Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2.DOI: http://dx.doi.org/10.7554/eLife.01213.001

Mutations in connexin26 (Cx26) underlie a range of serious human pathologies. Previously we have shown that Cx26 hemichannels are directly opened by CO2 (Meigh et al., 2013). However the effects of human disease-causing mutations on the CO2 sensitivity of Cx26 are entirely unknown. Here, we report the first connection between the CO2 sensitivity of Cx26 and human pathology, by demonstrating that Cx26 hemichannels with the mutation A88V, linked to Keratitis-Ichthyosis-Deafness syndrome, are both CO2 insensitive and associated with disordered breathing in humans.DOI: http://dx.doi.org/10.7554/eLife.04249.001

CO2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanisms. The mutation K125C gives potential for nitrosylation on Cys125 and formation of an SNO bridge to R104 of the neighbouring subunit. Unlike wild-type Cx26 hemichannels, which are insensitive to NO and NO2−, hemichannels comprising Cx26K125C can be opened by NO2− and NO donors. However, NO2− was unable to modulate the doubly mutated (K125C, R104A) hemichannels, indicating that an inter-subunit bridge between C125 and R104 is required for the opening action of NO2−. In a further test, we introduced two mutations into Cx26, K125C and R104C, to allow disulfide bridge formation across the inter-subunit boundary. These doubly mutated hemichannels open in response to changes in intracellular redox potential.

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.

A moderate increase in P CO 2 (55 mmHg) closes Cx26 gap junctions. r This effect of CO 2 is independent of changes in intra-or extracellular pH. r The CO 2-dependent closing effect depends on the same residues (K125 and R104) that are required for the CO 2-dependent opening of Cx26 hemichannels. r Pathological mutations of Cx26 abolish the CO 2-dependent closing of the gap junction. r Elastic network modelling suggests that the effect of CO 2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein.

AbstractBreathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally links the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.

Cx26 hemichannels are opened by modest levels of CO2 (PCO2 55 mmHg) acting via a novel carbamylation mechanism to bias the hemichannel to the open state. In the hemichannel, CO2 nonenzymatically carbamylates Lys125, and the resulting carbamate forms a salt bridge to Arg104 of the neighbouring subunit. By contrast, similar modest levels of CO2 cause Cx26 gap junction closure.Gap junctions of Cx31, a beta connexin that lacks the carbamylation motif, are insensitive to these levels of CO2. Mutations of the carbamylation motif, Lys125Arg and Arg104Ala, which abrogate hemichannel opening in Cx26, also prevent gap junction closing. Elastic network modelling suggests that the lowest entropy state, when CO2 is bound, is in the open configuration for the hemichannel and the closed configuration for the gap junction Surprisingly, we conclude that the opposing actions of CO2 on Cx26 gap junctions and hemichannels results from carbamylation of the same residues.

Carbamate bonds occur following the nucleophilic attack of CO2 on to an amine. In proteins, this can occur at lysine side chains or at the N-terminus. For CO2 binding to occur an amine must be present in the NH2 form and consequently carbamates represent a site-specific post-translational modification, occurring only in environments of reduced hydration. Due to the specific nature of these interactions, coupled with the inability of these bonds to survive protein preparation methods, carbamate reactions appear rare. However, more biologically important examples continue to emerge that use carbamates as key parts of their mechanisms. In this review, we discuss specific examples of carbamate bond formation and their biological consequences with an aim to highlight this important, and often forgotten, biochemical group.