biostudies-literatureNijjar SMedical Research CouncilLeverhulme Trust RPG-2015-090Biotechnology and Biological Sciences Research Council1071-1089https://www.ebi.ac.uk/biostudies/studies/S-EPMC11870076biostudies-literatureUnknown603(5)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 (GallThe Journal of physiologyMultiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO<sub>2</sub>.PMC11870076BB/T013346/1BB/S015132/1BB/T00746X/1MR/N014294/1MR/P010393/1F/00/128/AUNijjar SCann MDale NButler JGannon HGBrotherton DDospinescu VMCameron ALinthwaite VfalseMultiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO<sub>2</sub>.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 (Gall2025-01-01T00:00:00Z2025 Mar2026-06-02T08:06:03.376Z2025-04-04T13:46:52.577ZS-EPMC118700763990709610.1113/JP285885