Conformational changes and channel gating induced by CO₂binding to Connexin26

Abstract

CO2 is the inevitable by-product of oxidative metabolism. Many physiological processes such as breathing1 and cerebral blood flow2 are sensitive to CO2. Historically, the physiological actions of CO2 have been regarded as being mediated exclusively via changes in pH. Here, we change this consensus by showing that the gap junction protein Connexin26 (Cx26) acts as a receptor for CO2 showing sensitivity to modest changes in PCO2 around the physiological norm3-6. Mass spectrometry analysis7 shows that CO2 carbamylates specific lysines on a regulatory loop of Cx26 at high, but not at low levels of PCO2. By means of high resolution cryo-EM, we have solved structures of Cx26 gap junctions at 1.9, 2.2 and 2.1 Å for PCO2 of 90, 55 and 20 mmHg respectively, all at pH 7.4. Classification of the particles at each level of PCO2, shows the transmembrane helices and N-terminal helix flexing at the dynamic cytoplasmic side of the protein. Gating of Cx26 gap junctions by CO2 involves movements of the N-terminus to plug the channel at high PCO2. We therefore provide mechanistic detail for a new paradigm by which CO2 can directly control breathing8 and other key physiological functions9.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

“…Cx26 also plays a role in mediating CO 2 -dependent modulation of the excitability of neurons in the substantia nigra and ventral tegmental area (Hill et al 2020). We have also recently demonstrated that K125 is indeed carbamylated and produced new high resolution cryoEM structures of Cx26 at different levels of P CO 2 (Brotherton et al 2020). Thus, carbamylation of Cx26 occurs and is involved in mediating the CO 2 -dependent control of physiologically important functions.…”

“…We have also recently demonstrated that K125 is indeed carbamylated and produced new high resolution cryoEM structures of Cx26 at different levels of (Brotherton et al . 2020). Thus, carbamylation of Cx26 occurs and is involved in mediating the CO 2 ‐dependent control of physiologically important functions.…”

Biological insights from the direct measurement of purine release

“…While the mutational analysis strongly suggests carbamylation as a mechanism, it is not definitive. Recently however we have produced mass spectrometry evidence for the carbamylation of K125, and the consequent changes in channel gating in very high resolution cryoEM structures of Cx26 at different levels of PCO2 [70].…”

Real-time measurement of adenosine and ATP release in the central nervous system

“…There is a population of specialized glial cells present in a circumscribed nucleus-the caudal parapyramidal area that directly detect CO 2 via Cx26 and regulate breathing via the release of ATP [39]. Very recently, we have taken this down to the atomic level by solving cryoEM structures for Cx26 at different levels of PCO 2 to directly demonstrate the carbamylation mechanism and understand the conformational changes trigged by CO 2 -binding that lead to channel gating [40].…”