The Role of NH2-terminal Positive Charges in the Activity of Inward Rectifier KATP Channels

Cukras, Catherine A; Jeliazkova, Iana; Nichols, Colin G.

doi:10.1085/jgp.20028621

Cited by 71 publications

(82 citation statements)

References 47 publications

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“…The R27A mutant showed less marked but still significant reduction in ATP sensitivity. the channel ATP sensitivity, consistent with previous reports (11,12,(35)(36)(37)(38). (Fig.…”

Section: Resultssupporting

confidence: 92%

“…This is consistent with recent reports that SUR modifies the ATP binding pocket of Kir6.2 by increasing the width of the groove that binds the phosphate tail of ATP without changing the length of the groove and by enhancing the interaction with the adenine ring (40). Previous studies identified Lys-185 and Arg-201 on the C terminus and Arg-50 on the N terminus of Kir6.2 as particularly crucial for ATP binding (12,13,(35)(36)(37). Two processes have been proposed to describe the mechanism of K ATP inhibition by ATP.…”

Section: Discussionsupporting

confidence: 92%

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Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

Lü

Hong

Lee

2005

Journal of Biological Chemistry

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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, ...

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“…The R27A mutant showed less marked but still significant reduction in ATP sensitivity. the channel ATP sensitivity, consistent with previous reports (11,12,(35)(36)(37)(38). (Fig.…”

Section: Resultssupporting

confidence: 92%

Section: Discussionsupporting

confidence: 92%

Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

Lü

Hong

Lee

2005

Journal of Biological Chemistry

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“…Mutation of an eighth predicted residue, H226S, prevented activation of these channels by diC 8 phosphoinositides. Surprisingly, although the C-terminal cytoplasmic portion of Kir channels is longer than the N-terminal, and it contains the majority of PIP 2 -interacting residues (14,32), the most dramatic effects were seen with N-terminal mutations.…”

Section: Resultsmentioning

confidence: 97%

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Rohács

Lopes

Jin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

196

230

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Phosphoinositides are critical regulators of ion channel and transporter activity. Defects in interactions of inwardly rectifying potassium (Kir) channels with phosphoinositides lead to disease. ATP-sensitive K ؉ channels (KATP) are unique among Kir channels in that they serve as metabolic sensors, inhibited by ATP while stimulated by long-chain (LC) acyl-CoA. Here we show that KATP are the least specific Kir channels in their activation by phosphoinositides and we demonstrate that LC acyl-CoA activation of these channels depends on their low phosphoinositide specificity. We provide a systematic characterization of phosphoinositide specificity of the entire Kir channel family expressed in Xenopus oocytes and identify molecular determinants of such specificity. We show that mutations in the Kir2.1 channel decreasing phosphoinositide specificity allow activation by LC acyl-CoA. Our data demonstrate that differences in phosphoinositide specificity determine the modulation of Kir channel activity by distinct regulatory lipids.

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“…However, the nature of the phosphoinositide-channel interaction remains elusive. For one extensively studied group, the inward rectifier K (Kir) channels, there is an emerging consensus that a direct interaction occurs between cytoplasmic domains of the channel and inositol headgroups, based on electrophysiological analysis (5,(13)(14)(15)(16) and biochemical analysis of isolated channel domains (5,17,18). Direct interaction of functional channels with phospholipids in the membrane has been difficult to demonstrate unequivocally, and without this, quantification of the dose-response relationships for channel modulation by phospholipids is obviated, and further mechanistic understanding is limited (19,20).…”

mentioning

confidence: 99%

Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Enkvetchakul

Jeliazkova

Nichols

2005

Journal of Biological Chemistry

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Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86 Rb ؉ uptake assay. We demonstrate direct modulation by PIP 2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP 2 , KirBac1.1 is inhibited by PIP 2 incorporated in the membrane (K1 ⁄ 2 ‫؍‬ 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP 2 in the membrane.Phosphoinosotides constitute a major group of signaling molecules in eukaryotic membranes (1, 2) and modulate an ever growing list of ion channels, whether by application of exogenous phospholipids to the cytoplasmic membrane surface or by manipulation of endogenous phospholipids (3-12). However, the nature of the phosphoinositide-channel interaction remains elusive. For one extensively studied group, the inward rectifier K (Kir) channels, there is an emerging consensus that a direct interaction occurs between cytoplasmic domains of the channel and inositol headgroups, based on electrophysiological analysis (5, 13-16) and biochemical analysis of isolated channel domains (5,17,18). Direct interaction of functional channels with phospholipids in the membrane has been difficult to demonstrate unequivocally, and without this, quantification of the dose-response relationships for channel modulation by phospholipids is obviated, and further mechanistic understanding is limited (19, 20).The recent cloning and crystallization (21), as well as functional analysis of KirBac1.1 channels reconstituted in lipid membranes (22), provides the opportunity to examine channel activity using a highly purified protein preparation in membranes of defined composition and permits direct test of the nature of the channel-phosphoinositide interaction. EXPERIMENTAL PROCEDURESMethods are essentially as described previously (22). KcsA and KirBac1.1 in pQE60 vector were expressed in BL21* (DE3) cells after induction with isopropyl ␤-D-thiogalactopyranoside. Bacteria were lysed by sonication, incubated 2-4 h with 30 mM decylmaltoside (Anatrace), then centrifuged at 30,000 ϫ g for 30 min, and the supernatant was applied to a cobalt affinity column. The column was washed with 20 -30 volumes of wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM KCl, 10 mM imidazole, and 5 mM decylmaltoside) and eluted with 1-2 ml of wash buffer containing 500 mM imidazole. Protein was concentrated u...

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The Role of NH2-terminal Positive Charges in the Activity of Inward Rectifier KATP Channels

Cited by 71 publications

References 47 publications

Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

Specificity of activation by phosphoinositides determines lipid regulation of Kir channels

Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

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