Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

Abstract

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac K ATP channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the K ATP channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2⌬C26 with or without the coexpression of SUR2A and on Kir6. The cardiac ATP-sensitive K ϩ (K ATP ) 1 channels are biological sensors that respond to intracellular metabolic changes and play important roles in regulating cardiac functions (1). Although the role of the cardiac K ATP channels under normal physiological conditions remains unclear, it is crucial in ischemic preconditioning, and activation of K ATP channels occurs during cardiac ischemia and hypoxia, leading to reduced Ca 2ϩ influx and intracellular Ca 2ϩ overload (2). Some important insights were provided by studies using the Kir6.2 knock-out mice, which have compromised ability in modulating cardiac electrophysiological properties, contractility (3, 4), and in handling cardiac ischemia and stress (3,5,6). Recently, a form of human dilated cardiomyopathy was found to be associated with mutations of the cardiac K ATP channels (7).The cardiac K ATP channel is a heterooctamer containing four inward rectifier K ϩ channel (Kir6.2) and four sulfonylurea receptor (SUR2A) subunits. SUR contains two cytoplasmic nucleotide binding folds that were initially thought to be important for channel regulation by ATP (8). However, a truncation mutant of the Kir6.2 involving deletion of the C-terminal 26 residues (Kir6.2⌬C26) gives rise to channels that retain much of the ATP sensitivity in the absence of SUR (9). Also, mutations in the SUR produced only small effects on ATP inhibition of Kir6.2/SUR currents (10). Previous studies have demonstrated that both the N and C termini of Kir6.2 contribute to the site(s) that regulates ATP sensitivity, and they include Arg-50, Cys-166, Ile-167, Thr-171, Arg-176, Arg-177, Glu-179, Ile-182, Lys-185, Arg-192, Arg-201, and Gly-344 residues. Of these, Arg-50, Lys-185, and Arg-201 residues are particularly crucial for ATP sensitivity and are implicated for interaction with the ␣, ␤, and ␥ phosphates of ATP (11-13). However, the mechanism of Kir6.2 channel inhibition by ATP remains to be elucidated.Lipid metabolites are important modulators of the K ATP channels, and these include the long chain acyl-CoA esters (14), phosphatidylinositol 4,5-bisphosphate (PIP 2 ), phosphoinositides (15), L-palmitoylcarnitine (16), and epoxyeicosatrienoic acids (EETs) (17, 18). Arachidonic acid is converted by the cytochrome P450 epoxygenase into 4 EET regioisomers, 5.6-, 8,9-, 11,12-, and 14,15-EET (Fig. 1) (19). EETs are abundant endogenous constituents of the human and rat hearts, measured at 70 ng of total EETs per g of rat heart (20). 8,9-, 11,12-, and 14,15-EET contribute 39, 28, and 33%, respectively, ...

Action of epoxyeicosatrienoic acids on cellular function

“…Additional studies indicated that the EETs can directly interact with cellular proteins. Patch-clamp experiments have shown that EETs can directly interact with myocardial Na ϩ and ATP-sensitive K ϩ (K ATP ) channels (94,100), and the latter finding is consistent with the fact that an EET binding site has been detected in the K ATP ion channel (99). EETs also bind to intracellular proteins that contain fatty acid binding sites, including FABPs and PPAR␥ (97,161).…”

“…EETs bind to the myocardial K ATP channel and thereby reduce its sensitivity to ATP by an allosteric alteration of the ATP binding site (99,100). Activation of the mitochondrial K ATP channels protects the myocardium against ischemia-reperfusion injury (140), suggesting that myocardial preconditioning may occur through a direct interaction between EETs and the channel.…”

Molecular Mechanisms Mediating Inhibition of Human Large Conductance Ca 2+ -Activated K + Channels by High Glucose

“…22 hSlo cDNA and 2 recombinant adenoviruses, AdCAT carrying CAT and AdLacZ carrying ␤-galactosidase, were used for gene transfer. 23…”

Mechanism of rat mesenteric arterial KATP channel activation by 14,15-epoxyeicosatrienoic acid

“…Interaction between EET and cardiac K ATP channels is stereospecific with 11(S),12(R)-EET being the active enantiomer while 11(R),12(S)-EET is totally inactive (23). Recently, we have identified R192/R195 on Kir6.2, which encodes cardiac K ATP channels, as the critical EET sensitivity site (19). In contrast, we found that activation of vascular K ATP channels by EET is mediated by PKA-dependent mechanisms (37).…”