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 KATP 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 KATP channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2ΔC26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac KATP channel, 5 μm 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2ΔC26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP. We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac KATP 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 KATP channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2ΔC26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac KATP channel, 5 μm 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2ΔC26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP. The cardiac ATP-sensitive K+ (KATP) 1The abbreviations used are: KATP, ATP-sensitive potassium channel; Kir6.2, inward rectifier potassium channel 6.2; Kir6.2ΔC26, a deletion of the last C-terminal 26 residues of Kir6.2; SUR2A, sulfonylurea receptor 2A; KirBac1.1, inward rectifier potassium channel Bac1.1; EET, epoxyeicosatrienoic acid; PIP2, phosphatidylinositol 4,5-bisphosphate; Po, channel open probability; G, current conductance; IC50, the concentration at half-maximal inhibition; H, the Hill coefficient; HEK293 cells, human embryonic kidney 293 cells; wt, wild type. channels are biological sensors that respond to intracellular metabolic changes and play important roles in regulating cardiac functions (1Saito T. Sato T. Tamagawa M. Miki T. Seino S. Nakaya H. Circulation. 2003; 107: 682-685Crossref PubMed Scopus (114) Google Scholar). Although the role of the cardiac KATP channels under normal physiological conditions remains unclear, it is crucial in ischemic preconditioning, and activation of KATP channels occurs during cardiac ischemia and hypoxia, leading to reduced Ca2+ influx and intracellular Ca2+ overload (2Gross G.J. Peart J.N. Am. J. Physiol. Heart Circ. Physiol. 2003; 285: 921-930Crossref PubMed Scopus (321) Google Scholar). Some important insights were provided by studies using the Kir6.2 knock-out mice, which have compromised ability in modulating cardiac electrophysiological properties, contractility (3Suzuki M. Li R.A. Miki T. Uemura H. Sakamoto N. Ohmoto-Sekine Y. Tamagawa M. Ogura T. Seino S. Marban E. Nakaya H. Circ. Res. 2001; 88: 570-577Crossref PubMed Scopus (180) Google Scholar, 4Seino S. J. Diabetes Complications. 2003; 17: 2-5Crossref PubMed Scopus (29) Google Scholar), and in handling cardiac ischemia and stress (3Suzuki M. Li R.A. Miki T. Uemura H. Sakamoto N. Ohmoto-Sekine Y. Tamagawa M. Ogura T. Seino S. Marban E. Nakaya H. Circ. Res. 2001; 88: 570-577Crossref PubMed Scopus (180) Google Scholar, 5Hodgson D.M. Zingman L.V. Kane G.C. Perez-Terzic C. Bienengraeber M. Ozcan C. Gumina R.J. Pucar D. O'Coclain F. Mann D.L. Alekseev A.E. Terzic A. EMBO J. 2003; 22: 1732-1742Crossref PubMed Scopus (82) Google Scholar, 6Zingman L.V. Hodgson D.M. Bast P.H. Kane G.C. Perez-Terzic C. Gumina R.J. Pucar D. Bienengraeber M. Dzeja P.P. Miki T. Seino S. Alekseev A.E. Terzic A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13278-13283Crossref PubMed Scopus (262) Google Scholar). Recently, a form of human dilated cardiomyopathy was found to be associated with mutations of the cardiac KATP channels (7Bienengraeber M. Olson T.M. Selivanov V.A. Kathmann E.C. O'Cochlain F. Gao F. Karger A.B. Ballew J.D. Hodgson D.M. Zingman L.V. Pang Y.P. Alekseev A.E. Terzic A. Nat. Genet. 2004; 36: 382-387Crossref PubMed Scopus (300) Google Scholar). The cardiac KATP channel is a heterooctamer containing four inward rectifier K+ 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 (8Aguilar-Bryan L. Clement J.P.T. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (514) Google Scholar). However, a truncation mutant of the Kir6.2 involving deletion of the C-terminal 26 residues (Kir6.2ΔC26) gives rise to channels that retain much of the ATP sensitivity in the absence of SUR (9Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar). Also, mutations in the SUR produced only small effects on ATP inhibition of Kir6.2/SUR currents (10Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). 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 α, β, and γ phosphates of ATP (11Trapp S. Haider S. Jones P. Sansom M.S. Ashcroft F.M. EMBO J. 2003; 22: 2903-2912Crossref PubMed Scopus (75) Google Scholar, 12Ribalet B. John S.A. Weiss J.N. Biophys. J. 2003; 84: 266-276Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13John S.A. Weiss J.N. Xie L.H. Ribalet B. J. Physiol. (Lond.). 2003; 552: 23-34Crossref Scopus (49) Google Scholar). However, the mechanism of Kir6.2 channel inhibition by ATP remains to be elucidated. Lipid metabolites are important modulators of the KATP channels, and these include the long chain acyl-CoA esters (14Schulze D. Rapedius M. Krauter T. Baukrowitz T. J. Physiol. (Lond.). 2003; 552: 357-367Crossref Scopus (64) Google Scholar), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphoinositides (15Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (486) Google Scholar), l-palmitoylcarnitine (16Haruna T. Horie M. Takano M. Kono Y. Yoshida H. Otani H. Kubota T. Ninomiya T. Akao M. Sasayama S. Pfluegers Arch. Eur. J. Physiol. 2000; 441: 200-207Crossref PubMed Scopus (16) Google Scholar), and epoxyeicosatrienoic acids (EETs) (17Lu T. Hoshi T. Weintraub N.L. Spector A.A. Lee H.C. J. Physiol. (Lond.). 2001; 537: 811-827Crossref Scopus (72) Google Scholar, 18Lu T. VanRollins M. Lee H.C. Mol. Pharmacol. 2002; 62: 1076-1083Crossref PubMed Scopus (62) Google Scholar). 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) (19Zeldin D.C. J. Biol. Chem. 2001; 276: 36059-36062Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). EETs are abundant endogenous constituents of the human and rat hearts, measured at 70 ng of total EETs per g of rat heart (20Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. D.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). 8,9-, 11,12-, and 14,15-EET contribute and of the total EETs in the rat whereas is (20Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. D.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The of EETs is in and in the during cardiac ischemia and K. S. Gumina R.J. Falck J.R. G.J. 2001; PubMed Scopus Google Scholar). 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Res. 1997; PubMed Scopus Google Scholar, T. VanRollins M. Weintraub N.L. Spector A.A. Lee H.C. J. Physiol. (Lond.). 2001; Scopus Google Scholar). We have previously reported that EETs are potent activators of the cardiac KATP channels, reducing the channel sensitivity to ATP, and had of (17Lu T. Hoshi T. Weintraub N.L. Spector A.A. Lee H.C. J. Physiol. (Lond.). 2001; 537: 811-827Crossref Scopus (72) Google Scholar, 18Lu T. VanRollins M. Lee H.C. Mol. Pharmacol. 2002; 62: 1076-1083Crossref PubMed Scopus (62) Google Scholar). channel studies that EETs the effect of ATP on the KATP channel properties, the channel open and the channel (17Lu T. Hoshi T. Weintraub N.L. Spector A.A. Lee H.C. J. Physiol. (Lond.). 2001; 537: 811-827Crossref Scopus (72) Google Scholar). studies that only the but not the activate the cardiac KATP channels T. VanRollins M. Lee H.C. Mol. Pharmacol. 2002; 62: 1076-1083Crossref PubMed Scopus (62) Google Scholar). the the chain and the of on the EET were not the of the epoxide group is critical for KATP channel activation T. VanRollins M. Lee H.C. Mol. Pharmacol. 2002; 62: 1076-1083Crossref PubMed Scopus (62) Google Scholar). These results that is a and interaction EETs and cardiac KATP study we the role of SUR2A and the positively charged residues on the Kir6.2 that channel ATP sensitivity and open on channel sensitivity to Kir6.2 and and of Kir6.2 were using the containing the mutations were by The of the and the mutations were by of Kir6.2 and SUR2A in were provided by Kir6.2, SUR2A, and in were into HEK293 in of using a The were by the of under HEK293 were in with 5 and and channel were with and of the was at 5 and at as previously (17Lu T. Hoshi T. Weintraub N.L. Spector A.A. Lee H.C. J. Physiol. (Lond.). 2001; 537: 811-827Crossref Scopus (72) Google Scholar). currents of Kir6.2 channels were from using a to at with a of The was for and 5 for channel with the which to with the ATP of ATP to were to a containing potassium to with The was the ATP concentration in the was ATP sensitivity of the channel was measured currents and with the The effects of EETs on the ATP sensitivity of the channel were by the changes and of 5 μm 11,12-EET in the of ATP at the ATP IC50, the ATP and channel or at ATP was and using a Hill of the channel at ATP the concentration of ATP, IC50 is the concentration at half-maximal and is the Hill were at of Kir6.2 the of a channel, A. T. J. J. Ashcroft F.M. T. Science. 2003; PubMed Scopus Google Scholar), as the to the residues of in we a of The were from the The acid of and were with to the of the for The a N terminus that of inward rectifier potassium channel The Kir6.2 was by and the two residues was measured from the used were from 8,9-, and 14,15-EET were from in as 5 and at HEK293 and were from are as the was used to two of by a was to from using We the Kir6.2/SUR2A wild type channels are activated by We measured the ATP sensitivity of Kir6.2 in the and absence of 5 μm 11,12-EET to channel activation. of Kir6.2 ATP at IC50 concentration produced inhibition of the and effect was and to ATP. However, in the of 5 μm of ATP at IC50 failed to the of inhibition in the Kir6.2 The ATP effect was significantly by 11,12-EET as in native KATP channels in cardiac (17Lu T. Hoshi T. Weintraub N.L. Spector A.A. Lee H.C. J. Physiol. (Lond.). 2001; 537: 811-827Crossref Scopus (72) Google Scholar). channel that the effects of ATP on channel were reduced in the of 5 μm 11,12-EET (Fig. 5 μm 11,12-EET increased the ATP IC50 of the channel from a of to μm (Fig. The Hill at with not These results that the Kir6.2 channels in HEK293 retain the sensitivity to activation by EET, similar to native KATP channels in cardiac Kir6.2ΔC26 channels in HEK293 without the of SUR2A as previously reported (Fig. and (9Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar). 5 μm 11,12-EET increased the ATP IC50 of Kir6.2ΔC26 from μm at to μm without (Fig. However, in the of Kir6.2ΔC26 not only the channel ATP sensitivity was IC50 Kir6.2 but was significantly reduced to with in the Kir6.2 for The current in Kir6.2ΔC26 were much in Kir6.2 Kir6.2ΔC26 with SUR2A the channel ATP IC50 and to values similar to of the Kir6.2 (Fig. with (9Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (680) Google Scholar, S.J. Gribble F.M. P. Trapp S. T. F. Ashcroft F.M. EMBO J. 1998; 17: PubMed Scopus Google Scholar). These results that the SUR2A the Kir6.2 channel and ATP sensitivity, but activation of the KATP channel by EETs does not require the of Also, the last 26 acids of the Kir6.2 C terminus were not for sensitivity to the SUR2A is not with Kir6.2 channel sensitivity to EET, studies with Kir6.2 mutant channels were with the coexpression of of on channel ATP current from Kir6.2 wt, R50A, K185A, and R201A coexpressed with SUR2A in HEK293 using a to in the of of ATP as channel of the Kir6.2 wt, the mutants and the C-terminal mutants was the ATP the The R50A, K185A, and R201A mutants in ATP The mutant but in ATP EETs activate the KATP channels by reducing channel sensitivity to ATP and may a mechanism with other metabolites in KATP channel we to identify the molecular sites on the Kir6.2 that are important for EET sensitivity by the positively charged residues and to the effects of these mutations on EET We found that the Kir6.2 R27A, R50A, K185A, and R201A mutants significantly reduced the channel ATP sensitivity, with (11Trapp S. Haider S. Jones P. Sansom M.S. Ashcroft F.M. EMBO J. 2003; 22: 2903-2912Crossref PubMed Scopus (75) Google Scholar, 12Ribalet B. John S.A. Weiss J.N. Biophys. J. 2003; 84: 266-276Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, S.L. J. Nichols C.G. J. Physiol. 2000; PubMed Scopus Google Scholar, Nichols C.G. J. Physiol. 2002; PubMed Scopus Google Scholar, P. Gribble F.M. S.J. Ashcroft F.M. J. Physiol. (Lond.). Scopus Google Scholar, F. S.J. Ashcroft F.M. J. Physiol. (Lond.). Scopus Google Scholar). current of Kir6.2 wt, R50A, K185A, and R201A coexpressed with The ATP IC50 was increased from μm in the Kir6.2 to μm in to μm in K185A and to μm in R201A (Fig. The mutant produced a small but in ATP IC50 the mutant was with IC50 and currents be from the and mutants in as previously reported S.L. J. Nichols C.G. J. Physiol. 2000; PubMed Scopus Google Scholar, P. Li L. J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the effects of EETs on ATP inhibition of the Kir6.2 mutants, we the effects of 5 μm 11,12-EET in the of IC50 of ATP for mutant channel. We found that the mutants of positively charged and R50A, on the cytoplasmic N terminus of Kir6.2 were activated by with to ATP inhibition (Fig. 5 and These that EET and ATP at sites on the C-terminal of Kir6.2, sensitivity to 11,12-EET was in R192A, K207A, K222A, and However, the effects of 11,12-EET were in the K185A, R195A, and R201A mutants, and channel inhibition by ATP was not by 11,12-EET (Fig. of ATP sensitivity and 11,12-EET effects on Kir6.2 and positively charged Kir6.2 mutants is in the of the EET regioisomers are we the effects of 8,9- and 14,15-EET with 11,12-EET on Kir6.2 and mutant We found that 8,9-, 11,12-, and 14,15-EET are activators of Kir6.2 5 μm 8,9-, 11,12-, and 14,15-EET the channel ATP IC50 inhibition by and not (Fig. not affect the ATP inhibition on the Kir6.2 K185A, R195A, and R201A mutants and the critical sites with 11,12-EET (Fig. and Similar to 8,9- and the effect of 5 μm 14,15-EET was with the K185A and R201A However, 5 μm 14,15-EET not affect the ATP inhibition on the but the ATP effect on the Arg-192, instead of is a critical site for channel activation by 14,15-EET (Fig. These results that the residues Lys-185, and Arg-201 of the Kir6.2 C terminus are critical molecular for KATP channel sensitivity to study we reported on the molecular for activation of the KATP channels by we found that the SUR2A is not for EET to activate the Kir6.2 the positively charged residues of Kir6.2 are not important for EET sensitivity of the channel. C-terminal residues Lys-185, and Arg-201 are critical molecular sites for EET These results the EET sensitivity a region acid residues on the C terminus of Kir6.2, and may the EET binding These results indicate that the molecular for EET are from ATP and The of ATP inhibition of the cardiac KATP channels are and both Kir6.2 and SUR2A subunits. results that the of SUR2A the channel IC50 and to ATP and This is with that SUR the ATP binding of Kir6.2 by the of the that the of ATP without the of the and by the interaction with the M. A. Ashcroft F.M. J. Physiol. (Lond.). 2004; Scopus Google Scholar). Previous studies Lys-185 and Arg-201 on the C terminus and on the N terminus of Kir6.2 as particularly crucial for ATP binding B. John S.A. Weiss J.N. Biophys. J. 2003; 84: 266-276Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13John S.A. Weiss J.N. Xie L.H. Ribalet B. J. Physiol. (Lond.). 2003; 552: 23-34Crossref Scopus (49) Google Scholar, S.L. J. Nichols C.G. J. Physiol. 2000; PubMed Scopus Google Scholar, Nichols C.G. J. Physiol. 2002; PubMed Scopus Google Scholar, P. Gribble F.M. S.J. Ashcroft F.M. J. Physiol. (Lond.). Scopus Google Scholar). have to the mechanism of KATP inhibition by ATP. ATP may to a channel and in a in ATP may to the channel open and the open channel, reducing and the open B. John S.A. Weiss J.N. Biophys. J. 2003; 84: 266-276Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). studies that Lys-185 with the and Arg-201 with the of the interaction the channel open whereas the the channel into B. John S.A. Weiss J.N. Biophys. J. 2003; 84: 266-276Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 13John S.A. Weiss J.N. Xie L.H. Ribalet B. J. Physiol. (Lond.). 2003; 552: 23-34Crossref Scopus (49) Google Scholar). The role of is and interaction with the B. John S.A. Weiss J.N. Biophys. J. 2003; 84: 266-276Abstract Full Text Full Text PDF PubMed Scopus (40) Google and the γ (11Trapp S. Haider S. Jones P. Sansom M.S. Ashcroft F.M. EMBO J. 2003; 22: 2903-2912Crossref PubMed Scopus (75) Google of ATP ATP binding in Kir6.2 is not using of the cytoplasmic of Kir6.2 that ATP only to the cytoplasmic C terminus but not to the N terminus of the channel C.G. K. L. A.E. M. G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). study that Arg-50, Lys-185, and Arg-201 are critical sites for ATP sensitivity, and but significantly ATP Recently, reported that may the of Lys-185 and Arg-201 of the Kir6.2 to form ATP binding site by Haider S. P. Sansom M.S. Ashcroft F.M. EMBO J. PubMed Scopus Google Scholar). This study Lys-185, Arg-201, and residues as critical sites for the channel sensitivity to 8,9- and whereas is critical for the Kir6.2 channel sensitivity to This may be to the of the epoxide group and interaction with and produced the effect and in Kir6.2 channel that may the epoxide binding However, the mechanism EETs the ATP effects is not the and mutations ATP sensitivity without channel sensitivity to 11,12-EET. the or not change the to ATP, but channel activation by 11,12-EET was the K185A and R201A mutations both the ATP and EET effects on the channel. These results that the EET binding site on Kir6.2 is from that of ATP. for binding ATP and at the C terminus of Kir6.2 reported K. C.G. L. G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). Previous studies that the residues in two and in the C terminus of Kir6.2 are important for the effects S.L. J. Nichols C.G. J. Physiol. 2000; PubMed Scopus Google Scholar). Arg-176, Arg-177, and residues in are sites critical for Of the was as a for of ATP sensitivity in the KATP channels D. Krauter T. H. M. Baukrowitz T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google with the of Arg-201, of the residues with of ATP or Recently, long chain acyl-CoA esters were found to ATP inhibition of the KATP channels by the as (14Schulze D. Rapedius M. Krauter T. Baukrowitz T. J. Physiol. (Lond.). 2003; 552: 357-367Crossref Scopus (64) Google Scholar), these metabolites a site of interaction on the channel. it that is a for inward rectifier K+ channels Nichols C.G. J. Physiol. 2002; PubMed Scopus Google Scholar). The of is of residues and the is of and which that of the a known binding to charged residues Arg-176, Arg-177, Arg-192, and are on of the and are with interaction Nichols C.G. J. Physiol. 2002; PubMed Scopus Google Scholar), whereas and are important for EET EETs and with the KATP channels a similar mechanism that ATP sensitivity is at However, we to that are EET and on KATP channel activation. the EET effects are with in the of and the on the EET is not The EET is as as EET in reducing ATP sensitivity of the channel T. VanRollins M. Lee H.C. Mol. Pharmacol. 2002; 62: 1076-1083Crossref PubMed Scopus (62) Google Scholar). PIP2, EETs not activate the KATP channels or from with the of Arg-201, residues on the Kir6.2 channel that are important for sensitivity, as and are not important for EET we that EETs and acid a of metabolites that as KATP channel the role of the sites on Kir6.2, we a molecular of Kir6.2 on the of A. T. J. J. Ashcroft F.M. T. Science. 2003; PubMed Scopus Google (Fig. This ATP-sensitive channel with Kir6.2, and it contains a N terminus that of which used for Kir6.2 Nichols C.G. Mol. 2004; PubMed Scopus Google Scholar). the of two Kir6.2 and and the of residues that are to the current study are Lys-185, Arg-201, and are and may the ATP binding Based on the the to Lys-185 and to the Arg-201 chain is and the from Lys-185 to Arg-201 is similar to a S.A. Weiss J.N. Xie L.H. Ribalet B. J. Physiol. (Lond.). 2003; 552: 23-34Crossref Scopus (49) Google (Fig. that Arg-201 with the of ATP, and Lys-185 with the of ATP. to with the of ATP S.A. Weiss J.N. Xie L.H. Ribalet B. J. Physiol. (Lond.). 2003; 552: 23-34Crossref Scopus (49) Google Scholar). However, the of the molecular of the Kir6.2 N in the ATP-sensitive sites not The residues from Lys-185 to Arg-201 of with and of (Fig. The to is and may the the epoxide in 11,12-EET and the from to Lys-185, Arg-201, and are and whereas from to these residues are and and are from the from to is and are to be with ATP binding M. A. Ashcroft F.M. J. Physiol. (Lond.). 2004; Scopus Google Scholar). for the EET effects is that or the EET binding site and interaction with EETs in changes in the ATP binding Lys-185 and Arg-201 sites of EET binding (Fig. and mutations in these sites changes that the EET The of and are not in the of and Arg-177, which are important binding and may to EETs and PIP2, they are both metabolites that activate the cardiac KATP channels, we have provided insights on Kir6.2 channel regulation by positively charged Lys-185, and Arg-201, on the C terminus of Kir6.2 are crucial for EET and are not ATP-sensitive they may with in changes in the of the ATP binding and reducing the channel sensitivity to ATP. binding studies be to with