DESCRIPTION (Adapted from Applicant's Abstract): 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 is 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.
|Effective start/end date||9/30/00 → 7/31/05|
- National Heart, Lung, and Blood Institute: $24,974.00
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.