Project Outline

Connexins are large pore channels, which are regulated by various signals, such as pH, voltage or Ca²⁺. Research in Warwick has shown that connexin26 (Cx26) is also directly regulated by CO₂ independently of pH (1), and this can affect the control of breathing (2). Mutations of Cx26 are one of the main causes of congenital deafness, with rare mutations causing keratitis ichthyosis deafness syndrome (KIDS). Connexins function either as channels through a single membrane (a hemichannel) or as a channel between two neighbouring cells formed from two docked hemichannels (a gap junction). Interestingly whereas CO₂ closes gap junctions it opens hemichannels (3). The effect of CO₂ has been shown to be mediated through a specific lysine in the protein through carbamylation, a reversible covalent interaction that changes the charge on the lysine. Mutation of the lysine prevents the CO₂ response. Using cryo-EM we have shown the effect that CO₂ has on the structure of the gap junctions, modulating the aperture of the channel (4) and how this is linked to the lysine (5). However, we still do not understand this process fully or know how CO₂ can have a different effect on the hemichannels. The project would investigate these questions further, using cryo-EM and molecular dynamics to investigate how CO₂ affects channels or hemichannels and how this is affected by different disease-causing mutations. The project would be co-supervised by Prof Nicholas Dale who made the discovery of the direct effect of CO₂ on the activity of the channels with Phill Stansfeld supervising the molecular dynamics.

Relevant papers

1. Meigh L, Hussain N, Mulkey DK, & Dale N (2014) Connexin26 hemichannels with a mutation that causes KID syndrome in humans lack sensitivity to CO2. Elife 3, e04249. doi:10.7554/eLife.04249
2. van de Wiel J, Meigh L, Bhandare A, Cook J, Nijjar S, Huckstepp R, & Dale N (2020) Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata. Commun Biol 3, 521. doi:10.1038/s42003-020-01248-x
3. Sarbjit Nijjar DM, Louise Meigh, Elizabeth de Wolf, Thomas Rodgers, Martin Cann, Nicholas Dale (2020) Opposing modulation of Cx26 gap junctions and hemichannels by CO2
4. Brotherton DH, Savva CG, Ragan TJ, Dale N, & Cameron AD (2022) Conformational changes and CO2-induced channel gating in connexin26. Structure. doi:10.1016/j.str.2022.02.010
5. Brotherton DH, Nijjar S., Savva CG, Dale, N & Cameron A.D. (2023) Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO_2. bioRxiv 2023.08.22.554292; doi:https://doi.org/10.1101/2023.08.22.554292

Techniques

• Molecular biology
• Growth of bacterial cultures
• Solubilisation and purification of membrane proteins
• Cryo-EM
• Assays
• Molecular Dynamics Simulations