Project: Research project

Project Details


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 date9/30/007/31/05


  • Medicine(all)


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