title: Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids.
abstract: Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of K(ATP) channels, inhibiting binding of ATP to the channel.
authors.affiliation: Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. wang.xiaoli@mayo.edu
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Wang
authors.forename: Tong
authors.initials: T
authors.lastname: Lu
authors.forename: Sheng
authors.initials: S
authors.lastname: Cao
authors.forename: Vijay H
authors.initials: VH
authors.lastname: Shah
authors.forename: Hon-Chi
authors.initials: HC
authors.lastname: Lee
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D064233:Sulfonylurea Receptors; D000099161:Kcnj11 Channel
chemical_list: D015126:8,11,14-Eicosatrienoic Acid; D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D064233:Sulfonylurea Receptors; D000099161:Kcnj11 Channel; C410128:11,12-dihydroxyeicosatrienoic acid; C515338:11,12-epoxyeicosatrienoic acid methyl ester; C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; C050715:8,9-epoxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid