Multiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO 2

Nijjar, Sarbjit; Brotherton, Deborah; Butler, Jack; Dospinescu, Valentin‐Mihai; Gannon, Harry G.; Linthwaite, Victoria; Cann, Martin; Cameron, Alexander; Dale, Nicholas

Abstract

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. image 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.

Citation

Nijjar, S., Brotherton, D., Butler, J., Dospinescu, V., Gannon, H. G., Linthwaite, V., Cann, M., Cameron, A., & Dale, N. (online). Multiple carbamylation events are required for differential modulation of Cx26 hemichannels and gap junctions by CO 2. The Journal of Physiology, https://doi.org/10.1113/jp285885

• Journal Article Type: Article
• Acceptance Date: Jan 13, 2025
• Online Publication Date: Feb 5, 2025
• Deposit Date: Feb 10, 2025
• Publicly Available Date: Feb 10, 2025
• Journal: The Journal of Physiology
• Print ISSN: 0022-3751
• Electronic ISSN: 1469-7793
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• DOI: https://doi.org/10.1113/jp285885
• Keywords: carbamylation, connexin, cryo‐EM, gap junction channel, carboxylation, hemichannel
• Public URL: https://durham-repository.worktribe.com/output/3474709