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

Abstract

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. ATP-sensitive Kϩ (K ATP ) 7 channels are expressed in a variety of tissues, including smooth muscles, pancreatic ␤-cells, myocardium, and neurons, where they play an important role in cellular function (1, 2). Activity of the K ATP channels is tuned by physiological or pathophysiological stimuli, including hypoxia, hyperglycemia, ischemia, and oxidative stress, allowing a regulation of cellular excitability according to the metabolic state (3). The vascular smooth muscle (VSM) isoform of K ATP channels regulates vascular tones (4, 5). Activation of the channel by vasodilators produces hyperpolarization of VSM cells, reduces activity of the voltage-dependent Ca 2ϩ channels, and relaxes VSMs. Inhibition of the channel leads to constriction of VSMs. Disruption of the vascular K ATP channel in mice results in vasospasm in coronary arteries and sudden cardiac death (6, 7).Other studies have further shown that disruption of the vascular K ATP channel has drastic effects on the systemic response to septic stress. With a forward genetic approach by genomewide random chemical mutagenesis, Croker et al. (8) screened a large population of mice and found four strains that are highly susceptible to multiple septic pathogens, including lipopolysaccharides (LPSs). The LPS hypersensitivity phenotype of these mice is due to a null allele of Kcnj8, encoding the Kir6.1 subunit of the vascular K ATP channel (8). Similar septic susceptibility has been observed in Kcnj8-knock-out mice that also show coronary hypoperfusion and myocardial ischemia during LPS exposure (9). These studies thus indicate that the vascular K ATP channel not only contributes to the vascular tone regulation at physiological conditions but also affects critically systemic stress responses.Our recent studies have shown that the vascular K ATP channel is strongly inhibited in oxidative stress by S-glutathionylation (10). S-Glutathionylation is a post-translational modification mechanism occurring in a variety of physiological or pathophysiological conditions (11). This protein modulation mechanism is remarkable especially in vasculatures because oxidative stress is a major contributing factor to several cardiovascular diseases, in which S-glutathionylation plays an important role (12). Although S-glutathionylation is often associated with the adverse effects of oxidative stress, such a protein modulation is reversible under certain circumstances and can act as a functional modulation mechanism like protein phosphorylation (11). Thus, demonstration of how S-glutathionylation * This work was s...

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

“…(7,27,29,77,84,110,111,136,138). K ATP channels are made of four pore-forming Kir (inward rectifier K + channel) subunits and four regulatory sulphonylurea receptors of the ATP-binding cassette (SUR) subunits (102 (137,138). Functional studies show that the treatments with a variety of Sglutathionylation inducers including GSH/H 2 O 2 , (GSH: GSSG)/H 2 O 2 , GSSG, or GSH/diamide lead to functional channel inhibition.…”

“…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).…”

“…Structural modeling and molecular dynamics are useful tools to study ion channel structure and function (54,134,135) including S-glutathionylation-mediated structural changes of ion channels (55,137). Homologous ion channel crystal structures determined by X-ray are the most common templates for structural modeling.…”

“…If the proteins of interest have a homology that responds to S-glutathionylation differently, chimeras could also be created using these homologies to determine which domains or motifs are responsible for S-glutathionylation (137).…”

“…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.…”