Connexin26 mediates CO2-dependent regulation of breathing via glial cells of the medulla oblongata

Abstract

Breathing is highly sensitive to the PCO2 of arterial blood. Although CO2 is detected via the proxy of pH, CO2 acting directly via Cx26 may also contribute to the regulation of breathing. Here we exploit our knowledge of the structural motif of CO2-binding to Cx26 to devise a dominant negative subunit (Cx26DN) that removes the CO2-sensitivity from endogenously expressed wild type Cx26. Expression of Cx26DN in glial cells of a circumscribed region of the mouse medulla - the caudal parapyramidal area – reduced the adaptive change in tidal volume and minute ventilation by approximately 30% at 6% inspired CO2. As central chemosensors mediate about 70% of the total response to hypercapnia, CO2-sensing via Cx26 in the caudal parapyramidal area contributed about 45% of the centrally-mediated ventilatory response to CO2. Our data unequivocally link the direct sensing of CO2 to the chemosensory control of breathing and demonstrates that CO2-binding to Cx26 is a key transduction step in this fundamental process.

CO 2 sensing by connexin26 and its role in the control of breathing

“…Homomers of dnCx26 (as would be expected) are insensitive to CO 2 . We have shown, via fluorescence resonance energy transfer, that dnCx26 assembles very efficiently into hexamers with wild-type Cx26 [68]. HeLa cells that stably express Cx26 (and will dye load in response to CO 2 ), when transfected with dnCx26, lose their CO 2 sensitivity [68].…”

“…We have shown, via fluorescence resonance energy transfer, that dnCx26 assembles very efficiently into hexamers with wild-type Cx26 [68]. HeLa cells that stably express Cx26 (and will dye load in response to CO 2 ), when transfected with dnCx26, lose their CO 2 sensitivity [68]. Thus, dnCx26 does indeed act as a royalsocietypublishing.org/journal/rsfs Interface Focus 11: 20200029 dominant negative subunit and can remove CO 2 sensitivity from the endogenously expressed wild-type Cx26 [68].…”

“…HeLa cells that stably express Cx26 (and will dye load in response to CO 2 ), when transfected with dnCx26, lose their CO 2 sensitivity [68]. Thus, dnCx26 does indeed act as a royalsocietypublishing.org/journal/rsfs Interface Focus 11: 20200029 dominant negative subunit and can remove CO 2 sensitivity from the endogenously expressed wild-type Cx26 [68]. This makes it a very selective genetic tool to probe the contribution of CO 2 sensing via Cx26 to physiological processes; indeed, it links the motif of CO 2 binding to physiological action.…”

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

“…Cx26 is thus a CO 2 -gated channel capable of releasing ATP. We have recently used our structural understanding of how CO 2 interacts with Cx26 to develop a dominant negative construct t hat coassembles with endogenous wild-type Cx26 to remove CO 2 sensitivity from the resulting heteromeric channel [39]. This genetic approach has allowed us to demonstrate that direct CO 2 detection via Cx26 contributes about half of the adaptive response to hypercapnia generated by the central chemosensors.…”

“…This genetic approach has allowed us to demonstrate that direct CO 2 detection via Cx26 contributes about half of the adaptive response to hypercapnia generated by the central chemosensors. 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].…”