Mechanisms of permselectivity of connexin hemichannels to small molecules

Connexins can act either as hemichannels to facilitate ion and small-molecule movement from the cytosol to the extracellular space or as gap junction channels to provide a pathway for solute exchange between cells. Connexins are ubiquitously expressed throughout the body and are implicated in a wide range of processes. The permselectivity of connexin hemichannels for small neurochemicals remains poorly understood. By coexpressing genetically encoded fluorescent sensors for ATP, glutamate, and lactate with a range of connexins, we examined the ability of different hemichannels to permit the release of these compounds under physiological conditions and in response to physiological stimuli (small changes in partial pressure of CO₂ and transmembrane depolarization). We found that some connexin hemichannels were relatively nonselective (Cx26, Cx32, Cx43, and Cx31.1) allowing passage of ATP, glutamate, and lactate. By contrast, other connexin hemichannels (Cx36, Cx46, and Cx50) were highly selective. Cx36 and Cx46 hemichannels allowed the release of ATP but not glutamate or lactate. The size of the permeating molecule cannot be the sole determinant of permselectivity. By contrast, Cx50 hemichannels permitted the release of lactate and glutamate but not ATP. We also found that the nature of the opening stimulus could alter the permselectivity of the hemichannel-for some of the relatively nonselective connexins, hemichannel opening via depolarization was ineffective at allowing the release of lactate. By performing a mutational analysis, informed by the differential selectivity of the closely related Cx46 and Cx50 hemichannels, we found that the charge on the N terminus and N terminus-transmembrane 2 interactions are key contributors to permselectivity for ATP.