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

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

Research output: Contribution to journalArticle

5 Scopus citations

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 1 2006

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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