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

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) 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 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) 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 K(ATP) channels, inhibiting binding of ATP to the channel.     

Characterization of 5,6- and 8,9-epoxyeicosatrienoic acids (5,6- and 8,9-EET) as potent in vivo angiogenic lipids

The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.     

The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands

Cytochromes P450 of the CYP2C and CYP4A gene subfamilies metabolize arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) and to 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively. Abundant functional studies indicate that EETs and HETEs display powerful and often opposing biological activities as mediators of ion channel activity and regulators of vascular tone and systemic blood pressures. Incubation of 8,9-, 11,12-, and 14,15-EETs with microsomal and purified forms of rat CYP4A isoforms led to rapid NADPH-dependent metabolism to the corresponding 19- and 20-hydroxylated EETs. Comparisons of reaction rates and catalytic efficiency with those of arachidonic and lauric acids showed that EETs are one of the best endogenous substrates so far described for rat CYP4A isoforms. CYP4A1 exhibited a preference for 8,9-EET, whereas CYP4A2, CYP4A3, and CYP4A8 preferred 11,12-EET. In general, the closer the oxido ring is to the carboxylic acid functionality, the higher the rate of EET metabolism and the lower the regiospecificity for the EET omega-carbon. Analysis of cis-parinaric acid displacement from the ligand-binding domain of the human peroxisome proliferator-activated receptor-alpha showed that omega-hydroxylated 14,15-EET bound to this receptor with high affinity (K(i) = 3 +/- 1 nm). Moreover, at 1 microm, the omega-alcohol of 14,15-EET or a 1:4 mixture of the omega-alcohols of 8,9- and 11,12-EETs activated human and mouse peroxisome proliferator-activated receptor-alpha in transient transfection assays, suggesting a role for them as endogenous ligands for these orphan nuclear receptors.     

Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

Molecular basis for Kir6.2 channel inhibition by adenine nucleotides

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the alpha-phosphate with R201 and interaction of the beta-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the beta-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the alpha-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models.     

Epoxyeicosatrienoic and dihydroxyeicosatrienoic acids dilate human coronary arterioles via BK(Ca) channels: implications for soluble epoxide hydrolase inhibition

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.     

Effects of cytochrome P-450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC)

Sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron plays a central role in the regulation of body fluid volume. Previous studies have indicated that arachidonic acid (AA) and its metabolite 11,12-EET but not other regioisomers of EETs inhibit ENaC activity in the collecting duct. The goal of this study was to investigate the endogenous metabolism of AA in cultured mpkCCD(c14) principal cells and the effects of these metabolites on ENaC activity. Liquid chromatography/mass spectrometry analysis of the mpkCCD(c14) cells indicated that these cells produce prostaglandins, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12/8-HETE, and 15-HETE, but not 20-HETE. Single-channel patch-clamp experiments revealed that 8,9-EET, 14,15-EET, and 11,12-EET all decrease ENaC activity. Neither 5-, 12-, nor 15-HETE had any effect on ENaC activity. Diclofenac and ibuprofen, inhibitors of cyclooxygenase, decreased transepithelial Na(+) transport in the mpkCCD(c14) cells. Inhibition of cytochrome P-450 (CYP450) with MS-PPOH activated ENaC-mediated sodium transport when cells were pretreated with AA and diclofenac. Coexpression of CYP2C8, but not CYP4A10, with ENaC in Chinese hamster ovary cells significantly decreased ENaC activity in whole-cell experiments, whereas 11,12-EET mimicked this effect. Thus both endogenously formed EETs and their exogenous application decrease ENaC activity. Downregulation of ENaC activity by overexpression of CYP2C8 was PKA dependent and was prevented by myristoylated PKI treatment. Biotinylation experiments and single-channel analysis revealed that long-term treatment with 11,12-EET and overexpression of CYP2C8 decreased the number of channels in the membrane. In contrast, the acute inhibitory effects are mediated by a decrease in the open probability of the ENaC. We conclude that 11,12-EET, 8,9-EET, and 14,15-EET are endogenously formed eicosanoids that modulate ENaC activity in the collecting duct.     

11,12,20-Trihydroxy-eicosa-8(Z)-enoic acid: a selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries

Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5(Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa-8(Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa-8(Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15-EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H-11,12-EE8ZE (10(-5) M) inhibited relaxations to 14,15- and 11,12-EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10(-6) M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10(-5) M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system.     

Fibroblast growth factor-2 is a downstream mediator of phosphatidylinositol 3-kinase-Akt signaling in 14,15-epoxyeicosatrienoic acid-induced angiogenesis

To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.     

Multiple antiapoptotic targets of the PI3K/Akt survival pathway are activated by epoxyeicosatrienoic acids to protect cardiomyocytes from hypoxia/anoxia

Epoxyeicosatrienoic acids (EETs) reduce infarction of the myocardium after ischemia-reperfusion injury to rodent and dog hearts mainly by opening sarcolemmal and mitochondrial potassium channels. Other mediators for the action of EET have been proposed, although no definitive pathway or mechanism has yet been reported. Using cultured cells from two rodent species, immortalized myocytes from a mouse atrial lineage (HL-1) and primary myocytes derived from neonatal rat hearts, we observed that pretreatment with EETs (1 microM of 14,15-, 11,12-, or 8,9-EET) attenuated apoptosis after exposure to hypoxia and reoxygenation (H/R). EETs also preserved the functional beating of neonatal myocytes in culture after exposure to H/R. We demonstrated that EETs increased the activity of the prosurvival enzyme phosphatidylinositol 3-kinase (PI3K). In fact, cardiomyocytes pretreated with EET and exposed to H/R exhibited antiapoptotic changes in at least five downstream effectors of PI3K, protein kinase B (Akt), Bcl-x(L)/Bcl-2-associated death promoter, caspases-9 and -3 activities, and the expression of the X-linked inhibitor of apoptosis, compared with vehicle-treated controls. The PI3K/Akt pathway is one of the strongest intracellular prosurvival signaling systems. Our studies show that EETs regulate multiple molecular effectors of this pathway. Understanding the targets of action of EET-mediated protection will promote the development of these fatty acids as therapeutic agents against cardiac ischemia-reperfusion.     

Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.     

EETs relax airway smooth muscle via an EpDHF effect: BK(Ca) channel activation and hyperpolarization

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.     

Rat mesenteric arterial dilator response to 11,12-epoxyeicosatrienoic acid is mediated by activating heme oxygenase

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.     

N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (P(o)) and bursting patterns of activity observed in full-length K(ATP) channels. However, the nature of TMD0-Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in K(ATP) channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-K(ATP) channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced P(o), exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP(2). Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP(2). These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP(2). Moreover, they uncover a novel mechanism of K(ATP) channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.     

ATP activates ATP-sensitive potassium channels composed of mutant sulfonylurea receptor 1 and Kir6.2 with diminished PIP2 sensitivity

ATP-sensitive potassium (K(ATP)) channels are inhibited by ATP and activated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Both channel subunits Kir6.2 and sulfonylurea receptor 1 (SUR1) contribute to gating: while Kir6.2 interacts with ATP and PIP(2), SUR1 enhances sensitivity to both ligands. Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP(2) sensitivities resembling channels formed by Kir6.2 alone. We show here that when E128K SUR1 was co-expressed with Kir6.2 mutants known to disrupt PIP(2) gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. To explain this paradoxical gating behavior, we propose a model in which the open state of doubly mutant channels is highly unstable; ATP binding induces a conformational change in ATP-unbound closed channels that is conducive to brief opening when ATP unbinds, giving rise to the appearance of ATP-induced stimulation.     

Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.     

Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding

ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.     

ATP modulation of ATP-sensitive potassium channel ATP sensitivity varies with the type of SUR subunit

ATP-sensitive potassium (K(ATP)) channels comprise Kir and SUR subunits. Using recombinant K(ATP) channels expressed in Xenopus oocytes, we observed that MgATP (100 microm) block of Kir6.2/SUR2A currents gradually declined with time, whereas inhibition of Kir6.2/SUR1 or Kir6.2DeltaC36 currents did not change. The decline in Kir6.2/SUR2A ATP sensitivity was not observed in Mg(2+) free solution and was blocked by the phosphatidylinositol (PI) 3-kinase inhibitors LY 294002 (10 microm) and wortmannin (100 microm), and by neomycin (100 microm). These results suggest that a MgATP-dependent synthesis of membrane phospholipids produces a secondary decrease in the ATP sensitivity of Kir6.2/SUR2A. Direct application of the phospholipids PI 4,5-bisphosphate and PI 3,4,5-trisphosphate in the presence of 100 microm MgATP activated all three types of channel, but the response was faster for Kir6.2/SUR2A. Chimeric studies indicate that the different responses of Kir6.2/SUR2A and Kir6.2/SUR1 are mediated by the first six transmembrane domains of SUR. The MgATP-dependent loss of ATP sensitivity of Kir6.2/SUR2A was enhanced by the actin filament disrupter cytochalasin and blocked by phalloidin (which stabilizes the cytoskeleton). Phalloidin did not block the effect of PI 3,4,5-trisphosphate. This suggests that MgATP may cause disruption of the cytoskeleton, leading to enhanced membrane phospholipid levels (or better targeting to the K(ATP) channel) and thus to decreased channel ATP sensitivity.     

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.