ABCMdb

ABCC8 p.Lys719Ala

Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), L: D (91%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (91%), S: D (91%), T: D (91%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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115 K719A (NBD1 of SUR1) prevents ATP binding at both NBDs [73] and interferes with the stimulatory effect of KATP channel regulators which act through NBD2 [74-77]. Login to comment

[hide] Molecular basis for K(ATP) assembly: transmembrane... Neuron. 2000 Apr;26(1):155-67. Schwappach B, Zerangue N, Jan YN, Jan LY
Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter.
Neuron. 2000 Apr;26(1):155-67., [PMID:10798400]

Abstract [show]
K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.

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185 We chose junctions for the chimeras based on the previously char-are expected to reduce ATPase activity (SUR1 K719A and K1385M; Gribble et al., 1997b). Login to comment

[hide] Molecular aspects of ATP-sensitive K+ channels in ... Pharmacol Ther. 2000 Jan;85(1):39-53. Fujita A, Kurachi Y
Molecular aspects of ATP-sensitive K+ channels in the cardiovascular system and K+ channel openers.
Pharmacol Ther. 2000 Jan;85(1):39-53., [PMID:10674713]

Abstract [show]
ATP-sensitive K+ (K(ATP)) channels are inhibited by intracellular ATP (ATPi) and activated by intracellular nucleoside diphosphates and thus, provide a link between cellular metabolism and excitability. K(ATP) channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K(ATP) channel appears to be activated during ischemic or hypoxic conditions, and may be responsible for the increase of K+ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective, as well as proarrhythmic, effects. These channels are clearly heterogeneous. The cardiac K(ATP) channel is the prototype of K(ATP) channels possessing approximately 80 pS of single-channel conductance in the presence of approximately 150 mM extracellular K+ and opens spontaneously in the absence of ATPi. A vascular K(ATP) channel called a nucleoside diphosphate-dependent K+ (K(NDP)) channel exhibits properties significantly different from those of the cardiac K(ATP) channel. The K(NDP) channel has the single-channel conductance of approximately 30-40 pS in the presence of approximately 150 mM extracellular K+, is closed in the absence of ATPi, and requires intracellular nucleoside di- or triphosphates, including ATPi to open. Nevertheless, K(ATP) and K(NDP) channels are both activated by K+ channel openers, including pinacidil and nicorandil, and inhibited by sulfonylurea derivatives such as glibenclamide. It recently was found that the cardiac K(ATP) channel is composed of a sulfonylurea receptor (SUR)2A and a two-transmembrane-type K+ channel subunit Kir6.2, while the vascular K(NDP) channel may be the complex of SUR2B and Kir6.1. By precisely comparing the functional properties of the SUR2A/Kir6.2 and the SUR2B/Kir6.1 channels, we shall show that the single-channel characteristics and pharmacological properties of SUR/Kir6.0 channels are determined by Kir and SUR subunits, respectively, while responses to intracellular nucleotides are determined by both SUR and Kir subunits.

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565 Gribble et al. (1997b) found that the ATPi sensitivity of the SUR1/Kir6.2 channel was not modified by mutations on either or both of the two conserved lysine residues in the Walker A motifs in the first or the second NBF of SUR1 (K719A and K1384M, respectively). Login to comment
568 Because Ueda et al. (1997) and Gribble et al. (1997b) used different mutations (K719R, K719M, or D854N vs. K719A, respectively), it is not clear whether the ATPi binding found by Ueda et al. (1997) underlies the sensitization of Kir6.2 to ATPi by SUR1. Login to comment
629 Gribble et al. (1997b) showed that either the K719A or K1384M mutation of r-SUR1 abolished the stimulatory effects of ADPi on the partial rundown SUR1/Kir6.2 channel, both in the presence and in the absence of ATPi. Login to comment
569 Gribble et al. (1997b) found that the ATPi sensitivity of the SUR1/Kir6.2 channel was not modified by mutations on either or both of the two conserved lysine residues in the Walker A motifs in the first or the second NBF of SUR1 (K719A and K1384M, respectively). Login to comment
572 Because Ueda et al. (1997) and Gribble et al. (1997b) used different mutations (K719R, K719M, or D854N vs. K719A, respectively), it is not clear whether the ATPi binding found by Ueda et al. (1997) underlies the sensitization of Kir6.2 to ATPi by SUR1. Login to comment
633 Gribble et al. (1997b) showed that either the K719A or K1384M mutation of r-SUR1 abolished the stimulatory effects of ADPi on the partial rundown SUR1/Kir6.2 channel, both in the presence and in the absence of ATPi. Login to comment

[hide] Studies of the ATPase activity of the ABC protein ... FEBS J. 2007 Jul;274(14):3532-44. Epub 2007 Jun 11. de Wet H, Mikhailov MV, Fotinou C, Dreger M, Craig TJ, Venien-Bryan C, Ashcroft FM
Studies of the ATPase activity of the ABC protein SUR1.
FEBS J. 2007 Jul;274(14):3532-44. Epub 2007 Jun 11., [PMID:17561960]

Abstract [show]
The ATP-sensitive potassium (K(ATP)) channel couples glucose metabolism to insulin secretion in pancreatic beta-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and/or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide-binding domains (fused to maltose-binding protein to improve solubility). Electron microscopy showed that nucleotide-binding domains purified as ring-like complexes corresponding to approximately 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol P(i).s(-1).(nmol protein)(-1)] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and a mixture of nucleotide-binding domain 1 and nucleotide-binding domain 2, respectively. Corresponding K(m) values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower K(m) than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were found with GTP, but the K(m) values were lower. Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (K(i) 16 microm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide-binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may influence the activity of the protein.

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12 Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Login to comment
77 Mutating the WA lysine in NBD1 of SUR1 to alanine (K719A) reduced ATPase activity by approximately 60%, whereas mutating the WA lysine in NBD2 to methionine (K1385M) inhibited ATPase activity by about 80% (Fig. 2C) when compared to wild-type controls assayed in parallel (n ¼ 2). Login to comment
87 (C) ATPase activity of SUR1 containing the mutations K719A (triangles, n ¼ 2) or K1385M (diamonds, n ¼ 2). Login to comment
162 Mutation of the WA lysines (K719A, K1385M) reduced ATPase activity by 70-80%. Login to comment

[hide] Regulation of KATP channel activity by diazoxide a... J Gen Physiol. 1997 Dec;110(6):643-54. Shyng S, Ferrigni T, Nichols CG
Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor.
J Gen Physiol. 1997 Dec;110(6):643-54., [PMID:9382893]

Abstract [show]
KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945-951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4-. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.

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No. Sentence Comment
139 Gribble et al. (1997) have recently shown that mutation of the conserved lysine residues in the Walker A motifs of either NBF1 (mutation K719A) or NBF2 (mutation K1384M), which are predicted to reduce ATP hydrolytic activity (Azzaria et al., 1989; Carson et al., 1995; Ko and Pedersen, 1995), can block the stimulatory effect of MgADP. Login to comment
140 In that study, diazoxide stimulated K1384M (NBF2 mutant) channels, but failed to stimulate K719A (NBF1 mutant) channels, leading to the suggestion that hydrolysis at NBF1 was most critical for diazoxide stimulation. Login to comment

[hide] KATP channel interaction with adenine nucleotides. J Mol Cell Cardiol. 2005 Jun;38(6):907-16. Epub 2005 Feb 5. Matsuo M, Kimura Y, Ueda K
KATP channel interaction with adenine nucleotides.
J Mol Cell Cardiol. 2005 Jun;38(6):907-16. Epub 2005 Feb 5., [PMID:15910875]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels are regulated by adenine nucleotides to convert changes in cellular metabolic levels into membrane excitability. Hence, elucidation of interaction of SUR and Kir6.x with adenine nucleotides is an important issue to understand the molecular mechanisms underlying the metabolic regulation of the K(ATP) channels. We analyzed direct interactions with adenine nucleotides of each subunit of K(ATP) channels. Kir6.2 binds adenine nucleotides in a Mg(2+)-independent manner. SUR has two NBFs which are not equivalent: NBF1 is a Mg(2+)-independent high affinity nucleotide binding site, whereas NBF2 is a Mg-dependent low affinity site. Although SUR has ATPase activity at NBF2, it is not used to transport substrates against the concentration gradient unlike other ABC proteins. The ATPase cycle at NBF2 serves as a sensor of cellular metabolism. This may explain the low ATP hydrolysis rate compared to other ABC proteins. Based on studies of photoaffinity labeling, a model of K(ATP) channel regulation is proposed, in which K(ATP) channel activity is regulated by SUR via monitoring the intracellular MgADP concentration. K(ATP) channel activation is expected to be induced by the cooperative interaction of ATP binding at NBF1 and MgADP binding at NBF2.

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No. Sentence Comment
61 Mutations within the Walker A (K719A and K1385M) or Walker B (D853N, D1506A and D1506N) motifs of both NBFs of SUR1 abolished the activation of KATP channels by MgADP [43-45]. Login to comment
82 Gribble et al. [44] reported that mutations in the Walker A motif of both NBFs of SUR1 (K719A and K1385A) abolished the channel activation by MgADP. Login to comment

[hide] Potassium channel openers require ATP to bind to a... EMBO J. 1998 Oct 1;17(19):5529-35. Schwanstecher M, Sieverding C, Dorschner H, Gross I, Aguilar-Bryan L, Schwanstecher C, Bryan J
Potassium channel openers require ATP to bind to and act through sulfonylurea receptors.
EMBO J. 1998 Oct 1;17(19):5529-35., [PMID:9755153]

Abstract [show]
KATP channels are composed of a small inwardly rectifying K+ channel subunit, either KIR6.1 or KIR6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP-binding cassette superfamily. SUR1/KIR6.2 reconstitute the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular-smooth-muscle-type KATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUR2A and SUR2B requires ATP. Non-hydrolysable ATP-analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide-binding folds (NBFs) abolish or weaken [3H]P1075 binding to SUR2B, rendering reconstituted SUR2B/KIR6.2 channels insensitive towards KCOs. The C-terminus of SUR affects KCO affinity with SUR2B approximately SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [3H]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5-8-fold higher than their potencies for activation of SUR2B/KIR6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.

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112 For example, the substitution of an alanine for the conserved lysine in the Walker A motif in NBF1 (K719A) abolished diazoxide-induced activation of SUR1/ KIR6.2 channels, whereas substitution of a methionine at the equivalent position in NBF2 (K1384M) had a more subtle effect, eliminating activation by diazoxide in the presence of low (10 µM), but not high (100 µM) [ATP] (Gribble et al., 1997). Login to comment

[hide] Functional analysis of a mutant sulfonylurea recep... J Biol Chem. 2000 Dec 29;275(52):41184-91. Matsuo M, Trapp S, Tanizawa Y, Kioka N, Amachi T, Oka Y, Ashcroft FM, Ueda K
Functional analysis of a mutant sulfonylurea receptor, SUR1-R1420C, that is responsible for persistent hyperinsulinemic hypoglycemia of infancy.
J Biol Chem. 2000 Dec 29;275(52):41184-91., [PMID:10993895]

Abstract [show]
The ATP-sensitive potassium (K(ATP)(+)) channel is crucial for the regulation of insulin secretion from the pancreatic beta-cell, and mutations in either the sulfonylurea receptor type 1 (SUR1) or Kir6. 2 subunit of this channel can cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1. Mild tryptic digestion of SUR1 after photoaffinity labeling allowed analysis of the nucleotide-binding properties of NBF1 and NBF2. Labeling of NBF1 with 8-azido-[alpha-(32)P]ATP was inhibited by MgATP and MgADP with similar K(i) for wild-type SUR1 and SUR1-R1420C. However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1. MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide. However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC(50) for MgADP activation from 74 to 197 microm. We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller K(ATP)(+) current in R1420C-PHHI beta-cells and thereby to the enhanced insulin secretion. We also propose a new model for nucleotide activation of K(ATP)(+) channels.

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154 As Fig. 6A also shows, Kir6.2-R50G/SUR1-K719A currents were neither activated nor blocked by 100 ␮M MgATP, demonstrating that, at this ATP concentration, activation of Kir6.2-R50G/SUR1 and Kir6.2-R50G/SUR1-R1420C currents is not partially masked by an inhibitory effect of ATP. Login to comment
155 At ATP concentrations of 1 mM and above, however, Kir6.2-R50G/SUR1-K719A currents are inhibited. Login to comment
177 A, mean macroscopic currents recorded from oocytes coexpressing Kir6.2-R50G and either wild-type SUR1, SUR1-R1420C, or SUR1-K719A in the presence of 100 ␮M of the nucleotides indicated. The mean conductance in the presence of nucleotide (G) is expressed relative to the mean of that measured in control solution before and after removal of nucleotide (Gc indicated by the dashed line). Login to comment

[hide] Direct interaction of Na-azide with the KATP chann... Br J Pharmacol. 2000 Nov;131(6):1105-12. Trapp S, Ashcroft FM
Direct interaction of Na-azide with the KATP channel.
Br J Pharmacol. 2000 Nov;131(6):1105-12., [PMID:11082117]

Abstract [show]
1. The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type. 2. In inside-out patches from oocytes expressing Kir6.2 delta C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose. 3. Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2 delta C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+. 4. Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR. 5. We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit. The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration.

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138 To determine if the NBDs are also required for the ability of MgATP to support Na-azide activation we used a mutant SUR in which the conserved lysine in NBD1 was changed to alanine (SUR1-K719A). Login to comment
140 Inhibition of Kir6.2-R50G/SUR1-K719A currents by 3 mM Na-azide amounted to 23+1% (n=3) in the absence of ATP, which was not signi®cantly dierent (P40.05) from that observed for Kir6.2-R50G/SUR1 currents (Figure 6C). Login to comment
141 As previously reported (Gribble et al., 1998), 1 mM ATP did not activate Kir6.2-R50G/SUR1-K719A currents ± instead the current was blocked by 44+4% (n=6). Login to comment
177 Na-azide (3 mM) and ATP (1 mM) were added to the internal solution as indicated by the bars. (B) Macroscopic Kir6.2-R50G/ SUR1-K719A currents recorded from an inside-out patch in response to voltage ramps from 7110 mV to +100 mV. Na-azide (3 mM) and ATP (1 mM) were added to the internal solution as indicated by the bars. (C) Mean slope conductance (G) in the presence of Na-azide or Na-azide plus ATP is expressed relative to the mean conductance in control solution (lacking Na-azide) before and after addition of the test solution (Gc). Login to comment

[hide] ATP-sensitive potassium channels: a model of heter... Annu Rev Physiol. 1999;61:337-62. Seino S
ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies.
Annu Rev Physiol. 1999;61:337-62., [PMID:10099692]

Abstract [show]
ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. By cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the receptors for sulfonylureas (SUR1 and SUR2), researchers have clarified the molecular structure of KATP channels. KATP channels comprise two subunits: a Kir6.0 subfamily subunit, which is a member of the inwardly rectifying K+ channel family; and a SUR subunit, which is a member of the ATP-binding cassette (ABC) protein superfamily. KATP channels are the first example of a heteromultimeric complex assembled with a K+ channel and a receptor that are structurally unrelated to each other. Since 1995, molecular biological and molecular genetic studies of KATP channels have provided insights into the structure-function relationships, molecular regulation, and pathophysiological roles of KATP channels.

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204 However, mutations of lysine residues in the Walker A motifs in NBF-1 (K719A) and/or the equivalent mutation in NBF-2 (K1384M) of SUR1 do not prevent channel inhibition by ATP (36). Login to comment
207 In contrast, neither the NBF-1 mutant K719A channel nor the NBF-2 mutant K1384M channel is activated by MgADP (36). Login to comment
210 A mutation in the Walker A motif in NBF-1 (K719A) abolishes channel activation by diazoxide, but a mutation in the Walker A motif in NBF-2 (K1384M) does not affect the channel activation (36). Login to comment

[hide] Activation and inhibition of K-ATP currents by gua... Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8872-7. Trapp S, Tucker SJ, Ashcroft FM
Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits.
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8872-7., [PMID:9238070]

Abstract [show]
The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.

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14 We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. Login to comment
38 We examined the effects of mutating the WA lysine in either the first (K719A) or second (K1384M) NBD. Login to comment
95 We refer to these as K719A (wild-type Kir6.2 coexpressed with K719A-SUR1) and K1384M (wild-type Kir6.2 coexpressed with K1384M-SUR1). Login to comment
96 In contrast to wild-type K-ATP currents, neither K719A nor K1384M currents were activated by GTP (Fig. 2A). Login to comment
97 Instead, increasing GTP concentrations simply produced a dose-dependent inhibition of both K719A and K1384M currents (Fig. 2B). Login to comment
98 For K719A currents, the Ki for current inhibition was 2.7 Ϯ 0.6 mM and the Hill coefficient was 1.0 Ϯ 0.2 (n ϭ 5), and for K1384M currents, the Ki was 3.3 Ϯ 0.5 mM and the Hill coefficient was 1.4 Ϯ 0.2 (n ϭ 5). Login to comment
102 (A) Macroscopic currents recorded from three different inside-out patches in response to a series of voltage ramps from -110 to ϩ100 mV. Oocytes were coinjected with mRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, or K1384M-SUR1 mRNAs. Login to comment
111 Neither K719A nor K1384M currents were activated by 100 ␮M GDP. Login to comment
115 The lack of an inhibitory effect of 1 mM GDP on K719A or K1384M currents suggests that at this concentration GDP exerts only a stimulatory effect on wild-type K-ATP currents. Login to comment
116 MgGDP (10 mM) blocked K719A and K1384M currents by 30 Ϯ 5% and by 55 Ϯ 6%, respectively (Fig. 3C). Login to comment
117 The extent of this inhibition suggests that GDP does not interact as strongly as ADP with the inhibitory binding site, since as little as 100 ␮M ADP blocked K719A and K1384M currents by Ͼ60% (13). Login to comment
121 We therefore examined the ability of guanine nucleotides to support the action of diazoxide on wild-type, K719A, and K1384M currents. Login to comment
131 (A) Macroscopic currents recorded from three different inside-out patches in response to a series of voltage ramps from -110 to ϩ100 mV. Oocytes were coinjected with mRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, or K1384M-SUR1. Login to comment
136 The broken line indicates the control (GDP-free) conductance level. The number of patches is indicated above the bars. Diazoxide produced a slight (but significant, P Ͻ 0.05) block of K719A currents when either GTP or GDP was present. Login to comment
144 The efficacy of block was comparable to that observed for wild-type Kir6.2 when coexpressed with mutant SUR1: half-maximal inhibition was 6.0 mM for Kir6.2⌬C26 currents, 2.7 mM for K719A currents, and 3.3 mM for K1384M currents. Login to comment
148 This may explain why the K719A and K1384M currents appear slightly more sensitive to GTP than Kir6.2⌬C26 currents. Login to comment
155 (A) Macroscopic currents recorded from three different inside-out patches in response to a series of voltage ramps from -110 to ϩ100 mV. Oocytes were coinjected with mRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, or K1384M-SUR1. Login to comment
160 Inhibition of K719A currents by diazoxide was significant both in the presence of GTP (P Ͻ 0.05) or GDP (P Ͻ 0.005). Login to comment
167 One explanation for the inability of guanine nucleotides to enhance K719A or K1384M currents is that mutation of the WA lysines abolishes binding of either GTP or GDP. Login to comment
172 The fact that neither GTP nor GDP activate native K-ATP currents in the absence on Mg2ϩ (13), or K719A or K1384M currents in the presence of Mg2ϩ , is consistent with this hypothesis. Login to comment
174 We further show that mutation of the WA lysine at NBD1 (K719A) completely abolishes, while mutation of that at NBD2 (K1384M) very substantially reduces, the ability of GTP or GDP to support diazoxide activation. Login to comment

[hide] MgATP activates the beta cell KATP channel by inte... Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):7185-90. Gribble FM, Tucker SJ, Haug T, Ashcroft FM
MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit.
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):7185-90., [PMID:9618560]

Abstract [show]
ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.

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7 Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). Login to comment

[hide] The novel diazoxide analog 3-isopropylamino-7-meth... Diabetes. 2002 Jun;51(6):1896-906. Dabrowski M, Ashcroft FM, Ashfield R, Lebrun P, Pirotte B, Egebjerg J, Bondo Hansen J, Wahl P
The novel diazoxide analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide is a selective Kir6.2/SUR1 channel opener.
Diabetes. 2002 Jun;51(6):1896-906., [PMID:12031979]

Abstract [show]
ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.

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154 Specifically, we mutated lysine 719 to alanine [SUR1(K719A)] and lysine 1384 to methionine [SUR1(K1384M)]. Login to comment
230 A: Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to 100 mV from oocytes coexpressing Kir6.2 and either SUR1(K719A) or SUR1(K1384 mol/l). Login to comment
155 Specifically, we mutated lysine 719 to alanine [SUR1(K719A)] and lysine 1384 to methionine [SUR1(K1384M)]. Login to comment
231 A: Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from d1a;110 to 100 mV from oocytes coexpressing Kir6.2 and either SUR1(K719A) or SUR1(K1384 mol/l). Login to comment

[hide] Involvement of the n-terminus of Kir6.2 in couplin... J Physiol. 1999 Jul 15;518 ( Pt 2):325-36. Reimann F, Tucker SJ, Proks P, Ashcroft FM
Involvement of the n-terminus of Kir6.2 in coupling to the sulphonylurea receptor.
J Physiol. 1999 Jul 15;518 ( Pt 2):325-36., [PMID:10381582]

Abstract [show]
1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction with a high-affinity site on SUR1 and a low-affinity site on Kir6.2. MgADP and diazoxide interact with SUR1 to promote channel activity. 2. We examined the effect of N-terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2/SUR1. 3. A 14 amino acid N-terminal deletion (DeltaN14) did not affect the gating, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, Kir6.2DeltaC26, expressed in the absence of SUR1. Thus, the N-terminal deletion does not alter the intrinsic properties of Kir6.2. 4. When Kir6.2DeltaN14 was coexpressed with SUR1, the resulting KATP channels had a higher open probability (Po = 0.7) and a lower ATP sensitivity (Ki = 196 microM) than wild-type (Kir6.2/SUR1) channels (Po = 0.32, Ki = 28 microM). High-affinity tolbutamide block was also abolished. 5. Truncation of five or nine amino acids from the N-terminus of Kir6.2 also enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high-affinity tolbutamide block, although to a lesser extent than for the DeltaN14 deletion. Site-directed mutagenesis suggests that hydrophobic residues in Kir6. 2 may be involved in this effect. 6. The reduced ATP sensitivity of Kir6.2DeltaN14 may be explained by the increased Po. However, when the Po was decreased (by ATP), tolbutamide was unable to block Kir6. 2DeltaN14/SUR1-K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166S/SUR1-K719A,K1385M currents (which in the absence of ATP have a Po of > 0.8 and are not blocked by tolbutamide). Thus the N-terminus of Kir6.2 may be involved in coupling sulphonylurea binding to SUR1 to closure of the Kir6.2 pore.

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25 However, when the Pï was decreased (by ATP), tolbutamide was unable to block Kir6.2ÄN14ÏSUR1-K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166SÏSUR1-K719A,K1385M currents (which in the absence of ATP have a Pï of > 0·8 and are not blocked by tolbutamide). Login to comment
46 We use the abbreviation SUR1-KAKM to refer to mutation of the Walker A lysines in both NBD1 (K719A) and NBD2 (K1385M) of SUR1. Login to comment
169 Figure 9Ab shows that the ability of tolbutamide to block the channel in the presence of MgATP was also abolished when both Walker A lysines were mutated (SUR1-K719A,K1385M, abbreviated SUR1-KAKM). Login to comment

[hide] Interaction of vanadate with the cloned beta cell ... J Biol Chem. 1999 Sep 3;274(36):25393-7. Proks P, Ashfield R, Ashcroft FM
Interaction of vanadate with the cloned beta cell K(ATP) channel.
J Biol Chem. 1999 Sep 3;274(36):25393-7., [PMID:10464267]

Abstract [show]
Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (K(ATP)) channels in the absence of nucleotides. K(ATP) channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mM), in the absence and presence of magnesium nucleotides, on different types of cloned K(ATP) channels expressed in Xenopus oocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by approximately 50% but rapidly activated Kir6.2/SUR2A ( approximately 4-fold) and Kir6. 2/SUR2B ( approximately 2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6. 2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates K(ATP) channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1-6 of SUR.

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49 In SUR1, this corresponds to K719A and K1385M and in SUR2A to K707A and K1348A. Login to comment

[hide] The interaction of nucleotides with the tolbutamid... J Physiol. 1997 Oct 1;504 ( Pt 1):35-45. Gribble FM, Tucker SJ, Ashcroft FM
The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation.
J Physiol. 1997 Oct 1;504 ( Pt 1):35-45., [PMID:9350615]

Abstract [show]
1. We have examined the mechanism by which nucleotides modulate the tolbutamide block of the beta-cell ATP-sensitive K+ channel (KATP channel), using wild-type and mutant KATP channels heterologously expressed in Xenopus oocytes. This channel is composed of sulphonylurea receptor (SUR1) and pore-forming (Kir6.2) subunits. 2. The dose-response relation for tolbutamide block of wild-type KATP currents in the absence of nucleotide showed both a high-affinity (Ki = 2.0 microM) and a low-affinity (Ki = 1.8 mM) site. 3. The dose-response relation for tolbutamide block of Kir6.2 delta C36 (a truncated form of Kir6.2 which is expressed independently of SUR1) was best fitted with a single, low-affinity site (Ki = 1.7 mM). This indicates that the high-affinity site resides on SUR1, whereas the low-affinity site is located on Kir6.2. 4. ADP (100 microM) had a dual effect on wild-type KATP currents: the nucleotide enhanced the current in the presence of Mg2+, but was inhibitory in the absence of Mg2+. Kir6.2 delta C36 currents were blocked by 100 microM ADP in the presence of Mg2+. 5. For wild-type KATP currents, the blocking effect of 0.5 mM tolbutamide appeared greater in the presence of 100 microM MgADP (84 +/- 2%) than in its absence (59 +/- 4%). When SUR1 was mutated to abolish MgADP activation of KATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. 6. The Ki for tolbutamide interaction with either the high- or low-affinity site was unaffected by 100 microM MgADP, for both wild-type and K719A-K1384M currents. 7. MgGDP (100 microM) enhanced wild-type KATP currents and was without effect on K719A-K1384M currents. It did not affect the Ki for tolbutamide block at either the high- or low-affinity site. 8. Our results indicate that interaction of tolbutamide with the high-affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhibitory effect of ADP and leads to an apparent increase in channel inhibition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide inhibits the KATP channel.

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13 When SUR1 was mutated to abolish MgADP activation of KATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. Login to comment
15 The Ki for tolbutamide interaction with either the high- or low-affinity site was unaffected by 100 /SM MgADP, for both wild-type and K719A-K1384M currents. Login to comment
17 MgGDP (100 /M) enhanced wild-type KATP currents and was without effect on K719A-K1384M currents. Login to comment
55 The lysine residues at position 719 or 1384 of SUR1 were replaced by an alanine or methionine, respectively (K719A, K1384M). Login to comment
58 All SUR1 mutations were coexpressed with wild-type Kir6.2 and the resulting currents are referred to by the SUR1 mutation only (thus, wild-type Kir6.2 plus SUR1-K719A currents are referred to as K719A currents). Login to comment
107 Mutation of the WA lysine residues in either NBD1 (K719A) or NBD2 (K1384M), or both (K719A-K1384M) prevented the MgADP activation of KATP currents and unmasked the inhibitory effect of MgADP (Figs 2C and 4; Gribble et al. 1997 b). Login to comment
108 In the absence A 1 nA Tolbutamide B 30 s 0-5 nA MgADP Tolbutamide of nucleotides, the extent of block of K719A-K1384M, K719A and K1384M currents by 0 5 mm tolbutamide was similar to that of wild-type channels (Fig. 3). Login to comment
111 Effects of MgADP and tolbutamide on K719A-K1384M currents Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV (holding potential, 0 mV). Login to comment
112 The oocytes were coinjected with mRNAs encoding wild-type Kir6.2 and K719A-K1384M SUR1. Login to comment
122 K719A-K1384M currents by 67 % (Fig. 2C). Login to comment
124 Furthermore, MgADP did not enhance tolbutamide inhibition of K719A-K1384M currents (Figs 2 and 3). Login to comment
125 Similar results were observed for K719A and K1384M currents (Figs 3 and 4), which confirms that the WA lysine at each of the NBDs of SUR1 is needed both for the stimulatory effect of MgADP itself 150 - 100 -1. Login to comment
141 O (dashed line), K719A-K1384M currents (n = 6). Login to comment
144 K719A-K1384M channel currents: K11 = 1'6 /M, h1 = 0-7, K42 = 2-1 mM, h2==14, L = 0'40. Login to comment
152 O, K719A-K1384M currents, 100 /uM MgADP (n = 6). Login to comment
154 In the presence of 100 ,UM MgADP: Kj1 = 6-6 ,UM, h1 = 1.1, K12= 43 mM, h2= 1-0, L = 0-13,A =4-3, B= 0-33, for wild-type channel currents; and Kj1= 3-1 um, h1= 12, K12=5-5mM, h2=1,L=044,A=1 0,B=033,for K719A-K1384M currents. Login to comment
158 A, K719A-K1384M currents, 100 /uM MgGDP (n = 6). Login to comment
160 In the presence of 100 /uM MgGDP: Kj1 = 60 FuM, h1 = 1 0, K12 = 2-0 mm, h2 = 1 0, L = 0.39, A = 1 9, B = 0 9, for wild-type channel currents; and Ki = 3-8 FuM, h,= 10, K2= 28 mM, h2= 1-3, L= 0-56, A = 1 0, B= 0-9, for K719A-K1384M currents. Login to comment
161 1000 100000.01 0.1 1 10 100 [Tolbutamide] (uM) 40 J. Physiol.504.1 We next examined the dose-response curve for tolbutamide block of K719A-K1384M currents. Login to comment
172 Unlike wild-type KATP currents, K719A-K1384M channel currents were blocked by 100 /LM MgADP (by 63-4 + 2-8%, n = 17). Login to comment
175 The best fit to the mutant channel currents in 100 /M MgADP was obtained with a K1 of 3-1 uM (2-1-4-7 /tM, n = 5), which is not significantly different (ANOVA) from that of wild-type currents, or K719A-K1384M currents in the absence of ADP. Login to comment
178 This would account for the fact that MgADP has no effect on the tolbutamide block of K719A-K1384M currents, which are not stimulated by the nucleotide. Login to comment
189 In contrast to MgADP, 100 ,UM MgGDP had little effect on K719A-K1 384M currents, which were 90 + 3% (n = 6) of their amplitude in control solution. Login to comment
192 This confirms that MgGDP is able to act at the stimulatory nucleotide-binding site in wild-type, but not K719A-K1384M, channels. Login to comment
237 Second, the tolbutamide block of K719A-K1384M currents, which are not potentiated by MgADP, was not altered by MgADP. Login to comment
240 Since Mg2+ is required for the stimulatory effects of MgADP and MgGDP (Bokvist et al. 1991; Gribble et al. 1997 b), our results may also explain why nucleotide diphosphates only influence tolbutamide inhibition of KATP currents in the presence of Mg2+; in the absence of Mg2+, the effects of ADP would resemble those found for K719A-K1384M currents. Login to comment

[hide] The essential role of the Walker A motifs of SUR1 ... EMBO J. 1997 Mar 17;16(6):1145-52. Gribble FM, Tucker SJ, Ashcroft FM
The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.
EMBO J. 1997 Mar 17;16(6):1145-52., [PMID:9135131]

Abstract [show]
The ATP-sensitive K-channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg-ADP, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (W(A)) motifs of the first (K719A) and second (K1384M) nucleotide-binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the W(A) lysine of NBD1 (but not NBD2) is essential for activation of K-ATP currents by diazoxide. The potentiatory effects of Mg-ADP required the presence of the W(A) lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild-type currents. Metabolic inhibition led to activation of wild-type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K-ATP channel activity.

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10 In addition to its well- residues in the Walker A (WA) motifs of the first known inhibitory effect, Mg-ATP enhances channel (K719A) and second (K1384M) nucleotide-binding activity as evidenced by the fact that when Mg-ATP is domains (NBDs) of SUR1. Login to comment
19 When type and K1384M currents, but not K719A or K719A/ Mg2ϩ is present, however, high concentrations of ADP are K1384M currents, suggesting that there may be a inhibitory whereas low concentrations potentiate channel factor in addition to ATP and ADP which regulates activity (Dunne and Petersen, 1986; Kakei et al., 1986; K-ATP channel activity. Login to comment
45 We also found that metabolic inhibition led to activation ofγ and β phosphate groups of ATP and is essential for ATP hydrolysis (Azzaria et al., 1989; Saraste et al., 1990; Tian wild-type and K1384M currents, but not K719A or K719A/ K1384M currents. Login to comment
56 This may be attributed to relief of the blockingcritical lysine in the WA motifs of either NBD1 (K719A) or NBD2 (K1384M), or both (K719A/K1384M), of SUR1, effect of cytoplasmic ATP (Gribble et al., 1997). Login to comment
59 The mean current amplitudes at -100 mV following patch excision were: -3.9 Ϯ 0.8 nAseverely impair nucleotide hydrolysis without significantly affecting nucleotide binding (Azzaria et al., 1989; Saraste (n ϭ 12) for wild-type, -2.2 Ϯ 0.8 nA (n ϭ 12) for K719A, -5.0 Ϯ 1.9 nA (n ϭ 11) for K1384M and -2.6 Ϯ 0.9 nA (n ϭet al., 1990; Tian et al., 1990; Higgins, 1992; Carson et al., 1995; Ko and Pedersen, 1995). Login to comment
60 Our results indicate 8) for K719A/K1384M. Login to comment
68 Mutant (n ϭ 8) for K719A, 15.7 Ϯ 0.2 µM (n ϭ 6) for K1384M and 16.5 Ϯ 0.3 µM (n ϭ 5) for K719A/K1384M. Login to comment
79 Oocytes were coinjected with mutant channels showed 'refreshment` in the presence ofmRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, Mg2ϩ (Figure 2A), it is unlikely that the ATP hydrolysisK1384M-SUR1 or the double SUR1 mutant K719A/K1384M. Login to comment
81 channels (d, n ϭ 10) and the mutant K-ATP channels K719A (j, n ϭ 8), K1384M (u, n ϭ 6) or K719A/K1384M (s, n ϭ 5). Login to comment
86 Solid line fit to wild-type currents; dotted line fit to K719A/ inhibitory. Login to comment
92 The Hill coefficients were unaffected by mutation of the WA lysines, being Furthermore, both NBDs are required for channel activation: interaction of Mg-ADP with a single NBD is not1.03 Ϯ 0.06 (n ϭ 10) for currents formed from Kir6.2 and wild-type SUR1, 0.99 Ϯ 0.10 (n ϭ 8) for K719A, sufficient because neither K719A nor K1384M currents were enhanced by Mg-ADP. Login to comment
93 In this context, it is of interest1.35 Ϯ 0.13 (n ϭ 6) for K1384M and 0.99 Ϯ 0.14 (n ϭ 5) for K719A/K1384M (n.s., ANOVA). Login to comment
109 Figure 6A shows that, K719A-SUR1, K1384M-SUR1 or the double SUR1 mutant K719A/ in the presence of 100 µM Mg-ATP, diazoxide activatedK1384M; 100 µM MgATP and 100 µM MgADP were added to the both wild-type and K1384M currents but did not affectinternal solutions as indicated by the bars. Login to comment
110 K719A or K719A/K1384M currents. Login to comment
111 When Mg-ATP was reduced to 10 µM, wild-type currents were enhanced by diazoxide, K719A and K719A/K1384M were inhibited(Figure 5). Login to comment
119 The fact that the percentage block of the WA mutant currents by Mg-ADP is smaller in the presence K719A and K719A/K1384M currents. Login to comment
123 AMP (100 µM) blocked wild-type currents by 3.9 Ϯ 5.7% (n ϭ 6) and K719A/ that the channel open probability cannot exceed one. Login to comment
130 A third(measured between -20 and -100 mV) by 15.3 Ϯ 1.6% (n ϭ 8) in wild-type channels, by 17.4 Ϯ 0.6% (n ϭ 5) possibility, which cannot be completely excluded, is that our ADP solution contains a small quantity of ATP eitherin K719A channels, 18.2 Ϯ 1.0% (n ϭ 4) in K1384M channels and by 12.9 Ϯ 2.3% (n ϭ 6) in K719A/K1384M as a contaminant or formed from ADP by the action by enzymes present in the patch membrane.channels (n.s., ANOVA). Login to comment
136 The number of oocytes was: wild type (n ϭ 13), K719A (n ϭ 8), K1384M (n ϭ 12) and K719A/K1384M (n ϭ 5). Login to comment
162 Metabolic inhibition increased, and diazoxide further potentiated both wild-type and K1384M whole- ATP sensitivity reported for native K-ATP channels in the absence of Mg2ϩ (Ashcroft and Kakei, 1989).cell currents but was without effect in oocytes expressing K719A or K719A/K1384M. Login to comment
182 We Carson et al., 1995; Ko and Pedersen, 1995; Koronakis speculate that nucleotide binding, or hydrolysis, at NBD1 et al., 1995), a similar effect may be expected for K719A, potentiates or prolongs K-ATP channel opening and that K1384M and K719A/K1384M. Login to comment
188 A second possibility, therefore, is that to channel activation in intact oocytes, because whole- MgADP hydrolysis at the NBDs might produce some cell K1384M currents, but not K719A currents, were conformational change in SUR1 which enhances K-ATP activated by exposure to azide. Login to comment
189 Since both K719A and channel opening. Login to comment

[hide] MgADP antagonism to Mg2+-independent ATP binding o... J Biol Chem. 1997 Sep 12;272(37):22983-6. Ueda K, Inagaki N, Seino S
MgADP antagonism to Mg2+-independent ATP binding of the sulfonylurea receptor SUR1.
J Biol Chem. 1997 Sep 12;272(37):22983-6., [PMID:9287292]

Abstract [show]
Pancreatic beta-cell ATP-sensitive potassium (KATP) channels play an important role in the regulation of glucose-induced insulin secretion. The beta-cell KATP channel comprises two subunits, the sulfonylurea receptor SUR1, a member of the ATP-binding cassette (ABC) superfamily, and Kir6.2, a member of the inward rectifier K+ channel family. The activity of the KATP channel is under complex regulation by the intracellular ATP and ADP. To understand the roles of the two nucleotide-binding folds (NBFs) of SUR1 in the regulation of KATP channel activity, we introduced point mutations into the core consensus sequence of the Walker A or B motif of each NBF of SUR1 and characterized ATP binding and ADP or MgADP antagonism to it. SUR1 was efficiently photolabeled with 8-azido-[alpha-32P]ATP and 8-azido-[gamma-32P]ATP in the presence or absence of Mg2+ or vanadate. NBF1 mutations impaired ATP binding, but NBF2 mutations did not. MgADP strongly antagonized ATP binding, and the NBF2 mutation reduced MgADP antagonism. These results show that SUR1, unlike other ABC proteins, strongly binds ATP at NBF1 even in the absence of Mg2+ and that MgADP, through binding at NBF2, antagonizes the Mg2+-independent high affinity ATP binding at NBF1.

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95 Gribble et al. (28) reported recently that both NBFs in SUR1 are not essential for KATP channel inhibition by ATP, but that they are essential for channel activation by MgADP, using K719A and K1385M SUR1 mutants. Login to comment