This proposal aims to explore the mechanisms of EET on KATP channels in heart. It is hypothesized that EETs are endogenous activators of KATP channels, and these actions are mediated through effects on ATP mediated inhibition of the Kir6.2 subunit. Molecular studies indicate that KATP channels consist of at least two types of subunits: the K channel subunit is referred to as KIR6.2, and the other subunit is a sulfonylurea receptor subunit (SUR). KIR6.2 is a member of the inward rectifier family of potassium channels. The SUR subunit is a member of the ATP binding cassette family of proteins and confers channel sensitivity to the sulfonylurea drugs. The functional channel is assumed to be an octomer consisting of four KIR6.2 subunits and four SUR subunits. The pancreatic beta cell KATP channel is composed of KIR6.2 and SUR1. The cardiac channel consists of KIR6.2 and SUR2A whereas the smooth muscle channel consists of KIR6.2 and SUR2B. EETs are potent endothelium-derived vasodilators that modulate vascular tone by way of enhancement of calcium-activated potassium channels in vascular smooth muscle. Cytochrome P450 monooxygenases convert arachidonic acid to 4 epoxyeicosatrienoic acid regioisomers, including 5,6-, 8,9-, 11,12- and 14,15- EET, as well as the 19 and 20 hydroxyecosatetronoic acids (HETE). Studies have shown that rat heart contains substantial amounts of endogenous EET, and 11, 12 EET has been shown to enhance the recovery of cardiac function following global ischemia. Under normal conditions, EETs are present at nM concentrations in plasma. During conditions of ischemia, formation of cellular EETs may be enhanced, thus EET's may play a role in the modulation of cardiac electrophysiology and vascular tone during ischemia. These hypotheses will be addressed by testing EETs on KATP channels using electrophysiology. EC50s for channel activation and the effects of ATP dependent inhibition will be evaluated. The structural determinants of EETs required in modulating channel function will be explored. The stereoisomers of EETs and as well as carbon chain elongated and shortened variants will be studied. The molecular mechanisms of EET will be examined using mutant Kir6.2 and SUR2A to determine the subunit requirements for modulation and to map the sites of action. The first specific aim is to determine the effects of the four EET isomers on KATP channels in rat ventricular myocytes using patch clamp methods. The effects of EETs on the pharmacological and electrophysiological properties of cardiac KATP channels will be investigated. It is hypothesized that EETs are endogenous activators of the channel. Although this may be the case, these experiments will not be able to determine whether EETs are endogenous activators by studying rat myocytes. Nevertheless, these experiments will provide an important characterization of the native channels.
The second aim i s to identify the structural determinants of EETs important for modulating KATP channels. The PI will investigate 5,6-, 8,9-, 11,12- and 14,15- EETs to explore the chemical requirements for activity. These experiments seem well thought out and should provide novel insights into the mechanisms of activation and specificity.
A third aim will determine molecular mechanisms of EET effects on KATP channels by using cloned KIR6.2/SUR2A channels. The hypothesis is that EETs modulate the channel through altering the ATP interaction. It is believed from preliminary data that 11,12 EET caused a decrease in the ATP binding rate. This will be further explored through analysis of these actions on single KATP channels.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL063754-02
Application #
6390555
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Lathrop, David A
Project Start
2000-09-30
Project End
2002-07-31
Budget Start
2001-08-01
Budget End
2002-07-31
Support Year
2
Fiscal Year
2001
Total Cost
$24,974
Indirect Cost
Name
University of Iowa
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Lu, Tong; Ye, Dan; Wang, Xiaoli et al. (2006) Cardiac and vascular KATP channels in rats are activated by endogenous epoxyeicosatrienoic acids through different mechanisms. J Physiol 575:627-44
Zhou, Wei; Lu, Tong; Spector, Arthur A et al. (2006) Inhibition of PGI2 signaling by miconazole in vascular smooth muscle cells. Prostaglandins Other Lipid Mediat 80:28-34
Zhou, Wei; Wang, Xiao-Li; Lamping, Kathryn G et al. (2006) Inhibition of protein kinase Cbeta protects against diabetes-induced impairment in arachidonic acid dilation of small coronary arteries. J Pharmacol Exp Ther 319:199-207
Ye, Dan; Zhou, Wei; Lu, Tong et al. (2006) Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid. Am J Physiol Heart Circ Physiol 290:H1326-36
Wang, Xiao-Li; Lu, Tong; Cao, Sheng et al. (2006) Inhibition of ATP binding to the carboxyl terminus of Kir6.2 by epoxyeicosatrienoic acids. Biochim Biophys Acta 1761:1041-9
Lu, Tong; Wang, Xiao-Li; He, Tongrong et al. (2005) Impaired arachidonic acid-mediated activation of large-conductance Ca2+-activated K+ channels in coronary arterial smooth muscle cells in Zucker Diabetic Fatty rats. Diabetes 54:2155-63
Zhou, Wei; Wang, Xiao-Li; Kaduce, Terry L et al. (2005) Impaired arachidonic acid-mediated dilation of small mesenteric arteries in Zucker diabetic fatty rats. Am J Physiol Heart Circ Physiol 288:H2210-8
Ye, Dan; Zhou, Wei; Lee, Hon-Chi (2005) Activation of rat mesenteric arterial KATP channels by 11,12-epoxyeicosatrienoic acid. Am J Physiol Heart Circ Physiol 288:H358-64
Lu, Tong; Hong, Min-Pyo; Lee, Hon-Chi (2005) Molecular determinants of cardiac K(ATP) channel activation by epoxyeicosatrienoic acids. J Biol Chem 280:19097-104
Wang, Xiao-Li; Ye, Dan; Peterson, Timothy E et al. (2005) Caveolae targeting and regulation of large conductance Ca(2+)-activated K+ channels in vascular endothelial cells. J Biol Chem 280:11656-64

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