Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

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

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

Cx26 hemichannels open in response to moderate elevations of CO2 (PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2. Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 5.6 nS, 95% confidence interval, 3.2 to 11.9 nS) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification, but using 30 mM propionate we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 5.3 nS (2.8 to 8.3 nS). This effect of propionate was unaffected by the K125R mutation (median reduction 7.7 nS, 4.1 to 11.0 nS). pH-dependent and CO2-dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the Keratitis Ichthyosis Deafness syndrome (N14K, A40V and A88V) also abolished the CO2-dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound, is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction

On the modulation of connexin 26 by CO2

The mechanism through which changes in PCO2 in the blood are detected is much disputed. Although many believe the stimulus for CO2 detection to be the associated increase in H+, increasing evidence supports a role for direct CO2 detection. In a recent development, Huckstepp et al demonstrated that connexin 26 hemichannels open in response to elevated CO2 in the absence of a pH change. This model however remained incomplete, with the mechanism of CO2 interaction with the channel being unknown. In this work I have employed site directed mutagenesis and dye loading studies to identify the CO2 binding site of connexin 26. This was found to be lysine 125, with binding through the formation of a carbamate bond. Mutational and modelling studies support an intersubunit salt bridge between the subsequent negative charge and the positive arginine 104 as the mechanisms of channel opening. Using this model for connexin 26 channel opening I was also able to manipulate the channel to respond to novel stimuli. These new mutants act both to support our mechanism for channel opening and to provide tools for further research. The production of connexin 26 channels lacking the key residues of interest also provides the basis of a novel method for producing animal models lacking CO2-sensitvity for further research in vivo. The findings of this work confirm that connexin 26 interacts with CO2 through direct binding. Connexin 26 therefore represents the first established CO2 sensor in the chemosensitive areas of the brain and strongly supports the idea that CO2 itself participates in monitoring PCO2 levels. As this model requires no accessory proteins, this work also offers the intriguing possibility that CO2-sensitvity may be an important function of this protein in other tissues and supports the idea of connexin proteins as novel ligand gated channels

Rational design of new NO and redox sensitivity into connexin26 hemichannels

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

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

Cx26 hemichannels open in response to moderate elevations of CO2 (PCO2 55 mmHg) via a carbamylation reaction that depends on residues K125 and R104. Here we investigate the action of CO2 on Cx26 gap junctions. Using a dye transfer assay, we found that an elevated PCO2 of 55 mmHg greatly delayed the permeation of a fluorescent glucose analogue (NBDG) between HeLa cells coupled by Cx26 gap junctions. However, the mutations K125R or R104A abolished this effect of CO2. Whole cell recordings demonstrated that elevated CO2 reduced the Cx26 gap junction conductance (median reduction 66.7%, 95% confidence interval, 50.5 to 100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30 mM propionate, we found that acidification in the absence of a change in PCO2 caused a median reduction in the gap junction conductance of 41.7% (26.6 to 53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 28.0 to 86.3%). pH‐dependent and CO2‐dependent closure of the gap junction are thus mechanistically independent. Mutations of Cx26 associated with the Keratitis Ichthyosis Deafness syndrome (N14K, A40V and A88V), in combination with the mutation M151L, also abolished the CO2‐dependent gap junction closure. Elastic network modelling suggests that the lowest entropy state when CO2 is bound, is the closed configuration for the gap junction but the open state for the hemichannel. The opposing actions of CO2 on Cx26 gap junctions and hemichannels thus depend on the same residues and presumed carbamylation reaction