On the Use of Chemical Modification to Determine Connexin Hemichannel Topology and Function

Abstract

Listen

Translate article

Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful methodology to define pore-lining elements of channels and the changes in structure that accompany channel gating. To help identify the position of the gate of connexin26 (Cx26) hemichannels, using the Xenopus oocyte expression system, we performed cysteine-scanning mutagenesis of several residues within the pore, followed by chemical modification using methanesulfonate (MTS) reagents. Strikingly, we observed that modification with MTS reagents, at different pore lining residues, was reversed within minutes of washout of the reagents. This reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. We therefore tested whether the connexin pore was allowing cytosolic glutathione, a well-known cytosolic reducing agent, to access the sites. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulphoximine decreased the cytosolic glutathione in Xenopus oocytes and drastically reduced reversibility of MTS modification. In contrast to MTS reagents, maleimide reagents can chemically modify cysteines in a reaction that cannot be reversed by glutathione. As predicted, the maleimide modification did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx32, Cx26, Cx30 and heteromeric Cx26/Cx32 and Cx26/Cx30 hemichannels are permeable to glutathione, as others have shown for Cx43 channels. These results suggest that: (a) connexin hemichannels may mediate physiological glutathione release in diverse cell types; (b) maleimide-based modification is the more appropriate approach to perform chemical modification to study structure-function of connexin channels, and other channels and transporters that are permeable to large molecules, such as pannexin channels. Support: GM099490 & GM036044.