Structural Insights into the Regulation of Connexin26 Gap Junction Channels by Carbon Dioxide

Core Concepts

The K125E mutation in connexin26 biases the protein towards a constricted, closed conformation of the gap junction channel, mimicking the effects of elevated carbon dioxide levels.

Abstract

The content describes structural studies of the gap junction channel protein connexin26 (Cx26) and how it is regulated by carbon dioxide (CO2). Key insights: Improved cryo-EM data collection and analysis allowed the authors to observe distinct conformations of Cx26 associated with open and closed channel states. In the "NConst" conformation, the cytoplasmic loop containing the K125 residue adopts a more defined structure, with K125 positioned near R104 of a neighboring subunit. This conformation is associated with channel closure. Mutating K125 to glutamic acid (K125E) biases the protein towards the NConst, closed conformation, even in the absence of elevated CO2. This suggests the K125 residue and its potential carbamylation play a key role in CO2-dependent channel regulation. Comparisons to other connexin structures reveal that the NConst conformation is unique, with distinct positioning of transmembrane helices TM1 and TM2 that constrict the channel pore. The data support a model where CO2 sensing involves a conformational equilibrium between open and closed states, which can be shifted by the charge state of K125.

Stats

"Cx26K125E gap junctions are constitutively closed at a PCO2 of 35 mmHg." "For Cx26K125E expressing cells, no dye permeates into the neighbouring cell even after 10 minutes of recording at either 35 mmHg or 55 mmHg PCO2 despite the presence of morphological gap junctions."

Quotes

"Essentially the KVRIEG motif can be described as a break in TM3 as the helix extends at either side of this extended motif." "Glutamate is also much shorter than the carbamylated lysine, so that while the charges would be equivalent, the two residues might not be able to make the same specific interactions."

Key Insights Distilled From

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2

by Brotherton,D... at www.biorxiv.org 08-22-2023

https://www.biorxiv.org/content/10.1101/2023.08.22.554292v2

Deeper Inquiries

How might the conformational flexibility of Cx26 be exploited for the development of therapeutic interventions targeting CO2-dependent channel regulation?

The conformational flexibility of Cx26, as revealed in this study, provides a potential avenue for developing therapeutic interventions targeting CO2-dependent channel regulation. By understanding the structural changes that occur in response to CO2 levels, researchers can design molecules or compounds that specifically target and stabilize the closed conformation of Cx26. For example, small molecules or peptides could be developed to mimic the effect of the K125E mutation, biasing the channel towards a closed state even in the presence of elevated CO2 levels. This approach could be beneficial in conditions where dysregulation of Cx26 function is implicated, such as in certain forms of deafness or other diseases associated with connexin mutations.

What other factors, beyond the K125 residue, might contribute to the cooperative gating behavior observed in Cx26 hemichannels versus the apparent lack of cooperativity in the gap junction structures?

In addition to the K125 residue, several other factors may contribute to the cooperative gating behavior observed in Cx26 hemichannels compared to the lack of cooperativity in the gap junction structures. One key factor could be the presence of specific interacting residues within the channel that stabilize the closed conformation in response to CO2 levels. These residues may form intricate networks of interactions that are disrupted or altered in the gap junction structures, leading to a different gating behavior. Additionally, post-translational modifications or binding of regulatory molecules could play a role in modulating the conformational changes and cooperativity of the channel. The differences in the structural environment surrounding the K125 residue in hemichannels versus gap junctions may also contribute to the observed differences in cooperativity.

Could the insights into Cx26 structure-function relationships gleaned from this study inform our understanding of how other connexin isoforms or related large-pore channel proteins are regulated by diverse physiological stimuli?

The insights gained from this study on Cx26 structure-function relationships have the potential to inform our understanding of how other connexin isoforms or related large-pore channel proteins are regulated by diverse physiological stimuli. By elucidating the mechanisms by which Cx26 responds to CO2 levels and undergoes conformational changes, researchers can apply similar principles to study the regulation of other connexin isoforms or large-pore channel proteins. Understanding how specific residues, structural motifs, or post-translational modifications impact channel function in response to different stimuli can provide a framework for investigating the regulation of other channels. This knowledge can be valuable in uncovering common regulatory mechanisms across different connexin isoforms and related channel proteins, leading to a deeper understanding of intercellular communication and potential therapeutic targets for various diseases.