title: Molecular basis and structural insight of vascular K(ATP) channel gating by S-glutathionylation.
abstract: The vascular ATP-sensitive K(+) (K(ATP)) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular K(ATP) channel is inhibited in oxidative stress via S-glutathionylation. Here we show evidence for the molecular basis of the S-glutathionylation and its structural impact on channel gating. By comparing the oxidant responses of the Kir6.1/SUR2B channel with the Kir6.2/SUR2B channel, we found that the Kir6.1 subunit was responsible for oxidant sensitivity. Oxidant screening of Kir6.1-Kir6.2 chimeras demonstrated that the N terminus and transmembrane domains of Kir6.1 were crucial. Systematic mutational analysis revealed three cysteine residues in these domains: Cys(43), Cys(120), and Cys(176). Among them, Cys(176) was prominent, contributing to >80% of the oxidant sensitivity. The Kir6.1-C176A/SUR2B mutant channel, however, remained sensitive to both channel opener and inhibitor, which indicated that Cys(176) is not a general gating site in Kir6.1, in contrast to its counterpart (Cys(166)) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys(176) impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys(176), Cys(43) had only a modest contribution to S-glutathionylation, and Cys(120) was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state.
authors.affiliation: Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Weiwei
authors.initials: W
authors.lastname: Shi
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Chen
authors.forename: Ningren
authors.initials: N
authors.lastname: Cui
authors.forename: Anuhya S
authors.initials: AS
authors.lastname: Konduru
authors.forename: Yun
authors.initials: Y
authors.lastname: Shi
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Trower
authors.forename: Shuang
authors.initials: S
authors.lastname: Zhang
authors.forename: Chun
authors.initials: C
authors.lastname: Jiang
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D054086:KATP Channels; D051379:Mice; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013329:Structure-Activity Relationship; D000099161:Kcnj11 Channel
chemical_list: D005978:Glutathione; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; D000099161:Kcnj11 Channel; C092520:uK-ATP-1 potassium channel