Article thumbnail

Molecular Basis and Structural Insight of Vascular KATP Channel Gating by S-Glutathionylation*

By Yang Yang, Weiwei Shi, Xianfeng Chen, Ningren Cui, Anuhya S. Konduru, Yun Shi, Timothy C. Trower, Shuang Zhang and Chun Jiang

Abstract

The vascular ATP-sensitive K+ (KATP) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular KATP 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: Cys43, Cys120, and Cys176. Among them, Cys176 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 Cys176 is not a general gating site in Kir6.1, in contrast to its counterpart (Cys166) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys176 impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys176, Cys43 had only a modest contribution to S-glutathionylation, and Cys120 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

Topics: Membrane Biology
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:3059003
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)

  • To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.

    Suggested articles