Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids

Xiao Li Wang, Tong D Lu, Sheng Cao, Vijay Shah, Hon Chi Lee

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5 Citations (Scopus)

Abstract

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K+(KATP) channels. EETs activate KATP channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to KATP 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 μM) did not change TNP-ATP binding KD to GST-Kir6.2C, but Bmax 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 KATP channels, inhibiting binding of ATP to the channel.

Original languageEnglish (US)
Pages (from-to)1041-1049
Number of pages9
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1761
Issue number9
DOIs
StatePublished - Sep 2006

Fingerprint

Adenosine Triphosphate
Glutathione Transferase
Acids
KATP Channels
Arachidonic Acid
Epoxy Compounds
Protein C
Cytochrome P-450 Enzyme System
Carrier Proteins
Esters
Proteins
Fluorescence
11,12-epoxy-5,8,14-eicosatrienoic acid

Keywords

  • ATP
  • ATP-sensitive potassium channel
  • binding
  • Epoxyeicosatrienoic acid
  • Kir 6.2
  • TNP-ATP

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Biophysics

Cite this

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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+(KATP) channels. EETs activate KATP channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to KATP 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 μM) did not change TNP-ATP binding KD to GST-Kir6.2C, but Bmax 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 KATP channels, inhibiting binding of ATP to the channel.",
keywords = "ATP, ATP-sensitive potassium channel, binding, Epoxyeicosatrienoic acid, Kir 6.2, TNP-ATP",
author = "Wang, {Xiao Li} and Lu, {Tong D} and Sheng Cao and Vijay Shah and Lee, {Hon Chi}",
year = "2006",
month = "9",
doi = "10.1016/j.bbalip.2006.06.005",
language = "English (US)",
volume = "1761",
pages = "1041--1049",
journal = "Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids",
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TY - JOUR

T1 - Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids

AU - Wang, Xiao Li

AU - Lu, Tong D

AU - Cao, Sheng

AU - Shah, Vijay

AU - Lee, Hon Chi

PY - 2006/9

Y1 - 2006/9

N2 - Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K+(KATP) channels. EETs activate KATP channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to KATP 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 μM) did not change TNP-ATP binding KD to GST-Kir6.2C, but Bmax 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 KATP channels, inhibiting binding of ATP to the channel.

AB - Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K+(KATP) channels. EETs activate KATP channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to KATP 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 μM) did not change TNP-ATP binding KD to GST-Kir6.2C, but Bmax 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 KATP channels, inhibiting binding of ATP to the channel.

KW - ATP

KW - ATP-sensitive potassium channel

KW - binding

KW - Epoxyeicosatrienoic acid

KW - Kir 6.2

KW - TNP-ATP

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U2 - 10.1016/j.bbalip.2006.06.005

DO - 10.1016/j.bbalip.2006.06.005

M3 - Article

C2 - 16904368

AN - SCOPUS:33748475727

VL - 1761

SP - 1041

EP - 1049

JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

SN - 1388-1981

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