Arterial P CO 2 , a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of P CO 2 in the blood and brain, yet the mechanisms by which the brain senses CO 2 remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO 2 -dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO 2 -dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of P CO 2 . ATP release is independent of extracellular Ca 2+ and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO 2 -dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO 2 -dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO 2 -dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in P CO 2 is an important mechanism contributing to central respiratory chemosensitivity.

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We have previously shown connexin mediated CO 2 -dependent ATP release from the surface of the medulla oblongata. Given the localization of connexin 26 (Cx26) to the chemosensing areas of the medulla, we have tested in a heterologous expression system (HeLa cells) whether Cx26 may be sensitive to changes in P CO 2 . Cx26 responded to an increase in P CO 2 at constant extracellular pH by opening and to a decrease in P CO 2 by closing. Furthermore, Cx26 was partially activated at a physiological P CO 2 of around 40 mmHg. Cx26 in isolated patches responded to changes in P CO 2 , suggesting direct CO 2 sensitivity of the hemichannel to CO 2 . Heterologous expression of Cx26 in HeLa cells was sufficient to endow them with the capacity to release ATP in a CO 2 -sensitive manner. We have examined other heterologously expressed connexins for their ability to respond to changes in P CO 2 . The closely related β connexins Cx30 and Cx32 also displayed sensitivity to changes in P CO 2 , but with slightly different characteristics from Cx26. The more distant Cx43 exhibited CO 2 -dependent closing (possibly mediated through intracellular acidification), while Cx36 displayed no CO 2 sensitivity. These surprising findings suggest that connexins may play a hitherto unappreciated variety of signalling roles, and that Cx26 and related β connexins may impart direct sensitivity to CO 2 throughout the brain.