ABCMdb

ABCC8 p.Lys1384Met

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 (91%), L: D (95%), M: D (91%), N: D (95%), P: D (95%), Q: D (91%), R: D (80%), S: D (91%), T: D (91%), V: D (91%), 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] 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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No. Sentence Comment
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
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
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] 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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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] 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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No. Sentence Comment
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
115 On the other hand, substitution of a methionine in NBF2 of SUR1 (K1384M) does not reduce KCO binding at saturating ATP, but decreases the affinity for ATP (M.Schwanstecher et al., unpublished data). Login to comment

[hide] Regulation of cloned ATP-sensitive K channels by p... J Gen Physiol. 2000 Sep;116(3):391-410. Ribalet B, John SA, Weiss JN
Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation.
J Gen Physiol. 2000 Sep;116(3):391-410., [PMID:10962016]

Abstract [show]
Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.

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No. Sentence Comment
361 The hypothesis that phosphorylation of SUR1 favors binding of MgADP and thus functional coupling is supported by the observation that the K1384M mutation in SUR1`s second NBF completely suppress MgADP-dependent reactivation of ATP-inhibited channels (Gribble et al., 1997). 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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No. Sentence Comment
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
138 Diazoxide was without effect on K1384M currents in the presence of guanine nucleotides. 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
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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No. Sentence Comment
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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No. Sentence Comment
154 Specifically, we mutated lysine 719 to alanine [SUR1(K719A)] and lysine 1384 to methionine [SUR1(K1384M)]. Login to comment
161 In this respect, NNC 55-9216 differs from diazoxide, which is still able to induce some activation of Kir6.2/SUR1(K1384M) currents (33). Login to comment
155 Specifically, we mutated lysine 719 to alanine [SUR1(K719A)] and lysine 1384 to methionine [SUR1(K1384M)]. Login to comment
162 In this respect, NNC 55-9216 differs from diazoxide, which is still able to induce some activation of Kir6.2/SUR1(K1384M) currents (33). 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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No. Sentence Comment
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
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
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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No. Sentence Comment
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
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
112 The extent of this inhibition was comparable with that observed for wild-type channels when ADP was and K1384M was unaffected (Figure 6B). Login to comment
115 Diazoxide acted more slowly on K1384M than the wild-type channelwere also similar for wild-type and mutant channels. Login to comment
118 The reduced ATP sensitivity of the wild-type channel (Figure 2B) may thus The effects of diazoxide in the presence of the nucleotide diphosphate Mg-ADP are summarized in Figure 6C. Inresult from a simultaneous activation by Mg-ADP (formed from hydrolysis of Mg-ATP) which does not occur in WA the presence of 100 µM Mg-ADP, diazoxide slightly potentiated wild-type and K1384M currents and inhibitedmutant channels. 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
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
190 If this idea is correct, then hydrolysis at K1384M channels have similar ATP sensitivities and both NBDs must be required to sustain channel activation neither are upregulated by 100 µM Mg-ADP, this result because mutation of only one NBD removed the ability also argues that, in addition to ATP and ADP, there may of Mg-ADP to activate the current. Login to comment