pH/CO₂, especially near the highly vascularised ventral surface. For example a population of neurons highly sensitive to pH/CO₂ have been described in the retrotrapezoid nucleus (RTN) (Mulkey et al., 2004, 2006; Guyenet et al., 2008) and the medullary raphé nucleus (Richerson, 2004; Ray et al., 2011). Despite the acceptance of pH-sensing as the predominant mechanism by which PCO₂ is measured, there is substantial evidence for an additional and independent effect of molecular CO₂ (Eldridge et al., 1985; Shams, 1985; Huckstepp and Dale, 2011). For example, if pH is carefully controlled at the medullary surface, an increase in PCO₂ at constant pH will still enhance breathing by as much as a pH change at constant PCO₂ (Shams, 1985). We have recently shown that connexin 26 (Cx26) hemichannels, open in response to increases in PCO₂ at constant extracellular pH and are an important conduit for the CO₂-dependent, as opposed to pH-dependent, release of ATP (Huckstepp et al., 2010a). Cx26 hemichannels contribute to the chemosensory control of breathing (Huckstepp et al., 2010b; Wenker et al., 2012). Hemichannels of two closely related connexins, Cx30 and Cx32, also exhibited CO₂-sensitive opening (Huckstepp et al., 2010a). Despite this evidence, widespread acceptance of direct sensing of CO₂ requires a detailed molecular explanation of any putative transduction system.

One possible way that CO₂ can interact with proteins is via carbamylation—the formation of a covalent bond between the carbon of CO₂ and a primary amine group. For example, CO₂ forms carbamate bonds with haemoglobin (Kilmartin and Rossi-Bernardi, 1971) and the plant enzyme RuBisCo (Lundqvist and Schneider, 1991). Here we document an important new advance—the mechanism by which CO₂ binds directly to Cx26, most probably via carbamylation of a lysine residue, to cause hemichannel opening. Our work establishes a new field of direct CO₂ sensing that can be mediated by CO₂-dependent carbamylation of certain β connexins. As these are widely distributed in the brain and other tissues, it is likely that direct detection of CO₂ via this mechanism is important in many different physiological processes.

We have previously demonstrated that Cx26, and two related β connexins, Cx30 and Cx32, open when exposed to modest increases in PCO₂ at constant pH (Huckstepp et al., 2010a). This previous study demonstrated, in inside-out and outside-out excised membrane patches at a transmembrane potential of −40 mV, that Cx26 hemichannel gating respectively increased and decreased in response to increases and decreases of PCO₂. To reconfirm our previous findings that Cx26, and not some other hemichannel senses CO₂ (Huckstepp et al., 2010a), we demonstrated that the CO₂-dependent dye loading of HeLa cells expressing Cx26 was blocked by 100 µM carbenoxolone, but unaffected by 1 mM probenecid, a blocker of pannexin-1, (Silverman et al., 2008), and 20 µM ruthenium red, a blocker of calhm1 (Taruno et al., 2013), (Figure 1—figure supplement 1). Parental HeLa cells do not exhibit CO₂-dependent dye loading demonstrating that the heterologous expression of Cx26 is essential for this function (Huckstepp et al., 2010a) (Figure 3—figure supplement 1).

The extent of CO₂ chemosensitivity within the β connexins

To document the extent to which this sensitivity to CO₂ is limited within the β connexin family (Figure 1F), and to form the basis of a bioinformatic comparison to identify possible CO₂ binding motifs, we investigated whether another β connexin, Cx31, might also be sensitive to CO₂. We expressed, in HeLa cells, either rat Cx31 or rat Cx26 tagged at the C-terminal with mCherry and used a previously described dye loading assay (Huckstepp et al., 2010a) to test whether the cells could load with carboxyfluorescein (CBF) in a CO₂-dependent manner. As expected from our previous work, HeLa cells expressing the Cx26 readily loaded with CBF when exposed to this dye in the presence of elevated PCO₂ (55 mmHg, at pH 7.5, Figure 1A,B). However, HeLa cells expressing Cx31 failed to dye load in a CO₂-dependent manner (Figure 1A,B). As the connexins were tagged with mCherry, we could verify the presence of fluorescent puncta in both the Cx26 and Cx31 expressing HeLa cells (Figure 1—figure supplement 2). To check for the existence of functional hemichannels in the Cx31-expressing HeLa cells, we removed extracellular Ca²⁺ as a positive control. This manipulation will open all types of connexin hemichannel. Parental HeLa cells do not load with dye when Ca²⁺ is removed from the medium (Figure 3—figure supplement 1); they therefore possess virtually no endogenous hemichannels. The removal of extracellular Ca²⁺ readily caused loading of CBF into the Cx31-expressing HeLa cells (Figure 1A, inset), demonstrating the presence of functional Cx31 hemichannels.

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Insertion of the carbamylation motif into Cx31 creates a CO₂-sensitive mutant hemichannel

Our analysis predicts that if we were to introduce the putative carbamylation-motif of Cx26 into Cx31, the resulting mutant Cx31 (mCx31) should be sensitive to CO₂ as the lysine introduced into the sequence should be able to form a salt bridge with the native residue K104 in mCx31 once carbamylated (Figure 1C–E and 2A). We therefore made mCx31 in which the motif TQKVREI was introduced in place of K123H124 of the native connexin (Figure 2A). This insertion/substitution maintained the correct orientation of the K125 with respect to K104 of Cx31. HeLa cells expressing mCx31 displayed clear CO₂-dependent dye loading (Figure 2B,C). We confirmed the CO₂ sensitivity of mCx31 expressing HeLa cells by performing whole cell patch camp recordings. mCx31-expressing cells exhibited a conductance change of 3.3 ± 0.84 nS (mean ± SEM, n = 8, Supplementary file 1) when exposed to elevated PCO₂ (Figure 2D). Cells, expressing wild type Cx31 showed no CO₂-dependent changes in their whole cell conductance (mean conductance change −0.002 ± 0.023 nS, n = 6, Supplementary file 1, Figure 2D).

Figure 2

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K125 and R104 are essential for CO₂ sensitivity

To demonstrate that K125 is the key residue for interaction with CO₂, we made mCx31^K125R, by inserting TQRVREI into Cx31 in place of K123H124. Unlike lysine, the arginine side chain cannot be carbamylated by CO₂ as its pKa (12.5) is much higher than that of lysine (10.5), therefore this variant should have no sensitivity to CO₂. mCx31^K125R did indeed lack sensitivity to CO₂ (Figure 3A–C, Figure 3—source data 1). This was not because the mutant channel failed to assemble correctly, as the positive control of zero Ca²⁺ dye loading demonstrated the presence of functional hemichannels (Figure 3A, Figure 3—figure supplement 1). Next we investigated the relevant residues in Cx26 itself. The carbamate bridge that we propose must involve K125 (being carbamylated) and R104 (forming the salt bridge with the carbamylated lysine). We therefore made mutations that individually disrupted both of these functions: K125R to prevent carbamylation, and R104A to disrupt formation of the salt bridge. Neither Cx26^K125R nor Cx26^R104A exhibited sensitivity to CO₂ sensitivity. Nevertheless the positive controls demonstrated the presence of functional mutant hemichannels in the expressing HeLa cells (Figure 3A, Figure 3—figure supplement 1).

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Figure 3—source data 1

Median differences in pixel intensity between CO₂ and control dye loading experiments for the various connexin hemichannel variants in the histograms of Figure 3C and statistical analysis: Kruskal-Wallis anova, pairwise Mann-Whitney tests and false discovery rate procedure.

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Engineering an analogue of the carbamylated lysine into Cx26 makes it constitutively open

To test further our prediction that the carbamylated K125 forms a salt bridge with R104, we made the mutation K125E in Cx26. The insertion of glutamate in place of the lysine has the potential to act as an analogue of the carbamylated K125. If our hypothesis is correct, this mutated channel should be constitutively open, as the carboxyl group of the E125 should be able to form a salt bridge with R104. We found that HeLa cells expressing Cx26^K125E readily loaded with dye under control conditions and exhibited no sensitivity to CO₂ (Figure 4). The Cx26^K125E-expressing HeLa cells showed much greater loading under control conditions than parental HeLa cells (Figure 4, Figure 4—source data 1). To further confirm that the constitutive dye loading occurred via the misexpressed connexin, we demonstrated that carbenoxolone (100 µM) completely blocked CO₂-dependent dye loading in HeLa cells expressing Cx26^K125E (Figure 4, Figure 4—source data 1).

Figure 4

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Figure 4—source data 1

Median pixel intensity values for histogram in Figure 4C and statistical analysis: Kruskal-Wallis anova and pairwise Mann-Whitney tests.

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Reasoning that if bridge formation between subunits was key to opening the hemichannel, we also made the further mutation R104E. In the mutated channel E104 has the potential to form a salt bridge in the reverse direction with K125 and we predicted that if this were to happen such a mutant hemichannel should also be constitutively open. We found that HeLa cells expressing Cx26^R104E did indeed load with dye under control conditions and that this enhanced dye loading was blocked with carbenoxolone (Figure 5, Figure 5—source data 1).

Figure 5

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Figure 5—source data 1

Median pixel intensity values for histogram in Figure 5C and statistical analysis: Kruskal-Wallis anova and pairwise Mann-Whitney tests.

https://doi.org/10.7554/eLife.01213.013

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Elastic network model of Cx26 shows that carbamylation leads to hemichannel opening

Although our experimental data point to the importance of carbamylation of K125 and the formation of a salt bridge to R104 in the adjacent subunit, it is not clear how this would lead to opening of the Cx26 hemichannel. Course-grained modelling reduces protein atomistic complexity for more efficient computational studies of harmonic protein dynamics and is particularly suited to examining hemichannel opening over millisecond time scales (Sherwood et al., 2008). We therefore built coarse-grained elastic network models (ENM) to gain insight into the mechanism by which CO₂ maintains Cx26 in the open state. In an ENM the Cα-atom co-ordinates of an atomic resolution structure are used to represent a protein structure. The total global protein motion within the ENM consists of a defined number of modes, each of a characteristic frequency and representing a particular harmonic motion within the protein. ENMs are known to reproduce the global low frequency modes of protein motion well in comparison to experimental data (Delarue and Sanejouand, 2002; Valadie et al., 2003). We used the co-ordinates from a high-resolution crystal structure to construct an ENM (Tirion, 1996) for the Cx26 hexamer in the unbound and CO₂-bound states. CO₂ was represented in the ENM by the inclusion of six additional Hookean springs between residues K125 and R104 of neighbouring monomers (Figure 6A).

Figure 6

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Analysis of the model revealed that in the absence of CO₂ the lowest frequency mode (mode 1) represented an opening/closing motion that was able to fully occlude the hemichannel central pore (Video 1). Addition of springs representing CO₂-binding to the ENM restricted the closing motions of the hemichannel and thus connexin 26 was maintained in the open state (Video 2). We examined the overlap in the ordering of the modes in the unbound and CO₂ bound states to gain insight into how this occurs. A significant reordering of the lowest frequency modes to higher frequencies was observed in the presence of CO₂ rather than the removal of any modes from the total protein motion (Figure 6B). Mode 1, the lowest frequency mode that represents the opening/closing mode, represented about 40% of the total protein motion in the absence of CO₂. In the presence of CO₂ this closing mode is reordered through a change in its frequency as mode 9, which accounts for only about 2% of the total motion (Figure 6B,C). CO₂ therefore opens Cx26 through a reordering of the normal modes of global protein motion such that the normal closing motion of Cx26 no longer significantly contributes to the total motion of the hemichannel. We infer from this that the carbamate bridge formed between Cx26 monomers represents a constraining force that hinders hemichannel closure.

Video 1

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Video 2

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Evidence from six different experimental tests supports our hypothesis that CO₂ forms a carbamate bridge between K125 and R104 on the adjacent subunit to open the Cx26 hemichannel. Firstly, we demonstrated the sufficiency of the carbamylation motif to confer CO₂ sensitivity by inserting it into a CO₂-insensitive connexin, Cx31. Secondly, we showed that K125 of the carbamylation motif was essential for this motif to confer CO₂ sensitivity on Cx31. Thirdly and fourthly, we demonstrated that the mutations K125R and R104A in Cx26 (to prevent carbamate bridging at either end of the bridge) destroyed the CO₂ sensitivity of this connexin. Fifthly, by exploiting glutamate as an analogue of the carbamylated K125 (in Cx26^K125E), we demonstrated a gain of function—Cx26^K125E was constitutively open, yet had lost sensitivity to CO₂. Finally, we further tested the bridging concept by demonstrating that the bridge is in effect bidirectional: the mutated hemichannel Cx26^R104E, in which E104 can bridge to K125 in the reverse direction, was also constitutively open, but had no sensitivity to CO₂.

Although we have not directly demonstrated CO₂ binding to Cx26, our extensive testing of this hypothesis through selective mutations leads us to conclude that CO₂ interacts with Cx26 directly and that no other protein is required for CO₂ sensitivity. This interaction is most probably via carbamylation of K125. Interestingly, the mutations Cx26^K125E and Cx26^K125R can be considered respectively as open-state and closed-state analogues of the wild type channel. Collectively, our data strongly suggests that CO₂ binds to the intracellular surface of Cx26 and must therefore cross the membrane to reach this site. This could occur either direct diffusion through the membrane bilayer, potentially via Cx26 itself, or via other CO₂ permeable channels (Boron et al., 2011). Amongst its many other functions, Cx26 can therefore be regarded as a receptor for CO₂. Interestingly, this mechanism of modulation applies to both Cx30 and Cx32, which can both potentially form a carbamate bridge at equivalent residues to Cx26. In the case of Cx32 this would involve bridging to K104 rather than R104 (in Cx26 and 30). Cx26 can co-assemble with both Cx30 and Cx32 to form heteromeric hemichannels (Forge et al., 2003; Yum et al., 2007). Our structural studies predict that, as Cx30 and Cx32 have K125 and either R104 or K104, carbamate bridges could form in such heteromeric hemichannels and that they should also therefore be CO₂-sensitive.

Carbamylation involves formation of a labile covalent bond between the carbon of CO₂ and a primary amine. For this to occur the amine must be in a restricted hydration space so that it is not protonated. Some examples of physiologically significant carbamylation are known. The carbamylation of the N-terminal amines of haemoglobin contributes to the Bohr effect (Kilmartin and Rossi-Bernardi, 1971), whereby the affinity of haemoglobin for O₂ is reduced in the presence of elevated CO₂. However in mammalian systems no other examples of carbamylation by CO₂ have been described. In C3 photosynthetic plants, the enzyme RuBisCo, that participates in the Calvin cycle and carbon fixation is activated by carbamylation of a specific lysine residue (Lundqvist and Schneider, 1991). Several microbial enzymes are also carbamylated (Maveyraud et al., 2000; Golemi et al., 2001; Young et al., 2008).

Despite this precedent, the functional significance of CO₂-carbamylation and its potential as a transduction principle for the measurement of CO₂ has been almost completely overlooked in vertebrate physiology. The mechanism of formation of a salt bridge between a carbamylated lysine and an appropriately oriented arginine on the neighbouring subunit is a unique mechanism for modulation of an ion channel and establishes carbamylation as a mechanistic basis for the direct signalling of PCO₂ in mammalian physiology. This carbamylation of a lysine to transduce the concentration of CO₂ into a biological signal is somewhat equivalent to the nitrosylation of a cysteine residue by NO/nitrite. It establishes a CO₂-dependent signalling paradigm in which the concentration of CO₂ is signalled by ATP release via Cx26 from the chemosensory cell and consequent activation of neighbouring cells, or potentially by a Ca²⁺ influx through the Cx26 hemichannel (Fiori et al., 2012) to initiate a Ca²⁺ wave within the chemosensory cell itself and further Ca²⁺-dependent signalling processes.

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Author details

1. Louise Meigh

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   LM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Sophie A Greenhalgh

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   SAG, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Thomas L Rodgers

   1. Biophysical Sciences Institute, University of Durham, Durham, United Kingdom
   2. Department of Chemistry, University of Durham, Durham, United Kingdom

   Contribution

   TLR, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Martin J Cann

   1. Department of Chemistry, University of Durham, Durham, United Kingdom
   2. School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom

   Contribution

   MJC, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. David I Roper

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   DIR, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Nicholas Dale

   School of Life Sciences, University of Warwick, Coventry, United Kingdom

   Contribution

   ND, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   n.e.dale@warwick.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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