Multiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO2.

CO₂ directly modifies the gating of connexin26 (Cx26) gap junction channels and hemichannels. This gating depends upon Lys125, and the proposed mechanism involves carbamylation of Lys125 to allow formation of a salt bridge with Arg104 on the neighbouring subunit. We demonstrate via carbamate trapping and tandem mass spectrometry that five Lys residues within the cytoplasmic loop, including Lys125, are indeed carbamylated by CO₂. The cytoplasmic loop appears to provide a chemical microenvironment that facilitates carbamylation. Systematic mutation of these Lys residues to Arg shows that only carbamylation of Lys125 is essential for hemichannel opening. By contrast, carbamylation of Lys108 and Lys125 is essential for gap junction closure to CO₂. Chicken (Gallus gallus) Cx26 gap junction channels lack Lys108 and do not close to CO₂, as shown by both a dye transfer assay and a high-resolution cryogenic electron microscopy structure. The mutation Lys108Arg prevents CO₂-dependent gap junction channel closure in human Cx26. Our findings directly demonstrate carbamylation in connexins, provide further insight into the differential action of CO₂ on Cx26 hemichannels and gap junction channels, and increase support for the role of the N-terminus in gating the Cx26 channel. KEY POINTS: Direct evidence of carbamylation of multiple lysine residues in the cytoplasmic loop of Cx26. Concentration-dependent carbamylation at lysines 108, 122 and 125. Only carbamylation of lysine 125 is essential for hemichannel opening to CO₂. Carbamylation of lysine 108 along with lysine 125 is essential for CO₂-dependent gap junction channel closure.