Oxidative Stress Inhibits Vascular KATP Channels by S-Glutathionylation

Abstract

The K ATP channel is an important player in vascular tone regulation. Its opening and closure lead to vasodilation and vasoconstriction, respectively. Such functions may be disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrated that S-glutathionylation was a modulation mechanism underlying oxidant-mediated vascular K ATP channel regulation. An exposure of isolated mesenteric rings to hydrogen peroxide (H 2 O 2 ) impaired the K ATP channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, H 2 O 2 or diamide caused a strong inhibition of the vascular K ATP channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH). Similar channel inhibition was seen with oxidized glutathione (GSSG) and thiol-modulating reagents. The oxidant-mediated channel inhibition was reversed by the reducing agent dithiothreitol (DTT) and the specific deglutathionylation reagent glutaredoxin-1 (Grx1). Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester (BioGEE) showed incorporation of GSH to the Kir6.1 subunit in the presence of H 2 O 2 . These results suggest that S-glutathionylation is an important mechanism for the vascular K ATP channel modulation in oxidative stress.

S-Glutathionylation of Ion Channels: Insights into the Regulation of Channel Functions, Thiol Modification Crosstalk, and Mechanosensing

“…The combined treatments of GSH (100 lM-5 mM) with oxidants (e.g., H 2 O 2 , 10 lM-1 mM; diamide 100 lM-1 mM) are also commonly used (138,145). Both of these treatments are relatively specific to S-glutathionylation.…”

“…GSSG (1-10 mM) is often used to induce S-glutathionylation as well (28,129,138). The combined treatments of GSH (100 lM-5 mM) with oxidants (e.g., H 2 O 2 , 10 lM-1 mM; diamide 100 lM-1 mM) are also commonly used (138,145).…”

“…N-ethylmaleimide is often used to block the S-glutathionylation (129), and reactive disulfides (pyridine disulfides), for example, 2,2¢-dithiodipyridine and 2,2-dithiobis-5-nitropyridine are sometimes used to mimic the addition of the GSH moiety to the cysteine residues (137,138). These reagents, however, are also used for the study of S-nitrosylation (140).…”

“…The most popular method to test S-glutathionylation biochemically is to use BioGEE (a membrane permeable biotinylated glutathione ethyl ester) in western blot experiments (87,138). In principle, the cells expressing proteins of interest are loaded with BioGEE (100 lM-500 lM) first.…”

“…Protein S-glutathionylation has been extensively discussed in many excellent reviews with a variety of different emphases (31,44,45,75,76,88,93,100,107). Over the past few years, S-glutathionylation has been increasingly observed in many ion channels, such as voltage-gated calcium channels, ryanodine receptor (RyR), and ATP-sensitive potassium channels (K ATP channels), all of which contribute to critical cellular functions (4,123,137,138). In this review, we introduce redox-mediated thiol modulation first and then focus on S-glutathionylation of ion channels.…”