Abstract
A moderate increase in (55mmHg) closes Cx26 gap junctions. This effect of CO2 is independent of changes in intra- or extracellular pH. The CO2 -dependent closing effect depends on the same residues (K125 and R104) that are required for the CO2 -dependent opening of Cx26 hemichannels. Pathological mutations of Cx26 abolish the CO2 -dependent closing of the gap junction. Elastic network modelling suggests that the effect of CO2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein. Cx26 hemichannels open in response to moderate elevations of CO2 ( 55mmHg) 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 of 55mmHg 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% CI, 50.5-100.0%) but had no effect on Cx26K125R or Cx31 gap junctions. CO2 can cause intracellular acidification. Using 30mm propionate, we found that acidification in the absence of a change in caused a median reduction in the gap junction conductance of 41.7% (95% CI, 26.6-53.7%). This effect of propionate was unaffected by the K125R mutation (median reduction 48.1%, 95% CI, 28.0-86.3%). 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.
Highlights
The canonical function of connexins is to form intercellular junctions between cells – gap junctions – through the docking of hexameric connexons in the opposing membrane of each cell
We find that modest increases in PCO2 close complete gap junctions, and that this is most likely a direct effect mediated through CO2 binding to the same residues that result in the opening of the hemichannel
We adapted this type of assay to test whether increases in PCO2 could alter the permeation of a fluorescent glucose analogue (NBDG) through Cx26 gap junctions formed between HeLa cells
Summary
The canonical function of connexins is to form intercellular junctions between cells – gap junctions – through the docking of hexameric connexons in the opposing membrane of each cell. Hemichannels act as plasma membrane channels, which, in addition to mediating transmembrane ionic currents, permit the transmembrane fluxes of small molecules such as ATP (Stout et al 2002; Pearson et al 2005; Kang et al 2008; Huckstepp et al 2010a). CO2 opens the hemichannel and permits the efflux of ATP, which can act as a neurotransmitter. The resulting carbamylated amine bears a negative charge distributed over the oxygen atoms (and essentially converts the lysine side chain from a basic to an acidic moiety). This reaction differs from carbamylation involving cyanate, which results in a very stable modification of the amine. CO2 carbamylation is a highly reversible post-translational modification (Lorimer, 1983; Meigh, 2015)
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