ABCMdb

ABCC8 p.Lys719Met

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] Mutations in either nucleotide-binding site of P-g... Biochemistry. 1998 Mar 31;37(13):4592-602. Urbatsch IL, Beaudet L, Carrier I, Gros P
Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.
Biochemistry. 1998 Mar 31;37(13):4592-602., 1998-03-31 [PMID:9521779]

Abstract [show]
Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

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265 Point mutations in the Walker A and B motifs of NB1, K719R, K719M, and D854N impaired 8-azido[R-32 P]ATP binding, whereas NB2 mutations, K1385R, K1385M, and D1506N, retained their ability to bind low concentrations of 8-azido[R-32P]ATP in the presence or absence of Mg2+ (65). 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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567 The high-affinity binding site was saturated with 10 ␮M ATPi in the absence of Mg2ϩ i. Substitution of the conserved lysine residue in the Walker A motif (K719R and K719M) or the aspartate residue in the Walker B motif (D854N) in the first NBF all abolished the high-affinity ATPi-binding, while the corresponding mutations in the second NBF did not cause any significant effect. 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
571 The high-affinity binding site was saturated with 10 mM ATPi in the absence of Mg21 i. Substitution of the conserved lysine residue in the Walker A motif (K719R and K719M) or the aspartate residue in the Walker B motif (D854N) in the first NBF all abolished the high-affinity ATPi-binding, while the corresponding mutations in the second NBF did not cause any significant effect. 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

[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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74 Mutations of either theWalkerA orWalker B motifs of NBF1, K719M and D854N abolished the high-affinity 8-azido-ATP labeling of SUR1, whereas the equivalent mutations in NBF2 did not affect it [47]. Login to comment
83 We found that the K719M mutation of SUR1 affects 8-azido-ATP binding not only to NBF1 but also to NBF2 (Matsuo M, et al., unpublished data). Login to comment

[hide] Sulfonylurea receptor 1 mutations that cause oppos... J Biol Chem. 2009 Mar 20;284(12):7951-9. Epub 2009 Jan 16. Pratt EB, Yan FF, Gay JW, Stanley CA, Shyng SL
Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.
J Biol Chem. 2009 Mar 20;284(12):7951-9. Epub 2009 Jan 16., [PMID:19151370]

Abstract [show]
The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.

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124 We therefore tested the effect of R74W or E128K on channel ATP sensitivity in the background of SUR1-NBD mutations such as G1479D and G1479R in NBD2 and K719M in NBD1, which are known to abolish channel response to MgADP stimulation (28). Login to comment
126 We also combined E128K with NBD1 mutation K719M (E128K/K719M), and the resulting channels were as insensitive to ATP as E128K (not shown). Login to comment

[hide] Zinc is both an intracellular and extracellular re... J Physiol. 2004 Aug 15;559(Pt 1):157-67. Epub 2004 Jun 24. Prost AL, Bloc A, Hussy N, Derand R, Vivaudou M
Zinc is both an intracellular and extracellular regulator of KATP channel function.
J Physiol. 2004 Aug 15;559(Pt 1):157-67. Epub 2004 Jun 24., [PMID:15218066]

Abstract [show]
Extracellular Zn(2+) has been identified as an activator of pancreatic K(ATP) channels. We further examined the action of Zn(2+) on recombinant K(ATP) channels formed with the inward rectifier K(+) channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn(2+), applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of K(ATP) channels. External Zn(2+), at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn(2+) dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 microm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn(2+) on the SUR subunit. Effects of internal Zn(2+) were not due to Zn(2+) leaking out, since they were unaffected by the presence of a Zn(2+) chelator on the external side. Similarly, internal chelators did not affect activation by external Zn(2+). Therefore, Zn(2+) is an endogenous K(ATP) channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting K(ATP) channels, and suggest a new role for Zn(2+) as an intracellular signalling molecule.

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162 KATP channels with impaired nucleotide binding domains are still activated by intracellular Zn2+ Channel activity was measured at -30 mV in inside-out patches excised from Xenopus oocytes coinjected with Kir6.2 and SUR1(K719M,K1385M) or SUR2A(D832N,D1469N). Login to comment
163 A, activation by 20 µM Zn2+ of SUR1(K719M,K1385M)/Kir6.2 channels. B, average currents in 100 µM ATP measured before or during application of 20 µM Zn2+. 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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230 Both mutations of the lysine in the Walker A motif (K719R, K719M) and a mutation of the aspartic acid in the Walker B motif (D854N) of SUR1 impair Mg2+ -independent high-affinity ATP binding (124). Login to comment

[hide] ATP binding properties of the nucleotide-binding f... J Biol Chem. 1999 Dec 24;274(52):37479-82. Matsuo M, Kioka N, Amachi T, Ueda K
ATP binding properties of the nucleotide-binding folds of SUR1.
J Biol Chem. 1999 Dec 24;274(52):37479-82., [PMID:10601323]

Abstract [show]
Pancreatic beta cell ATP-sensitive potassium (K(ATP)) channels regulate glucose-induced insulin secretion. The activity of the K(ATP) channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[(32)P]ATP. The 35-kDa fragment was strongly labeled with 5 microM 8-azido-[(32)P]ATP even in the absence of Mg(2+) and was immunoprecipitated with the antibody against NBF1. The 65-kDa fragment labeled with 100 microM 8-azido-[alpha-(32)P]ATP in the presence of Mg(2+) was immunoprecipitated with anti-NBF2 and anti-C terminus antibodies. These results indicate that NBF1 of SUR1 binds 8-azido-ATP strongly in a magnesium-independent manner and that NBF2 binds 8-azido-ATP weakly in a magnesium-dependent manner. Furthermore, the 65-kDa tryptic fragment was not photoaffinity-labeled with 8-azido-[gamma-(32)P]ATP at 37 degrees C, whereas the 35-kDa tryptic fragment was, suggesting that NBF2 of SUR1 may have ATPase activity and that NBF1 has none or little.

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12 We have reported previously that mutations of either the Walker A or B motifs of NBF1, K719M, and D854N abolish the high-affinity 8-azido-ATP binding of SUR1, whereas the equivalent mutations in NBF2 do not affect ATP binding (15). Login to comment

[hide] Syntaxin-1A interacts with distinct domains within... J Biol Chem. 2011 Jul 1;286(26):23308-18. Epub 2011 May 3. Chang N, Liang T, Lin X, Kang Y, Xie H, Feng ZP, Gaisano HY
Syntaxin-1A interacts with distinct domains within nucleotide-binding folds of sulfonylurea receptor 1 to inhibit beta-cell ATP-sensitive potassium channels.
J Biol Chem. 2011 Jul 1;286(26):23308-18. Epub 2011 May 3., [PMID:21540180]

Abstract [show]
The ATP-sensitive potassium (K(ATP)) channel regulates pancreatic beta-cell function by linking metabolic status to electrical activity. Syntaxin-1A (Syn-1A), a SNARE protein mediating exocytotic fusion, binds and inhibits the K(ATP) channel via the nucleotide-binding folds (NBFs) of its sulfonylurea receptor-1 (SUR1) regulatory subunit. In this study, we elucidated the precise regions within the NBFs required for Syn-1A-mediated K(ATP) inhibition, using in vitro binding assays, whole cell patch clamp and FRET assay. Specifically, NBF1 and NBF2 were each divided into three subregions, Walker A (W(A)), signature sequence linker, and Walker B (W(B)), to make GST fusion proteins. In vitro binding assays revealed that Syn-1A associates with W(A) and W(B) regions of both NBFs. Patch clamp recordings on INS-1 and primary rat beta-cells showed that Syn-1A-mediated channel inhibition was reversed by co-addition of NBF1-W(B) (not NBF1-W(A)), NBF2-W(A), and NBF2-W(B). The findings were corroborated by FRET studies showing that these truncates disrupted Syn-1A interactions with full-length SUR1. To further identify the binding sites, series single-site mutations were made in the Walker motifs of the NBFs. Only NBF1-W(A) (K719M) or NBF2-W(A) (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of K(ATP) channels. These data suggest that NBF1-W(A) (Lys-719) and NBF2-W(A) (Lys-1385) are critical for Syn-1A-K(ATP) channel interaction. Taken together, Syn-1A intimately and functionally associates with the SUR1-NBF1/2 dimer via direct interactions with W(A) motifs and sites adjacent to W(B) motifs of NBF1 and NBF2 but transduces its inhibitory actions on K(ATP) channel activity via some but not all of these NBF domains.

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8 Only NBF1-WA (K719M) or NBF2-WA (K1385M) mutant no longer bound to Syn-1A; K1385M failed to disrupt Syn-1A-mediated inhibition of KATP channels. Login to comment
53 Mutants on WA and WB motifs in these truncated NBF1 (K719M and D854N, respectively) and NBF2 (K1385M and D1506N, respectively) regions were also generated (see Fig. 5A) using QuikChange௡ site-directed mutagenesis according to the manufacturer`s instructions (Stratagene, La Jolla, CA). Login to comment
191 The mutations were made by substitutions of lysine in WA and aspartic acid in WB with methionine and asparagine, respectively, and the consequent constructs are GST-NBF1-WA (K719M) (24), GST-NBF1-WB (D854N) (24, 25), GST-NBF2-WA (K1385M) (26), and GST-NBF2-WB (D1506N) (27). Login to comment
193 The WA motif mutations in NBF1-WA (K719M) (Fig. 5B, panel (i)) and NBF2-WA (K1385M) (Fig. 5B, panel (ii)) did not bind Syn-1A in contrast to its respective wild type proteins (Fig. 1B). Login to comment
194 Because NBF1-WA (wt) domain did not disrupt Syn-1A/ SUR1 interactions on our FRET or patch clamp studies (Figs. 2-4 and supplemental Fig. S1), we did not further examine NBF1-WA (K719M). Login to comment
246 Conserved lysine residues in WA motifs were substituted with methionine, generating NBF1-WA (K719M) and NBF2-WA (K1385M) truncated proteins. Login to comment
249 Panel (i), NBF-1: WA motif mutant NBF1-WA (K719M) could not bind Syn-1A, whereas WB motif mutant NBF1-WB (D854N) remained able to bind Syn-1A. Full-length NBF1, WT NBF1-WA, and WT NBF1-WB are positive controls. Login to comment
189 The mutations were made by substitutions of lysine in WA and aspartic acid in WB with methionine and asparagine, respectively, and the consequent constructs are GST-NBF1-WA (K719M) (24), GST-NBF1-WB (D854N) (24, 25), GST-NBF2-WA (K1385M) (26), and GST-NBF2-WB (D1506N) (27). Login to comment
192 Because NBF1-WA (wt) domain did not disrupt Syn-1A/ SUR1 interactions on our FRET or patch clamp studies (Figs. 2-4 and supplemental Fig. S1), we did not further examine NBF1-WA (K719M). Login to comment
252 Panel (i), NBF-1: WA motif mutant NBF1-WA (K719M) could not bind Syn-1A, whereas WB motif mutant NBF1-WB (D854N) remained able to bind Syn-1A. Full-length NBF1, WT NBF1-WA, and WT NBF1-WB are positive controls. Login to comment

[hide] NEM modification prevents high-affinity ATP bindin... FEBS Lett. 1999 Sep 24;458(3):292-4. Matsuo M, Tucker SJ, Ashcroft FM, Amachi T, Ueda K
NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1.
FEBS Lett. 1999 Sep 24;458(3):292-4., [PMID:10570926]

Abstract [show]
Pancreatic beta-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 microM 8-azido-[alpha-32P]ATP or 8-azido-[gamma-32P]ATP was inhibited by NEM with Ki of 1.8 microM and 2.4 microM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 microM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1.

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25 Hamster SUR1 (K719M) cDNA was generously provided by Dr Susumu Seino (Chiba University, Japan). Login to comment
63 However, the K719M mutant form of SUR1 (in which the lysine residue within the Walker A motif of NBF1 was replaced with methionine) was not photoa¤nity labeled either in the absence or in the presence of NEM. These results indicate that cysteine-717 within NBF1 of SUR1 is responsible for inhibition of high-a¤nity 8-azido-ATP binding by NEM, and suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1. Login to comment
64 We have reported previously that mutations in either the Walker A or B motifs of NBF1, K719M and D854N, abolish Fig. 1. Login to comment
85 Acknowledgements: We thank Dr S. Seino (Chiba University, Japan) for providing hamster SUR1 (K719M) cDNA and the antibody against SUR1. Login to comment

[hide] A mutation in the TMD0-L0 region of sulfonylurea r... Diabetes. 2007 May;56(5):1357-62. Epub 2007 Feb 22. Masia R, De Leon DD, MacMullen C, McKnight H, Stanley CA, Nichols CG
A mutation in the TMD0-L0 region of sulfonylurea receptor-1 (L225P) causes permanent neonatal diabetes mellitus (PNDM).
Diabetes. 2007 May;56(5):1357-62. Epub 2007 Feb 22., [PMID:17317760]

Abstract [show]
OBJECTIVE: We sought to examine the molecular mechanisms underlying permanenent neonatal diabetes mellitus (PNDM) in a patient with a heterozygous de novo L225P mutation in the L0 region of the sulfonylurea receptor (SUR)1, the regulatory subunit of the pancreatic ATP-sensitive K(+) channel (K(ATP) channel). RESEARCH DESIGN AND METHODS: The effects of L225P on the properties of recombinant K(ATP) channels in transfected COS cells were assessed by patch-clamp experiments on excised membrane patches and by macroscopic Rb-flux experiments in intact cells. RESULTS: L225P-containing K(ATP) channels were significantly more active in the intact cell than in wild-type channels. In excised membrane patches, L225P increased channel sensitivity to stimulatory Mg nucleotides without altering intrinsic gating or channel inhibition by ATP in the absence of Mg(2+). The effects of L225P were abolished by SUR1 mutations that prevent nucleotide hydrolysis at the nucleotide binding folds. L225P did not alter channel inhibition by sulfonylurea drugs, and, consistent with this, the patient responded to treatment with oral sulfonylureas. CONCLUSIONS: L225P underlies K(ATP) channel overactivity and PNDM by specifically increasing Mg-nucleotide stimulation of the channel, consistent with recent reports of mechanistically similar PNDM-causing mutations in SUR1. The mutation does not affect sulfonylurea sensitivity, and the patient is successfully treated with sulfonylureas.

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117 Consistent with this, the effects of L225P on channel activity in the intact cell were abolished by combination with either of two NBF mutations (K719M or D1506A) that abolish Mg-nucleotide stimulation of the channel (19,20) (Fig. 2D). Login to comment
118 Consistent with this, the effects of L225P on channel activity in the intact cell were abolished by combination with either of two NBF mutations (K719M or D1506A) that abolish Mg-nucleotide stimulation of the channel (19,20) (Fig. 2D). Login to comment

[hide] The structure and function of the ATP-sensitive K+... J Mol Endocrinol. 1999 Apr;22(2):113-23. Miki T, Nagashima K, Seino S
The structure and function of the ATP-sensitive K+ channel in insulin-secreting pancreatic beta-cells.
J Mol Endocrinol. 1999 Apr;22(2):113-23., [PMID:10194514]

Abstract [show]
ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion.

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97 Mutations of Walker A (K719R and K719M) in NBF-1 and Walker B (D854N) in NBF-1 of SUR1 severely impair Mg2+ -independent high-affinity ATP binding. 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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61 Substitutions of the conserved lysine in Walker A, K719R and K719M (lanes 2 and 3), or the aspartate in Walker B, D854N (lane 4), abolished the binding of 5 ␮M 8-azido-[␣-32 P]ATP, although substitutions at equivalent sites in NBF2, K1385R, K1385M, or D1506N (lanes 5, 6, and 7) did not affect it. SUR1 with mutations in NBF1 binds ATP only slightly even when incubated with 40 ␮M 8-azido-[␣-32 P]ATP. Login to comment
96 However, we have observed that while K719M and K719R mutants severely impair functional expression of KATP channels, K1385M and K1385R mutants do not.2 Although whether or not SUR1 has ATP hydrolysis activity is unknown, ATP binding to NBF1 of SUR1 might be important in maintaining KATP channels in the operative state. Login to comment
103 Lane 1, wild-type SUR1; lane 2, K719R; lane 3, K719M; lane 4, D854N; lane 5, K1385R; lane 6, K1385M; lane 7, D1506N. Login to comment