ABCMdb

PMID: 22451668

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., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. Login to comment 13 ABCC8 p.Gln1178Arg The KD for ATP4d1a; is b03;1 versus 12 mM, Q1178R versus wild type, respectively. Login to comment 14 ABCC8 p.Gln1178Arg The linkage constant is b03;10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Login to comment 17 ABCC8 p.Gln1178Arg 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. Login to comment 47 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln Here we focus on two mutations, Q1178R and R1182Q, located in the 15th helix (transmembrane helix) of the second transmembrane domain (TMD2). Login to comment 48 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln ABCC8 p.Cys1174Phe ABCC8 p.Ser1185Ala ABCC8 p.Ala1184Glu These substitutions are in a cluster of mutations that cause either neonatal diabetes (Q1178R, R1182Q, and A1184E) or hyperinsulinism (C1174F and S1185A). Login to comment 58 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 101 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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). Login to comment 105 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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). Login to comment 107 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 109 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 115 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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). Login to comment 117 ABCC8 p.Gln1178Arg 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. Login to comment 126 ABCC8 p.Arg1182Gln The parameters for WT and R1182Q receptors are given in Table 2. Login to comment 139 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln ABCC8 p.Cys1174Phe 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). Login to comment 143 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 146 ABCC8 p.Gln1178Arg 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; . Login to comment 148 ABCC8 p.Gln1178Arg 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. Login to comment 156 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 163 ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 166 ABCC8 p.Gln1178Arg ABCC8 p.Gln1178Arg ABCC8 p.Arg1182Gln 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. Login to comment 192 ABCC8 p.Gln1178Arg Q1178R Receptor Has Higher Affinity for MgADP and ADP3afa; -MgADP stimulates KATP channel activity (49-53). Login to comment 199 ABCC8 p.Gln1178Arg Q1178R retains a high affinity for ATP at NBD1. Login to comment 203 ABCC8 p.Arg1182Gln ABCC8 p.Arg1182Gln A, the curvesarebestfitstoalogisticequation.TheIC50 forQ1178Ris39afe;8òe;M.The IC50 for the averaged values of WT and R1182Q is 9.8 afe; 0.3 òe;M; the individual values are 10.1 afe; 0.5 òe;M (WT) and 9.2 afe; 0.8 òe;M (R1182Q). Login to comment 205 ABCC8 p.Gln1178Arg B, example autoradiograms comparing the labeling of WT and Q1178R receptors. Login to comment 228 ABCC8 p.Gln1178Arg The WT and Q1178R data and curves are taken from Fig. 2B. Login to comment 232 ABCC8 p.Gln1178Arg The IC50 values are 146 afe; 47 and 24 afe; 3 òe;M for WT and Q1178R, respectively. Login to comment 233 ABCC8 p.Gln1178Arg 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. Login to comment 241 ABCC8 p.Gln1178Arg 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. Login to comment 246 ABCC8 p.Arg1182Gln The same argument holds for the R1182Q substitution (see Table 2). Login to comment