gamma-Glutamyl transpeptidase (gamma-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH (GSH(o)). The objective of this study was to identify the role of gamma-GT in reversing pathogenic K(+) channel remodeling in the diseased heart. Chronic ventricular dysfunction was induced in rats by myocardial infarction (MI), and studies were done after 6-8 wk. Biochemical assays of tissue extracts from post-MI hearts revealed significant increases in gamma-GT activity in left ventricle (47%) and septum (28%) compared with sham hearts, which paralleled increases in protein abundance and mRNA. Voltage-clamp studies of isolated left ventricular myocytes from post-MI hearts showed that downregulation of transient outward K(+) current (I(to)) was reversed after 4-5 h by 10 mmol/l GSH(o) or N-acetylcysteine (NAC(o)), and that the effect of GSH(o) but not NAC(o) was blocked by the gamma-GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine did not prevent upregulation of I(to) by GSH(o), suggesting that intracellular synthesis of GSH was not directly involved. However, pretreatment of post-MI myocytes with an SOD mimetic [manganese (III) tetrapyridylporphyrin] and catalase completely blocked recovery of I(to) by GSH(o). Confocal microscopy using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate confirmed that GSH(o) increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, GSH(o)-mediated upregulation of I(to) was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, and AG1024) and thioredoxin reductase (auranofin and 13-cis-retinoic acid). These data suggest that GSH(o) elicits gamma-GT- and ROS-dependent transactivation of tyrosine kinase signaling that upregulates K(+) channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by gamma-GT catalysis of GSH(o) may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart.