Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.

Original publication

DOI

10.1085/jgp.201511375

Type

Journal article

Journal

J Gen Physiol

Publication Date

09/2015

Volume

146

Pages

245 - 254

Keywords

Animals, Cell Membrane Permeability, Cells, Cultured, Connexin 26, Connexins, Gap Junctions, Glutathione, Mutagenesis, Site-Directed, Oocytes, Xenopus laevis