Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules

The Journal of General Physiology
Xuhui TongJorge E Contreras

Abstract

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 G...Continue Reading

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Citations

Feb 10, 2016·Frontiers in Physiology·Mauricio A RetamalCarlos González
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Apr 4, 2020·Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids·Mauricio A RetamalGuillermo A Altenberg

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