CO₂-dependent opening of Connexin 43 hemichannels

Sequence and structure comparisons between alpha and beta connexins, Cx43 and Cx26, revealed that Cx43 has a motif, the carbamylation motif, that confers CO₂-sensitivity on a subset of beta connexins. By using a fluorescent dye loading assay, whole cell patch clamp recordings and real time measurement of ATP release via GRAB_ATPwe have demonstrated that Cx43 hemichannels open in a highly CO₂sensitive manner over the range 20 to 70 mmHg. Mutational analysis confirms that the equivalent residues to those in Cx26 that have been shown to be involved in mediating the effects of CO₂on gating of hemichannels and gap junction channels, also mediate Cx43 hemichannel gating. Our data predicts that Cx43 will be partially open and able to release ATP at resting physiological levels of PCO₂. We have tested this prediction in acute hippocampal slices, by showing that CO₂-dependent enhancement of synaptic transmission can be blocked by the Cx43-selective mimetic peptide Gap26. Our data resolves an inconsistency in the literature betweenin vivostudies suggesting that Cx43 hemichannels are at least partially open at rest, andin vitrostudies, performed in the absence of HCO₃⁻/CO₂buffering that show Cx43 hemichannels are shut. Our evidence suggests that the ancestral gene that duplicated to give the alpha and beta connexin clades must have possessed the carbamylation motif. CO₂sensitivity is thus a fundamental ancient characteristic of several connexins that has been lost in more recently derived members of this gene family.