ABCC8 p.Gln1178Arg
| Predicted by SNAP2: | A: D (53%), C: D (66%), D: D (75%), E: D (71%), F: D (53%), G: D (63%), H: D (63%), I: N (53%), K: D (75%), L: N (57%), M: N (66%), N: D (63%), P: D (75%), R: D (75%), S: D (59%), T: D (59%), V: D (53%), W: D (80%), Y: D (63%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] A novel ABCC8 (SUR1)-dependent mechanism of metabo... J Biol Chem. 2008 Apr 4;283(14):8778-82. Epub 2008 Feb 15. Babenko AP
A novel ABCC8 (SUR1)-dependent mechanism of metabolism-excitation uncoupling.
J Biol Chem. 2008 Apr 4;283(14):8778-82. Epub 2008 Feb 15., [PMID:18281290]
Abstract [show]
ATP/ADP-sensing (sulfonylurea receptor (SUR)/K(IR)6)(4) K(ATP) channels regulate the excitability of our insulin secreting and other vital cells via the differential MgATP/ADP-dependent stimulatory actions of their tissue-specific ATP-binding cassette regulatory subunits (sulfonylurea receptors), which counterbalance the nearly constant inhibitory action of ATP on the K(+) inwardly rectifying pore. Mutations in SUR1 that abolish its stimulation have been found in infants persistently releasing insulin. Activating mutations in SUR1 have been shown to cause neonatal diabetes. Here, analyses of K(IR)6.2-based channels with diabetogenic receptors reveal that MgATP-dependent hyper-stimulation of mutant SUR can compromise the ability of K(ATP) channels to function as metabolic sensors. I demonstrate that the channel hyperactivity rises exponentially with the number of hyperstimulating subunits, so small subpopulations of channels with more than two mutant SUR can dominate hyperpolarizing currents in heterozygous patients. I uncovered an attenuated tolbutamide inhibition of the hyperstimulated mutant, which is normally sensitive to the drug under non-stimulatory conditions. These findings show the key role of SUR in sensing the metabolic index in humans and urge others to (re)test mutant SUR/K(IR)6 channels from probands in physiologic MgATP.
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No. Sentence Comment
67 The more significant (p Ͻ 0.005) effect of the mutation at these higher MgATP/ADP ratios indicates that the Q1178R markedly amplifies the stimulatory action of [MgATP] Ͼ 0.5 mM (see later).
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ABCC8 p.Gln1178Arg 18281290:67:114
status: NEW84 Consistent with the latter report and my model, I hypothesize that the Q1178R mutation reduces the ability of the sulfonylurea-binding TM domain to destabilize the magnesium⅐nucleotide-bound state of the receptor.
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ABCC8 p.Gln1178Arg 18281290:84:71
status: NEW[hide] Two neonatal diabetes mutations on transmembrane h... J Biol Chem. 2012 May 25;287(22):17985-95. doi: 10.1074/jbc.M112.349019. Epub 2012 Mar 27. Ortiz D, Voyvodic P, Gossack L, Quast U, Bryan J
Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2.
J Biol Chem. 2012 May 25;287(22):17985-95. doi: 10.1074/jbc.M112.349019. Epub 2012 Mar 27., [PMID:22451668]
Abstract [show]
K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic beta-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, beta-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.
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No. Sentence Comment
9 Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants.
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ABCC8 p.Gln1178Arg 22451668:9:121
status: NEW13 The KD for ATP4d1a; is b03;1 versus 12 mM, Q1178R versus wild type, respectively.
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ABCC8 p.Gln1178Arg 22451668:13:49
status: NEW14 The linkage constant is b03;10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R.
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ABCC8 p.Gln1178Arg 22451668:14:122
status: NEW17 An eight-state model describes linkage between diazoxide and ATP4d1a; binding; diazoxide markedly increases the affinity of Q1178R for ATP4d1a; and ATP4d1a; augments diazoxide binding.
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ABCC8 p.Gln1178Arg 22451668:17:127
status: NEW47 Here we focus on two mutations, Q1178R and R1182Q, located in the 15th helix (transmembrane helix) of the second transmembrane domain (TMD2).
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ABCC8 p.Gln1178Arg 22451668:47:32
status: NEW48 These substitutions are in a cluster of mutations that cause either neonatal diabetes (Q1178R, R1182Q, and A1184E) or hyperinsulinism (C1174F and S1185A).
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ABCC8 p.Gln1178Arg 22451668:48:87
status: NEW58 The results imply that outward facing conformations with dimerized NBDs bind GBC and diazoxide with low and high affinity, respectively, and that the enhanced stimulatory action of Q1178R and R1182Q is due to their increased affinity for ATP and ADP.
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ABCC8 p.Gln1178Arg 22451668:58:181
status: NEW101 Table 1 and Table S1 compare the dissociation constants, determined from saturation binding data, for the WT, Q1178R, and R1182Q aporeceptors (i.e. without nucleotides).
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ABCC8 p.Gln1178Arg 22451668:101:110
status: NEW105 Q1178R and R1182Q Neonatal Diabetes Mutations Potentiate Negative Allosteric Action of MgATP on GBC Binding to SUR1-To assess quantitatively the effect of MgATP on GBC binding, membranes were incubated with increasing ATP concentrations maintained constant by a regenerating system (34); the level of ATP was constant over the 30-min time period of the incubation, as determined using luciferase assays (supplemental Fig. S1).
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ABCC8 p.Gln1178Arg 22451668:105:0
status: NEW107 The IC50 values for MgATP are b03;850, 37, and 19 òe;M for the WT, R1182Q, and Q1178R receptors, respectively, an increase in the apparent affinity of SUR1Q1178R for ATP of b03;45-fold.
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ABCC8 p.Gln1178Arg 22451668:107:86
status: NEW109 Q1178R and R1182Q Receptors Have Higher Affinity for ATP4afa; than WT-All ABC proteins have Walker-type nucleotide binding sites whose physiologic substrate is MgATP.
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ABCC8 p.Gln1178Arg 22451668:109:0
status: NEW115 In terms of an enzyme-substrate model where hydrolysis is blocked, the results show that the mutant receptors have at least a 10-fold greater affinity for ATP4afa; , with Q1178R having a somewhat higher affinity than R1182Q (Tables 2 and S2).
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ABCC8 p.Gln1178Arg 22451668:115:174
status: NEW117 Simplified Allosteric Model-Previous studies using affinity labeling with 8-N3-[32 P]ATP show that NBD1 has a dissociation constant for NBD1 in the low micromolar range in the absence of Mg2af9; (18), confirmed below for WT and Q1178R receptors.
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ABCC8 p.Gln1178Arg 22451668:117:231
status: NEW139 E, neonatal diabetes (Q1178R in red and R1182Q in green) and hyperinsulinemia (C1174F in blue) causing mutations are clustered on transmembrane helix 15 (yellow), which feeds into NBD1 (light gray).
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ABCC8 p.Gln1178Arg 22451668:139:22
status: NEW143 Values are means afe; S.E. SUR1 KG nM WT 0.25 afe; 0.02 Q1178R 1.0 afe; 0.1 R1182Q 0.50 afe; .15 SUR1 Nucleotide Affinities in Neonatal Diabetes 17988 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287ߦNUMBER 22ߦMAY 25, 2012 equation for the four-state model can be expressed as a simple binding isotherm (see supplemental material), G afd; ᐵGᐶ ᐵGᐶ af9; Kobs (Eq. 4) where Kobs, the apparent dissociation constant at a specified concentration of ATP4afa; , is given by the following: Kobs afd; betaKG ᐳᐵTᐶ af9; KTᐴ ᐳᐵTᐶ af9; betaKTᐴ (Eq. 5) Kobs values at increasing concentrations of ATP4afa; were determined by homologous competition experiments (Fig. 3, A-D) and used to estimate KT and beta.
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ABCC8 p.Gln1178Arg 22451668:143:62
status: NEW146 To determine if the Q1178R mutation affects binding at NBD1, WT and SUR1Q1178R receptors were labeled with 8-N3-[ॹ-32 P]ATP plus increasing unlabeled ATP4afa; .
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ABCC8 p.Gln1178Arg 22451668:146:20
status: NEW148 The results indicate the Q1178R mutation somewhat reduces the apparent affinity of NBD1 for ATP4afa; , but NBD1 will be nearly saturated in both WT and SUR1Q1178R before there is a significant effect of ATP4afa; on GBC binding (compare Figs.
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ABCC8 p.Gln1178Arg 22451668:148:25
status: NEW156 A, the curves through the data are logistic equations with IC50 values of 849 afe; 195, 37 afe; 13, and 19 afe; 7 òe;M for WT, R1182Q, and Q1178R, respectively.
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ABCC8 p.Gln1178Arg 22451668:156:152
status: NEW163 The allosteric and ATP4afa; dissociation constants (beta/KD) (òe;M) are 14/12,200, 6.8/1250, and 9.3/1000 for the WT, R1182Q, and Q1178R receptors, respectively.
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ABCC8 p.Gln1178Arg 22451668:163:137
status: NEW166 TABLE 2 KT and beta values for WT and neonatal diabetes SUR1 are shown d1e; S.E. (p values in parentheses) SUR1 beta KT òe;M WT 14 afe; 10 (0.2) 12,200 afe; 4030 (0.02) R1182Q 6.8 afe; 0.9 (b0d;0.001) 1250 afe; 258 (0.003) Q1178R 9.3 afe; 1.8 (b0d;0.001) 1000 afe; 179 (b0d;0.001) Q1178R (Kobs method) 10.5 afe; 0.8 (0.005) 846 afe; 142 (0.03) SUR1 Nucleotide Affinities in Neonatal Diabetes MAY 25, 2012ߦVOLUME 287ߦNUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 17989 An eight-state model that describes the linkage between the diazoxide, GBC, and nucleotide binding sites under equilibrium, non-hydrolysis conditions is given in Fig. 5.
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ABCC8 p.Gln1178Arg 22451668:166:245
status: NEWX
ABCC8 p.Gln1178Arg 22451668:166:315
status: NEW192 Q1178R Receptor Has Higher Affinity for MgADP and ADP3afa; -MgADP stimulates KATP channel activity (49-53).
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ABCC8 p.Gln1178Arg 22451668:192:0
status: NEW199 Q1178R retains a high affinity for ATP at NBD1.
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ABCC8 p.Gln1178Arg 22451668:199:0
status: NEW205 B, example autoradiograms comparing the labeling of WT and Q1178R receptors.
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ABCC8 p.Gln1178Arg 22451668:205:59
status: NEW228 The WT and Q1178R data and curves are taken from Fig. 2B.
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ABCC8 p.Gln1178Arg 22451668:228:11
status: NEW232 The IC50 values are 146 afe; 47 and 24 afe; 3 òe;M for WT and Q1178R, respectively.
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ABCC8 p.Gln1178Arg 22451668:232:72
status: NEW233 Mg2af9; strongly potentiates ADP binding; ADP3afa; has a small, but significant, effect on Q1178R above 10 mM, but no detectable effect on WT at 100 mM.
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ABCC8 p.Gln1178Arg 22451668:233:97
status: NEW241 The use of a simplified equilibrium model with binding of a single ATP is justified by the finding that the Q1178R substitution has only minor effects on the binding of ATP4afa; to NBD1.
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ABCC8 p.Gln1178Arg 22451668:241:108
status: NEW[hide] Reinterpreting the action of ATP analogs on K(ATP)... J Biol Chem. 2013 Jun 28;288(26):18894-902. doi: 10.1074/jbc.M113.476887. Epub 2013 May 12. Ortiz D, Gossack L, Quast U, Bryan J
Reinterpreting the action of ATP analogs on K(ATP) channels.
J Biol Chem. 2013 Jun 28;288(26):18894-902. doi: 10.1074/jbc.M113.476887. Epub 2013 May 12., [PMID:23665564]
Abstract [show]
Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting beta-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPgammaS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPgammaS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.
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No. Sentence Comment
81 The E1507Q and Q1178R substitutions do not have large effects on the affinities of the apo-receptors for GBC, given by the dissociation constants (KG), which are determined in independent experiments (Table 1).
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ABCC8 p.Gln1178Arg 23665564:81:15
status: NEW89 Dashed curves are fits to a logistic equation used to estimate the IC50 values (in òe;M): WT: 1163 afe; 439, slope afd; 0.8 afe; 0.1; Q1178R: 17 afe; 3, slope afd; 0.8 afe; 0.1; E1507Q: 2.7 afe; 0.4, slope afd; 0.8 afe; 0.1.
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ABCC8 p.Gln1178Arg 23665564:89:147
status: NEW92 The parameters are KT afd; 1 òe;M and beta afd; 40 with a defined valueforKG afd;0.63nM.B,theeffectofATP4afa; onGBCbinding.Thecurvesarethebest fitstothefour-statemodel.TheparametersareKT afd;94,2550,and13,400òe;Mandbetaafd; 40,9.2,and15.1withKGdefinedas0.63,1.0,and0.25nM,forE1507Q,Q1178R,andWT, respectively.
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ABCC8 p.Gln1178Arg 23665564:92:308
status: NEW94 TABLE 1 Binding parameters (KG, IC50, and KT values) for WT, Q1178R, and E1507Q KG values are nM; all IC50 and KT values are òe;M.
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ABCC8 p.Gln1178Arg 23665564:94:61
status: NEW96 KG IC50 KT MgATP MgADP MgATP (beta) ATP4d1a; (beta) MgATPॹS (beta) ATPॹS4d1a; (beta) WT 0.25 afe; 0.02 1163 afe; 439 161 afe; 28 - 13,400 afe; 3360 (15.1 afe; 9.4) - - Q1178R 1.0 afe; 0.1 17 afe; 3 25 afe; 2 - 2550 afe; 290 (9.2 afe; 1.4) - - E1507Q 0.63 afe; 0.16 2.7 afe; 0.4 - 1 afe; 0.2 (40 afe; 3) 94 afe; 8 (40 afe; 1) 29 afe; 7 (11.6 afe; 3) 234 afe; 31 (3.2 afe; 0.1) Action of ATP Analogs on SUR1 18896 four-state model.
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ABCC8 p.Gln1178Arg 23665564:96:201
status: NEW135 The solid lines are fits to a logistic equation: IC50 values: WT: 161 afe; 28, slope afd; 1.7 afe; 0.4; Q1178R: 25 afe; 2, slope afd; 1.5 afe; 0.1.
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ABCC8 p.Gln1178Arg 23665564:135:113
status: NEW193 Substi- TABLE 2 Binding parameters (S0.5 and K1, K2, and KA values) for Q1178R and E1507Q Values are òe;M; K1 and K2, the affinities for MgATP at NBD1 and NBD2, respectively and KA, the affinity for MgAMP-PNP at NBD1, were estimated using an eight-state model.
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ABCC8 p.Gln1178Arg 23665564:193:72
status: NEW195 SUR1 S0.5 K1 K2 KA MgAMP-PNP MgAMP-PCP AMP-PNP4d1a; Q1178R 530 afe; 124 - - - - - E1507Q 573 afe; 103 3360 afe; 410 31100 afe; 2240 0.7 afe; 0.2 1.5 afe; 0.1 1.2 afe; 0.2 FIGURE 5.
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ABCC8 p.Gln1178Arg 23665564:195:55
status: NEW[hide] Neonatal Diabetes and Congenital Hyperinsulinism C... Front Endocrinol (Lausanne). 2015 Apr 15;6:48. doi: 10.3389/fendo.2015.00048. eCollection 2015. Ortiz D, Bryan J
Neonatal Diabetes and Congenital Hyperinsulinism Caused by Mutations in ABCC8/SUR1 are Associated with Altered and Opposite Affinities for ATP and ADP.
Front Endocrinol (Lausanne). 2015 Apr 15;6:48. doi: 10.3389/fendo.2015.00048. eCollection 2015., [PMID:25926814]
Abstract [show]
ATP-sensitive K(+) (KATP) channels composed of potassium inward-rectifier type 6.2 and sulfonylurea receptor type 1 subunits (Kir6.2/SUR1)4 are expressed in various cells in the brain and endocrine pancreas where they couple metabolic status to membrane potential. In beta-cells, increases in cytosolic [ATP/ADP]c inhibit KATP channel activity, leading to membrane depolarization and exocytosis of insulin granules. Mutations in ABCC8 (SUR1) or KCNJ11 (Kir6.2) can result in gain or loss of channel activity and cause neonatal diabetes (ND) or congenital hyperinsulinism (CHI), respectively. SUR1 is reported to be a Mg(2+)-dependent ATPase. A prevailing model posits that ATP hydrolysis at SUR1 is required to stimulate openings of the pore. However, recent work shows nucleotide binding, without hydrolysis, is sufficient to switch SUR1 to stimulatory conformations. The actions of nucleotides, ATP and ADP, on ND (SUR1E1506D) and CHI (SUR1E1506K) mutants, without Kir6.2, were compared to assess both models. Both substitutions significantly impair hydrolysis in SUR1 homologs. SUR1E1506D has greater affinity for MgATP than wildtype; SUR1E1506K has reduced affinity. Without Mg(2+), SUR1E1506K has a greater affinity for ATP(4-) consistent with electrostatic attraction between ATP(4-), unshielded by Mg(2+), and the basic lysine. Further analysis of ND and CHI ABCC8 mutants in the second transmembrane and nucleotide-binding domains (TMD2 and NBD2) found a relation between their affinities for ATP (+/-Mg(2+)) and their clinical phenotype. Increased affinity for ATP is associated with ND; decreased affinity with CHI. In contrast, MgADP showed a weaker relationship. Diazoxide, known to reduce insulin release in some CHI cases, potentiates switching of CHI mutants from non-stimulatory to stimulatory states consistent with diazoxide stabilizing a nucleotide-bound conformation. The results emphasize the greater importance of nucleotide binding vs. hydrolysis in the regulation of KATP channels in vivo.
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No. Sentence Comment
122 to the current regulatory model, both E1506 substitutions have reduced affinity for MgADP (Figure 4), consistent with electrophysiological data demonstrating that SUR1E1506D/Kir6.2 and 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0.0 0.2 0.4 0.6 0.8 1.0 E1506Q Q1178R E1506D R1182Q I1424V WT S1185A C1174F E1506K G1479R Specific Bound GBC [MgATP] (&#b5;M) 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0.0 0.2 0.4 0.6 0.8 1.0 E1506Q E1506K Q1178R I1424V E1506D R1182Q WT S1185A C1174F G1479R Specific Bound GBC [ATP 4- ] (&#b5;M) B A FIGURE 3 | Comparison of nucleotide-induced conformational switching in WT and SUR1 mutants.
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ABCC8 p.Gln1178Arg 25926814:122:252
status: NEWX
ABCC8 p.Gln1178Arg 25926814:122:421
status: NEW128 To support this hypothesis we analyzed additional mutations including I1424V (ND) and G1479R (CHI) in NBD2 and a cluster of disease causing mutations in TMD2: C1174F (CHI), S1185A (CHI), Q1178R (ND), and R1182Q (ND).
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ABCC8 p.Gln1178Arg 25926814:128:187
status: NEW151 Figure 5 shows that diazoxide potentiates the 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 Q1178R I1424V R1182Q S1185A C1174F WT E1506Q E1506D G1479R E1506K Specific Bound GBC [MgADP] (&#b5;M) FIGURE 4 | MgADP-induced conformational switching in WT and SUR1 mutants.
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ABCC8 p.Gln1178Arg 25926814:151:84
status: NEW