Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids.
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.
Wang
Xiao-Li
XL
Department of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. wang.xiaoli@mayo.edu
Lu
Tong
T
Cao
Sheng
S
Shah
Vijay H
VH
Lee
Hon-Chi
HC
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
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