ABCMdb

ABCC8 p.Arg1420Cys

Predicted by SNAP2:	A: D (75%), C: D (66%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (66%), I: D (85%), K: N (78%), L: D (85%), M: D (75%), N: D (71%), P: D (91%), Q: D (66%), S: D (71%), T: D (80%), V: D (80%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Defective trafficking and function of KATP channel... Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2882-7. Cartier EA, Conti LR, Vandenberg CA, Shyng SL
Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2882-7., [PMID:11226335]

Abstract [show]
The ATP-sensitive potassium channel (K(ATP)) regulates insulin secretion in pancreatic beta cells. Loss of functional K(ATP) channels because of mutations in either the SUR1 or Kir6.2 channel subunit causes persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We investigated the molecular mechanism by which a single phenylalanine deletion in SUR1 (DeltaF1388) causes PHHI. Previous studies have shown that coexpression of DeltaF1388 SUR1 with Kir6.2 results in no channel activity. We demonstrate here that the lack of functional expression is due to failure of the mutant channel to traffic to the cell surface. Trafficking of K(ATP) channels requires that the endoplasmic reticulum-retention signal, RKR, present in both SUR1 and Kir6.2, be shielded during channel assembly. To ask whether DeltaF1388 SUR1 forms functional channels with Kir6.2, we inactivated the RKR signal in DeltaF1388 SUR1 by mutation to AAA (DeltaF1388 SUR1(AAA)). Inactivation of similar endoplasmic reticulum-retention signals in the cystic fibrosis transmembrane conductance regulator has been shown to partially overcome the trafficking defect of a cystic fibrosis transmembrane conductance regulator mutation, DeltaF508. We found that coexpression of DeltaF1388 SUR1(AAA) with Kir6.2 led to partial surface expression of the mutant channel. Moreover, mutant channels were active. Compared with wild-type channels, the mutant channels have reduced ATP sensitivity and do not respond to stimulation by MgADP or diazoxide. The RKR --> AAA mutation alone has no effect on channel properties. Our results establish defective trafficking of K(ATP) channels as a molecular basis of PHHI and show that F1388 in SUR1 is critical for normal trafficking and function of K(ATP) channels.

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196 This mechanism has been shown to be responsible for the reduced channel sensitivity to MgADP in another PHHI-associated SUR1 mutation R1420C, also located in NBF2 (17). 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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151 A missense SUR1 mutation (R1420C) was identified in Japanese PHHI patients [86]. Login to comment
154 R1420 is located between the Walker A motif and the ABC signature motif of NBF2.An 86 Rb+ efflux study revealed that KATP channels composed of SUR1-R1420C and Kir6.2 are not activated by metabolic inhibition as much as wild-type channels. Login to comment
156 This may be related to the fact that the expression level of SUR1-R1420C was only about half that of the wild-type channel when it was transiently expressed in COS-7 cells [86]. Login to comment
157 Photoaffinity labeling experiments revealed that the R1420C PHHI mutation does not affect the affinity of NBF1 for nucleotides, but lowers the affinity of NBF2 for ATP and ADP [88]. Login to comment
158 The R1420C mutation impairs the cooperative nucleotide binding of two NBFs, although it does not show direct effects on the high-affinity 8-azido-ATP binding to NBF1. Login to comment
161 Structural modeling of NBFs of SUR1 showed that R1420 is proximal to the a-helical subdomain in NBF2, suggesting that the R1420C mutation affects the interaction of NBFs with nucleotides indirectly [89]. Login to comment

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

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

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1 We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1. Login to comment
3 Labeling of NBF1 with 8-azido-[␣-32 P]ATP was inhibited by MgATP and MgADP with similar Ki for wild-type SUR1 and SUR1-R1420C. Login to comment
4 However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1. Login to comment
5 MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Login to comment
6 Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide. Login to comment
7 However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC50 for MgADP activation from 74 to 197 ␮M. Login to comment
8 We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller KATP ؉ current in R1420C-PHHI beta-cells and thereby to the enhanced insulin secretion. Login to comment
27 Recently, we identified a missense SUR1 mutation (R1420C) in Japanese PHHI patients (31). Login to comment
42 An 86 Rbϩ efflux study revealed that KATP ϩ channels composed of SUR1-R1420C and Kir6.2 are not activated by metabolic inhibition as much as wild-type channels. Login to comment
43 This may be related to the fact that the expression level of SUR1-R1420C was only about half that of the wild-type channel when it was transiently expressed in COS-7 cells (31). Login to comment
44 We also reported that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes the binding of 8-azido-ATP at NBF1 of wild-type SUR1 (19), and that the R1420C mutation impairs this effect without altering high affinity 8-azido-ATP binding to NBF1 (31). Login to comment
46 In this study, we examined the nucleotide-binding properties of the two NBFs of SUR1-R1420C by photoaffinity labeling experiments and studied the properties of Kir6.2/SUR1-R1420C channels by electrophysiological analysis. Login to comment
51 Crude membranes, containing similar amounts of wild-type SUR1 and SUR1-R1420C as determined by Western blotting, were incubated with 50 ␮M 8-azido-[32 P]ATP in the presence or absence of 0.1-1000 ␮M ATP or ADP in 3 ␮l of TEM buffer (40 mM Tris-Cl (pH 7.5), 0.1 mM EGTA, 1 mM MgSO4) containing 2 mM ouabain. Login to comment
78 RESULTS Photoaffinity Labeling of SUR1-R1420C with 8-Azido-[␣- 32 P]ATP and 8-Azido-[␥-32 P]ATP-To examine the biochemical properties of SUR1-R1420C, we investigated 8-azido-[32 P]ATP photoaffinity labeling of NBF1 and NBF2 (20). Login to comment
79 Crude membranes from COS-7 cells expressing SUR1 or SUR1-R1420C were incubated with 50 ␮M 8-azido-[␣-32 P]ATP followed by mild trypsin digestion. Login to comment
81 This indicates that both NBFs of SUR1-R1420C can bind 8-azido-ATP, as is found for wild-type SUR1. Login to comment
83 We therefore examined whether SUR1-R1420C is photoaffinity-labeled with 8-azido-[␥-32 P]ATP. Login to comment
84 Both NBF1 of SUR1 and of SUR1-R1420C were photoaffinity-labeled with 8-azido-[␥- 32 P]ATP (Fig. 1, lanes 5 and 7), indicating that NBF1 either does not have ATPase activity or has little ATPase activity under our experimental conditions. Login to comment
85 In contrast, NBF2s of SUR1 and SUR1-R1420C were not photoaffinity-labeled with 8-azido-[␥-32 P]ATP (Fig. 1, lanes 6 and 8), although they were photoaffinity-labeled by 8-azido-[␣-32 P]ATP (Fig. 1, lanes 2 and 4). Login to comment
86 These results suggest that the ␥-phosphate dissociates from 8-azido-[32 P]ATP bound at NBF2 and thus that NBF2 of SUR1- R1420C might have ATPase activity as is found for wild-type SUR1. Login to comment
87 Affinity of NBFs of SUR1-R1420C for ATP and ADP-To characterize the biochemical properties of SUR1-R1420C further, we investigated the affinities of both NBF1 and NBF2 for ATP and ADP. Login to comment
88 When crude membranes from COS-7 cells expressing SUR1 or SUR1-R1420C were incubated with 50 ␮M 8-azido-[␣-32 P]ATP in the presence of ATP or ADP followed by mild trypsin digestion, photoaffinity labeling of tryptic fragments containing either NBF1 or NBF2 was inhibited in a concentration-dependent manner (Fig. 2). Login to comment
89 This indicates that SUR1 and SUR1-R1420C can bind ATP and ADP. Login to comment
91 The Ki values of NBF1 of SUR1 for ATP and ADP were 1.6 Ϯ 0.64 (n ϭ 3) and 17 Ϯ 7.9 ␮M (n ϭ 3) respectively, and those of SUR1-R1420C were 1.5 Ϯ 0.26 (n ϭ 3) and 13 Ϯ 6.8 ␮M (n ϭ 3), respectively. Login to comment
92 This indicates that the affinity of NBF1 for nucleotides is not significantly different between wild-type SUR1 and SUR1-R1420C and that the affinity for ATP is significantly higher than that for ADP. Login to comment
95 However, the Ki values of NBF2 of SUR1-R1420C for ATP and ADP (350 Ϯ 36 (n ϭ 3) and 290 Ϯ 66 ␮M (n ϭ 3), respectively) were significantly higher than those of wild-type SUR1 (64 Ϯ 4.7 (n ϭ 3) and 65 Ϯ 16 ␮M (n ϭ 3), respectively). Login to comment
96 These results demonstrate that the R1420C mutation decreases the affinity of NBF2 for both ATP and ADP. Login to comment
97 Cooperative Nucleotide Binding of SUR1-R1420C-To explore the possibility that the impaired cooperative nucleotide binding observed for SUR1-R1420C is due to the low nucleotide-binding affinity of NBF2, we examined the dependence of cooperative binding on nucleotide concentration. Login to comment
102 In contrast, neither ATP nor ADP (1 mM) stabilized 8-azido-[32 P]ATP binding at NBF1 of SUR1-R1420C despite the fact that 1 mM MgATP and MgADP inhibited photoaffinity labeling of NBF2 (68 Ϯ 3.2 (n ϭ 3) and 76 Ϯ 2.9% (n ϭ 3), respectively) as efficiently as that of wild-type SUR1 (74 Ϯ 10 (n ϭ 3) and 73 Ϯ 1.5% (n ϭ 3), FIG. 1. Login to comment
103 Photoaffinity labeling of the NBFs of SUR1 and SUR1-R1420C with 8-azido-[␣-32 P]ATP and 8-azido-[␥-32 P]ATP. Login to comment
104 Membrane proteins (10-20 ␮g) from COS-7 cells expressing wild-type SUR1 (lanes 1, 2, 5, and 6) or SUR1-R1420C (lanes 3, 4, 7, and 8) were incubated with 50 ␮M 8-azido-[␣-32 P]ATP (lanes 1-4) or 8-azido-[␥- 32 P]ATP (lanes 5-8) for 10 min at 37 °C and then UV-irradiated. Login to comment
110 Inhibition of photoaffinity labeling of the NBFs of SUR1 and SUR1-R1420C by nucleotides. Login to comment
111 Membrane proteins (10-20 ␮g) from COS-7 cells expressing wild-type SUR1 or SUR1-R1420C were incubated with 50 ␮M 8-azido-[␣-32 P]ATP in the presence of 0.1-1000 ␮M ATP or ADP for 10 min at 0 °C and then UV-irradiated. Login to comment
113 Relative photoaffinity labeling of wild-type SUR1 in the presence of ATP (open squares) or ADP (closed squares) and of SUR1-R1420C in the presence of ATP (open circles) or ADP (closed circles) are expressed as percentages of that obtained in the absence of cold nucleotides. Login to comment
115 The symbols show the mean values and the S.E. TABLE I Ki values for interaction of ATP and ADP with the NBFs of SUR1 and SUR1-R1420C The data of Fig. 2 were fit by the Hill equation, and Ki values were obtained. Login to comment
116 Ki Wild-type R1420C ␮M ␮M NBF1-ATP 1.6 Ϯ 0.64 1.5 Ϯ 0.26 NBF1-ADP 17 Ϯ 7.9 13 Ϯ 6.8 NBF2-ATP 64 Ϯ 4.7 350 Ϯ 36 NBF2-ADP 65 Ϯ 16 290 Ϯ 66 respectively) (Fig. 2). Login to comment
117 These results suggest that neither MgATP nor MgADP bound at NBF2 can stabilize 8-azido-ATP binding at NBF1 of SUR1-R1420C. Login to comment
118 Electrophysiological Analysis-To examine the functional properties of the mutant KATP ϩ channels, we coexpressed SUR1-R1420C with Kir6.2 in Xenopus oocytes. As observed for the wild-type channel (Kir6.2/SUR1), no significant current was detected for Kir6.2/SUR1-R1420C channels in the cell-attached configuration, but large currents developed following patch excision into nucleotide-free solution. Login to comment
119 The mean current amplitude at -100 mV was 3.0 Ϯ 1.0 nA (n ϭ 10) for Kir6.2/SUR1 and 1.5 Ϯ 0.6 nA (n ϭ 15) for Kir6.2/SUR1-R1420C channels. Login to comment
121 A quantitatively similar reduction in SUR1-R1420C protein expression was observed in COS-7 cells, indicating that the lower expression of mutant SUR1 is confined to the oocyte expression system. Login to comment
122 Pharmacological Regulation-We first examined the effect of the R1420C mutation on the activation of the channel by the potassium channel opener diazoxide (Fig. 4) and its inhibition by the sulfonylurea tolbutamide. Login to comment
125 Diazoxide increased Kir6.2/SUR1 currents by 694 Ϯ 40% (n ϭ 4) and Kir6.2/SUR1-R1420C currents by 499 Ϯ 126% (n ϭ 7). Login to comment
126 The sulfonylurea tolbutamide blocked Kir6.2/SUR1 currents by 53 Ϯ 2% (n ϭ 3) and Kir6.2/SUR1-R1420C currents by 68 Ϯ 7% (n ϭ 3). Login to comment
127 Thus the R1420C mutation does not affect the pharmacological properties of the channel. Login to comment
128 Nucleotide Regulation-Inhibition of the channel by ATP was not affected by the R1420C mutation. Login to comment
129 Kir6.2/SUR1 currents were inhibited with an IC50 of 13 Ϯ 2 ␮M and a Hill coefficient of 0.99 Ϯ 0.18 (n ϭ 5), whereas Kir6.2/SUR1-R1420C currents were blocked with an IC50 of 12 Ϯ 2 ␮M and a Hill coefficient of 0.90 Ϯ 0.14 (n ϭ 6; Fig. 5). Login to comment
130 ADP produced a mean current increase of 402 Ϯ 82% (n ϭ 3) and 347 Ϯ 52% (n ϭ 6) for Kir6.2/SUR1 and Kir6.2/SUR1-R1420C, respectively, when tested in the presence of 100 ␮M MgATP. Login to comment
132 Cooperative binding of nucleotides and 8-azido-[␣- 32 P]ATP to SUR1 and SUR1-R1420C. Login to comment
133 Membrane proteins (10-20 ␮g) from COS-7 cells expressing wild-type SUR1 or SUR1-R1420C were preincubated with 10 ␮M 8-azido-[␣-32 P]ATP for 3 min at 37 °C. Free 8-azido-[␣-32 P]ATP was then removed by washing the membranes with excess cold buffer. Login to comment
134 Proteins were UV-irradiated after a postincubation for 15 min at 37 °C in the absence of nucleotide (SUR1, square with cross; SUR1-R1420C, circle with cross), in the presence of ATP (SUR1, open squares; SUR1-R1420C, open circles), or in the presence of ADP (SUR1, closed squares; SUR1-R1420C, closed circles). Login to comment
137 Diazoxide and nucleotide modulation of Kir6.2/SUR1-R1420C. Login to comment
138 A, macroscopic currents recorded from a giant patch on an oocyte coinjected with Kir6.2 and SUR1-R1420C mRNA. Login to comment
141 B, mean macroscopic currents recorded from oocytes coexpressing Kir6.2 and either wild-type SUR1 or SUR1-R1420C in the presence of the nucleotides indicated. The mean conductance in the presence of nucleotide (G) is expressed relative to the mean of that measured in control solution before and after removal of nucleotide (Gc; indicated by the dashed line). Login to comment
144 ATP sensitivity of Kir6.2/SUR1-R1420C. Login to comment
145 Mean MgATP concentration-response relationships for Kir6.2/SUR1 (filled squares, n ϭ 5) and Kir6.2-SUR1-R1420C (open squares, n ϭ 6) currents are shown. Login to comment
151 Therefore, to quantify the difference in the extent of nucleotide activation of wild-type and mutant KATP ϩ channels, we coexpressed either SUR1 or SUR1-R1420C with a mutant form of Kir6.2, Kir6.2-R50G, which exhibits a greatly reduced ATP sensitivity (18, 36). Login to comment
152 Both Kir6.2-R50G/SUR1 and Kir6.2-R50G/SUR1-R1420C currents were activated by 100 ␮M MgATP and by 100 ␮M MgGTP (Fig. 6A), and in both cases, the extent of activation was greater for Kir6.2-R50G/SUR1-R1420C channels. Login to comment
154 As Fig. 6A also shows, Kir6.2-R50G/SUR1-K719A currents were neither activated nor blocked by 100 ␮M MgATP, demonstrating that, at this ATP concentration, activation of Kir6.2-R50G/SUR1 and Kir6.2-R50G/SUR1-R1420C currents is not partially masked by an inhibitory effect of ATP. Login to comment
157 Fig. 6B shows the relationship between MgADP concentration and activation of Kir6.2-R50G/SUR1 and Kir6.2-R50G/ SUR1-R1420C channels. Login to comment
159 The EC50 for ADP activation was also increased by the SUR1-R1420C mutation from 74 Ϯ 30 ␮M (n ϭ 5) for Kir6.2-R50G/SUR1 channels to 197 Ϯ 20 ␮M (n ϭ 6) for Kir6.2-R50G/SUR1-R1420C channels. Login to comment
160 These values are comparable with those found for ADP displacement of 8-azido-ATP binding to NBF2 of SUR1 and SUR1-R1420C, respectively (Table I). Login to comment
161 One possible explanation for the fact that ADP causes greater activation of Kir6.2-R50G/SUR1-R1420C channels is that their open probability (Po) is less than that of Kir6.2-R50G/SUR1 channels. Login to comment
164 Kir6.2/SUR1 and Kir6.2/SUR1-R1420C channels had Po of 0.29 Ϯ 0.09 (n ϭ 4) and 0.32 Ϯ 0.03 (n ϭ 5), respectively. Login to comment
165 When SUR1 or SUR1-R1420C was coexpressed with Kir6.2-R50G, the Po was 0.35 Ϯ 0.07 (n ϭ 6) and 0.34 Ϯ 0.07 (n ϭ 7), respectively. Login to comment
167 Whole-cell Studies-To explore the effect of metabolic inhibition on Kir6.2/SUR1 and Kir6.2/SUR1-R1420C channels, we recorded whole-cell KATP ϩ currents from intact oocytes and used 3 mM azide as a metabolic poison. Login to comment
168 In control solution, oocytes expressing both types of channels exhibited very small current amplitudes, similar to those of water-injected oocytes. As shown in Fig. 8, metabolic poisoning produced a large increase in both Kir6.2/SUR1 and Kir6.2/SUR1-R1420C currents. Login to comment
170 The fact that Kir6.2/SUR1-R1420C currents induced by metabolic inhibition are smaller than those of the wild-type channel may reflect, at least in part, a lower level of expression of the mutant channel because in inside-out membrane patches the currents activated on patch excision were also smaller (Fig. 8B). Login to comment
171 DISCUSSION We analyzed the functional properties of SUR1 containing a PHHI missense mutation, R1420C, and demonstrated that the mutation lowers the affinities of NBF2 for ATP and ADP and impairs the ability of MgATP and MgADP to stabilize ATP binding at NBF1. Login to comment
172 This cooperative nucleotide binding was not observed for SUR1-R1420C even with 1 mM MgATP or MgADP, although 1 mM MgATP and MgADP inhibited the photoaffinity labeling of NBF2 of wild-type SUR1 and SUR1-R1420C with similar efficiency. Login to comment
173 The affinities of NBF1s for ATP and ADP are similar for wild-type SUR1 and SUR1-R1420C. Login to comment
174 These results suggest that the R1420C mutation not only decreases the nucleotide-binding affinity of NBF2 but also impedes the transduction of a conformational change at NBF2 and thereby its ability to stabilize ATP binding at NBF1. Login to comment
176 Nucleotide activation of Kir6.2/SUR1-R1420C. Login to comment
177 A, mean macroscopic currents recorded from oocytes coexpressing Kir6.2-R50G and either wild-type SUR1, SUR1-R1420C, or SUR1-K719A in the presence of 100 ␮M of the nucleotides indicated. The mean conductance in the presence of nucleotide (G) is expressed relative to the mean of that measured in control solution before and after removal of nucleotide (Gc indicated by the dashed line). Login to comment
178 Numbers of oocytes are given above the bars. B, relationship between the MgADP concentration and the KATP ϩ current for Kir6.2-R50G/SUR1 (circles, n ϭ 4) and Kir6.2-R50G/ SUR1-R1420C (squares, n ϭ 5) channels. Login to comment
184 As found for binding studies, the EC50 for channel activation by ADP increased to 197 Ϯ 20 ␮M when arginine 1420 was mutated to cysteine, which compares favorably with the Ki of 290 Ϯ 66 ␮M found for ADP binding to NBF2 of SUR1-R1420C. Login to comment
190 Second, the R1420C mutation may reduce the affinity of MgADP binding yet at the same time enhance the transduction of the conformational change in response to MgADP. Login to comment
192 This might occur if, for example, channel activation is greater when MgADP binds to both NBFs than when MgATP binds to NBF1 and MgADP to NBF2. Our results suggest that the level of expression of Kir6.2/ SUR1-R1420C KATP ϩ channels in Xenopus oocytes is about half that of wild-type KATP ϩ channels because mutant currents in excised patches were about half those of wild-type currents. Login to comment
193 We have previously reported that Rbϩ efflux from COS-7 cells expressing Kir6.2/SUR1-R1420C KATP ϩ channels is about half that of cells expressing wild-type KATP ϩ channels when intracellular ATP is depleted by metabolic inhibition with 2-deoxyglucose and oligomycin. Login to comment
195 Thus the R1420C mutation results in a reduced KATP ϩ current whether expressed in oocytes or in mammalian cells. Login to comment
196 However, this cannot be the only explanation for the ability of the R1420C mutation to cause PHHI because heterozygotes carrying other PHHI mutations that result in a total loss of protein, which would also be expected to produce KATP ϩ currents of about half the normal amplitude, do not develop PHHI. Login to comment
201 We have proposed that the greater ability of SUR1 to stimulate opening of KATP ϩ channels when metabolism falls is linked to the higher nucleotide-binding affinities of NBF1 and/or NBF2. Our finding that the PHHI missense mutation R1420C lowers the affinity of NBF2 for ATP and ADP and increases the EC50 for ADP activation of channel activity adds further support to the idea that ADP binding to NBF2 of SUR1 is important for KATP ϩ channel stimulation. Login to comment
208 Single Kir6.2/SUR1 and Kir6.2/ SUR1-R1420C channel currents. Login to comment
209 Single channel currents recorded at -60 mV from patches excised from oocytes expressing Kir6.2/SUR1 and Kir6.2/SUR1-R1420C channels are shown. Login to comment
211 Metabolic regulation of Kir6.2/SUR1-R1420C. Login to comment
213 Oocytes were coinjected with mRNA encoding Kir6.2 and SUR1 or SUR1-R1420C. Login to comment
215 Oocytes were coinjected with mRNA encoding Kir6.2 and SUR1 or SUR1-R1420C. Login to comment
225 In this study, we have demonstrated that the R1420C PHHI mutation strongly affects the biochemical properties of SUR1; it lowers the affinity of NBF2 for ATP and ADP and abolishes the ability of nucleotide binding at NBF2 to stabilize 8-azido-ATP binding at NBF1. Login to comment

[hide] Physiological and pathophysiological roles of ATP-... Prog Biophys Mol Biol. 2003 Feb;81(2):133-76. Seino S, Miki T
Physiological and pathophysiological roles of ATP-sensitive K+ channels.
Prog Biophys Mol Biol. 2003 Feb;81(2):133-76., [PMID:12565699]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.

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1007 Some mutations, such as SUR1-R1420C, which gives rise to a mild-form of patients with PHHI, have been shown to affect KATP channels only slightly (Matsuo et al., 2000). Login to comment
1008 The homozygous mutation SUR1-R1420C was found in a PHHI patient with focal adenomatous hyperplasia of pancreatic islets (Verkarre et al., 1998), and was shown to cause a mild-form of PHHI with clinical remission (Tanizawa et al., 2000) that is resistant to diazoxide treatment. Login to comment
999 Some mutations, such as SUR1-R1420C, which gives rise to a mild-form of patients with PHHI, have been shown to affect KATP channels only slightly (Matsuo et al., 2000). Login to comment
1000 The homozygous mutation SUR1-R1420C was found in a PHHI patient with focal adenomatous hyperplasia of pancreatic islets (Verkarre et al., 1998), and was shown to cause a mild-form of PHHI with clinical remission (Tanizawa et al., 2000) that is resistant to diazoxide treatment. Login to comment

[hide] K(ATP) channels and insulin secretion disorders. Am J Physiol Endocrinol Metab. 2002 Aug;283(2):E207-16. Huopio H, Shyng SL, Otonkoski T, Nichols CG
K(ATP) channels and insulin secretion disorders.
Am J Physiol Endocrinol Metab. 2002 Aug;283(2):E207-16., [PMID:12110524]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider implications for future understanding of the mechanisms of control of insulin secretion in normal and diseased states.

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79 One HI mutation (SUR1[R1420C]) lowers the affinity of NBF2 for ATP and ADP and abolishes the cooperative binding between the two NBFs (36). Login to comment
82 Consistent with the biochemical data, the EC50 for MgADP activation of the SUR1[R1420C] ϩ Kir6.2[R50G] mutant channel is about three times higher than that of wild-type SUR1 ϩ Kir6.2[R50G] channels. Login to comment
68 One HI mutation (SUR1[R1420C]) lowers the affinity of NBF2 for ATP and ADP and abolishes the cooperative binding between the two NBFs (36). Login to comment
71 Consistent with the biochemical data, the EC50 for MgADP activation of the SUR1[R1420C] af9; Kir6.2[R50G] mutant channel is about three times higher than that of wild-type SUR1 af9; Kir6.2[R50G] channels. Login to comment

[hide] Genotypes of the pancreatic beta-cell K-ATP channe... Clin Endocrinol (Oxf). 2005 Apr;62(4):458-65. Ohkubo K, Nagashima M, Naito Y, Taguchi T, Suita S, Okamoto N, Fujinaga H, Tsumura K, Kikuchi K, Ono J
Genotypes of the pancreatic beta-cell K-ATP channel and clinical phenotypes of Japanese patients with persistent hyperinsulinaemic hypoglycaemia of infancy.
Clin Endocrinol (Oxf). 2005 Apr;62(4):458-65., [PMID:15807877]

Abstract [show]
OBJECTIVE: Persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) is a disorder of glucose metabolism that is characterized by dysregulated secretion of insulin from pancreatic beta-cells. This disease has been reported to be associated with mutations of the sulfonylurea receptor SUR1 (ABCC8) or the inward-rectifying potassium channel Kir6.2 (KCNJ11), which are two subunits of the pancreatic beta-cell ATP-sensitive potassium channel. PATIENTS AND METHODS: In 14 Japanese PHHI patients, all exons of SUR1 and Kir6.2 genes were analysed by polymerase chain reaction (PCR) and direct sequencing. Four patients responded to diazoxide, and nine patients underwent a subtotal pancreatectomy. Histologically, seven patients were diagnosed to have a focal form and two a diffuse form of the disease. RESULTS: We found nine novel mutations in the SUR1 gene and two in the Kir6.2 gene. In the SUR1 gene mutations, three were nonsense mutations (Y512X, Y1354X and G1469X), one was a one-base deletion in exon 7, and two were missense mutations in the nucleotide-binding domain 2 (K1385Q, R1487K). The other three mutations occurred in introns 14, 29 and 36, which might cause aberrant splicing of RNA. Two siblings in one family were heterozygotes for a missense mutation, K1385Q, which was maternally inherited. In Kir6.2 gene screening, one patient was found to be a compound heterozygote of a missense mutation (R34H) and a one-base deletion (C344fs/ter). CONCLUSION: The novel mutations reported here could be pathological candidates for PHHI in Japan. They also reveal that SUR1 and Kir6.2 mutations in the Japanese population exhibit heterogeneity and that they occurred at a frequency similar to other genetic populations.

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122 To date, only three kinds of SUR1 mutations (I446fsdelT, R1420C and R1436Q) have been reported in Japanese PHHI patients.20,42 We defined 11 novel mutations, and the SUR1 and Kir6.2 mutations account for about 80% of the PHHI cases in this study, although Tanizawa et al.20 reported only about 20% (three kinds of mutations in four patients from the screening of 17 PHHI patients) and Someya et al.42 did not detect any mutations (in eight PHHI patients). Login to comment
130 For example, the patients having some kinds of missense mutations in the SUR1 gene such as F591L,13 R1420C, 20 R1506K 6 and K1385Q in this report showed mild hypoglycaemia, which is known to be responsive to drug therapy, and thus they do not need an operation. Login to comment

[hide] Genetic analysis of Japanese patients with persist... Diabetes. 2000 Jan;49(1):114-20. Tanizawa Y, Matsuda K, Matsuo M, Ohta Y, Ochi N, Adachi M, Koga M, Mizuno S, Kajita M, Tanaka Y, Tachibana K, Inoue H, Furukawa S, Amachi T, Ueda K, Oka Y
Genetic analysis of Japanese patients with persistent hyperinsulinemic hypoglycemia of infancy: nucleotide-binding fold-2 mutation impairs cooperative binding of adenine nucleotides to sulfonylurea receptor 1.
Diabetes. 2000 Jan;49(1):114-20., [PMID:10615958]

Abstract [show]
To elucidate the genetic etiology of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) in the Japanese population, we conducted a polymerase chain reaction-single-strand conformation polymorphism analysis of the sulfonylurea receptor 1 (SUR1) and Kir6.2 genes in 17 Japanese PHHI patients, including a pair of siblings from a consanguineous family. We also analyzed the glutamate dehydrogenase gene for the exons encoding an allosteric regulatory domain of the enzyme. In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. None of these mutations were found in control Japanese subjects. Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Functional consequences of these mutations on K(ATP) function were evaluated using 86Rb+ efflux studies in COS-7 cells. SUR1-446fsdelT and SUR1-1436Q did not form a functional K(ATP). Western blot analysis after transient expression in COS-7 cells revealed the expression of SUR1-1436Q protein to be markedly reduced, suggesting SUR1-1436Q to be unstable in these cells. K(ATP)(SUR1-1420C) showed reduced responses to metabolic inhibition by oligomycin and 2-deoxyglucose. K(ATP) channels are under complex regulation by intracellular ATP and ADP. ATP both inhibits and activates these channels. The inhibition is probably mediated through direct ATP interaction with a pore-forming subunit Kir6.2, whereas the activation is likely to be through a regulatory subunit SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in regulating the K(ATP) channel. In SUR1-1420C, high-affinity binding of ATP to the nucleotide-binding fold (NBF)-1 was indistinguishable from that of wild-type SUR1. However, stabilization of ATP binding to NBF-1 by MgATP or MgADP was impaired, suggesting that this defect may account for impaired K(ATP)(SUR1-1420C) function. This is the first direct biochemical evidence that the cooperativity of nucleotide binding to SUR1 is impaired in a SUR1 mutant causing PHHI. No mutations were identified in the Kir6.2 and glutamate dehydrogenase genes. The genetic etiology of PHHI appears to be heterogeneous. SUR1 mutations may account for no more than 20% of PHHI cases in Japanese patients. Mutations of Kir6.2 and glutamate dehydrogenase genes are likely to be even less common.

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2 In the SUR1 gene, we identified one frameshift (I446fsdelT) and two missense (R1420C, R1436Q) mutations. Login to comment
4 Siblings homozygous for the R1420C mutation had a mild form, whereas two patients heterozygous for the I446fsdelT and R1436Q mutations, respectively, exhibited a severe form of PHHI. Login to comment
62 The I446fsdelT and R1420C mutations created an NlaIII restriction site (CATG). Login to comment
67 Site-directed mutagenesis was performed using mutagenesis primers (for I446fsdelT: 5 -acg ctc cgt tca tgc ctc tcc atc atc c-3 ; for R1420C: 5 -gcc agt aca gat cat gtg ggc gtg atc ctc c-3 ; and for R1436Q: 5 -ttc agc ggc acc atc caa ttc aacctg gac cc-3 ) and a GeneEditor in vitro Site-DirectedMuta- genesisSystem (Promega, Madison, WI). Login to comment
81 We identified three potential disease-causing mutations (I446fsdelT, R1420C, and R1436Q). Login to comment
90 Two other mutations (R1420C and R1436Q) were missense mutations, both in NBF-2, a functionally important domain of SUR1. Login to comment
93 Two patients (patients 2 and 3), siblings from a consanguineous family, were homozygous for the R1420C mutation. Login to comment
111 A sibling pair (patients 2 and 3), who were homozygous for the R1420C mutation, had a milder form of PHHI. Login to comment
120 Therefore, we introduced I446fsdelT, R1420C, and R1436Q mutations by site-directed mutagenesis to the corresponding positions of mouse SUR1 cDNA, and mutant SUR1 and mouse Kir6.2 were transiently coexpressed in COS-7 cells to reconstitute the mutant KATP channel. Login to comment
128 Activation ofthe channel by diazoxide also appeared to be impaired in the KATP(SUR1-1420C) mutant, TABLE 3 Profiles of patients with SUR1 gene mutations Onset Birth Treatment/ Patient Mutation (day) weight (g) outcome 1 I446fsdelT 0 5,014 Partial pancreatectomy 2 R1420C 0 5,254 Remission 3 R1420C 0 5,080 Remission 4 R1436Q 0 3,410 Partial pancreatectomy FIG. 1. Login to comment
131 Means of measurements made in triplicate for the representative experiment are plotted. , SUR1 (wild-type); , SUR1-R1420C; , SUR1-446delT; , LacZ. Login to comment
140 Effect of R1420C mutation on cooperative binding of adenine nucleotides in SUR1. There is a cooperative regulation of ATP and ADP binding to SUR1, and this cooperativity may be involved in MgATP and MgADP regulation of the KATP channel (18,19). Login to comment
143 Effects of the R1420C mutation in NBF-2 on cooperative binding of ATP and ADP were assessed. Login to comment
196 This may also be the case for the R1420C mutation, although we did not test this possibility directly. Login to comment
199 In our 86 Rb+ efflux studies, impairment of KATP channel function was modest with the R1420C mutation, while no channel activities were observed for the other two mutations, R1436C and I446fsdelT. Login to comment
200 This parallels clinical disease severities of these patients: patients with the R1420C mutation achieved seemingly spontaneous remission after a few months of medical therapy, whereas patients who had R1436C or I446fsdelT mutations required partial pancreatectomy. Login to comment
66 Site-directed mutagenesis was performed using mutagenesis primers (for I446fsdelT: 5 -acg ctc cgt tca tgc ctc tcc atc atc c-3 ; for R1420C: 5 -gcc agt aca gat cat gtg ggc gtg atc ctc c-3 ; and for R1436Q: 5 -ttc agc ggc acc atc caa ttc aacctg gac cc-3 ) and a GeneEditor in vitro Site-DirectedMuta- genesisSystem (Promega, Madison, WI). Login to comment
80 We identified three potential disease-causing mutations (I446fsdelT, R1420C, and R1436Q). Login to comment
89 Two other mutations (R1420C and R1436Q) were missense mutations, both in NBF-2, a functionally important domain of SUR1. Login to comment
92 Two patients (patients 2 and 3), siblings from a consanguineous family, were homozygous for the R1420C mutation. Login to comment
110 A sibling pair (patients 2 and 3), who were homozygous for the R1420C mutation, had a milder form of PHHI. Login to comment
119 Therefore, we introduced I446fsdelT, R1420C, and R1436Q mutations by site-directed mutagenesis to the corresponding positions of mouse SUR1 cDNA, and mutant SUR1 and mouse Kir6.2 were transiently coexpressed in COS-7 cells to reconstitute the mutant KATP channel. Login to comment
127 Activation ofthe channel by diazoxide also appeared to be impaired in the KATP(SUR1-1420C) mutant, TABLE 3 Profiles of patients with SUR1 gene mutations Onset Birth Treatment/ Patient Mutation (day) weight (g) outcome 1 I446fsdelT 0 5,014 Partial pancreatectomy 2 R1420C 0 5,254 Remission 3 R1420C 0 5,080 Remission 4 R1436Q 0 3,410 Partial pancreatectomy FIG. 1. Login to comment
130 Means of measurements made in triplicate for the representative experiment are plotted. , SUR1 (wild-type); , SUR1-R1420C; , SUR1-446delT; , LacZ. Login to comment

[hide] Potassium channel regulation. EMBO Rep. 2003 Nov;4(11):1038-42. Campbell JD, Sansom MS, Ashcroft FM
Potassium channel regulation.
EMBO Rep. 2003 Nov;4(11):1038-42., [PMID:14593442]

Abstract [show]
The sulphonylurea receptor (SUR) is a member of the ATP-binding cassette (ABC) family of membrane proteins. It functions as the regulatory subunit of the ATP-sensitive potassium (KATP) channel, which comprises SUR and Kir6.x proteins. Here, we review data demonstrating functional differences between the two nucleotide binding domains (NBDs) of SUR1. In addition, to explain the structural basis of these functional differences, we have constructed a molecular model of the NBD dimer of human SUR1. We discuss the experimental data in the context of this model, and show how the model can be used to design experiments aimed at elucidating the relationship between the structure and function of the KATP channel.

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107 Although some of these mutations prevent targeting of the protein to the plasma membrane, others, such as G1381S, R1420C, E1506K and L1551V (Fig. 3) cause CHI by impairing KATP channel activation in response to metabolic inhibition or MgADP (Huopio et al., 2000). Login to comment
114 The main effect of the CHI mutation R1420C is to prevent cooperative nucleotide binding (Matsuo et al., 2000b). Login to comment

[hide] The genetic basis of congenital hyperinsulinism. J Med Genet. 2009 May;46(5):289-99. Epub 2009 Mar 1. James C, Kapoor RR, Ismail D, Hussain K
The genetic basis of congenital hyperinsulinism.
J Med Genet. 2009 May;46(5):289-99. Epub 2009 Mar 1., [PMID:19254908]

Abstract [show]
Congenital hyperinsulinism (CHI) is biochemically characterised by the dysregulated secretion of insulin from pancreatic beta-cells. It is a major cause of persistent hyperinsulinaemic hypoglycaemia (HH) in the newborn and infancy period. Genetically CHI is a heterogeneous condition with mutations in seven different genes described. The genetic basis of CHI involves defects in key genes which regulate insulin secretion from beta-cells. Recessive inactivating mutations in ABCC8 and KCNJ11 (which encode the two subunits of the adenosine triphosphate sensitive potassium channels (ATP sensitive K(ATP) channels)) in beta-cells are the most common cause of CHI. The other recessive form of CHI is due to mutations in HADH (encoding for-3-hydroxyacyl-coenzyme A dehydrogenase). Dominant forms of CHI are due to inactivating mutations in ABCC8 and KCNJ11, and activating mutations in GLUD1 (encoding glutamate dehydrogenase) and GCK (encoding glucokinase). Recently dominant mutations in HNF4A (encoding hepatocyte nuclear factor 4alpha) and SLC16A1 (encoding monocarboxylate transporter 1) have been described which lead to HH. Mutations in all these genes account for about 50% of the known causes of CHI. Histologically there are three (possibly others which have not been characterised yet) major subtypes of CHI: diffuse, focal and atypical forms. The diffuse form is inherited in an autosomal recessive (or dominant manner), the focal form being sporadic in inheritance. The diffuse form of the disease may require a near total pancreatectomy whereas the focal form requires a limited pancreatectomy potentially curing the patient. Understanding the genetic basis of CHI has not only provided novel insights into beta-cell physiology but also aided in patient management and genetic counselling.

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53 Several mutations in ABCC8 (for example, R1420C, T1139M and R1215Q) have now been described that result in the loss of ADP dependent gating properties of the channel.75 77 78 Loss of ADP dependent gating results in the constitutive inhibition of KATP channels by ATP. Login to comment

[hide] Different binding properties and affinities for AT... J Biol Chem. 2000 Sep 15;275(37):28757-63. Matsuo M, Tanabe K, Kioka N, Amachi T, Ueda K
Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B.
J Biol Chem. 2000 Sep 15;275(37):28757-63., [PMID:10893240]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K(ATP) channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K(ATP) channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[alpha-(32)P]ATP or 8-azido-[gamma-(32)P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg(2+) dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of K(ATP) channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K(ATP) channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs.

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105 Cooperative Binding of Nucleotides to NBF1 and NBF2-We reported previously that ADP stabilizes the binding of prebound 8-azido-[32 P]ATP at NBF1 on SUR1, either by direct binding to NBF2 or hydrolysis of bound ATP at NBF2 (21), and this effect was impaired in mutant SUR1 (R1420C) found in Japanese persistent hyperinsulinemic hypoglycemia of infancy patients (24). Login to comment

[hide] ATP-binding cassette proteins involved in glucose ... Biosci Biotechnol Biochem. 2010;74(5):899-907. Epub 2010 May 7. Matsuo M
ATP-binding cassette proteins involved in glucose and lipid homeostasis.
Biosci Biotechnol Biochem. 2010;74(5):899-907. Epub 2010 May 7., [PMID:20460728]

Abstract [show]
Glucose and lipids are essential to the body, but excess glucose or lipids lead to metabolic syndrome. ATP-binding cassette (ABC) proteins are involved in the homeostasis of glucose and lipid in that they regulate insulin secretion and remove excess cholesterol from the body. Sulfonylurea receptor (SUR) is a subunit of the ATP-sensitive potassium channels, which regulate insulin secretion from pancreatic beta-cells by sensing cellular metabolic levels. ABCG1 removes excess cholesterol from peripheral tissues and functions in reverse cholesterol transport to the liver. ABCG5 and ABCG8 suppress the absorption of cholesterol in the intestine and exclude cholesterol from the liver to the bile duct. ABCG1 and ABCG4, expressed in the central nervous system, play roles in lipid metabolism in the brain. These ABC proteins are targets of drugs and functional foods to cure and prevent diabetes, hyperlipidemia, and neurodegenerative diseases. In this review, recent knowledge of the physiological function and regulation of ABC proteins in the homeostasis of glucose and lipids is discussed.

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77 Several mutations have been reported to impair KATP channel activity by reducing MgADP activation.33,34) A missense SUR1 mutation (R1420C) has been identified in Japanese patients with a mild form of PHHI.35) Verkarre et al. also reported this Fig. 2. Login to comment
86 We found that the mutation does not affect the affinity of NBF1 for nucleotides, but lowers the affinity of NBF2 for ATP and for ADP.37) The R1420C mutation impairs cooperative nucleotide binding of the two NBFs, although it does not show direct effects on high-affinity 8-azido-ATP binding to NBF1. Login to comment

[hide] Nateglinide is Effective for Diabetes Mellitus wit... Clin Pediatr Endocrinol. 2012 Jul;21(3):45-52. doi: 10.1297/cpe.21.45. Epub 2012 Jul 25. Saito-Hakoda A, Yorifuji T, Kanno J, Kure S, Fujiwara I
Nateglinide is Effective for Diabetes Mellitus with Reactive Hypoglycemia in a Child with a Compound Heterozygous ABCC8 Mutation.
Clin Pediatr Endocrinol. 2012 Jul;21(3):45-52. doi: 10.1297/cpe.21.45. Epub 2012 Jul 25., [PMID:23926410]

Abstract [show]
ABCC8 encodes the sulfonylurea receptor 1 (SUR1) subunits of the beta-cell ATP-sensitive potassium (K-ATP) channel playing a critical role in the regulation of insulin secretion, and inactivating mutations in ABCC8 cause congenital hyperinsulinism. Recently, ABCC8 inactivating mutations were reported to be involved in the development of diabetes mellitus later in life. We report a girl who was born macrosomic with transient hypoglycemia and thereafter developed diabetes mellitus accompanied by severe reactive hypoglycemia at the age of 11 yr. An OGTT (oral glucose tolerance test) revealed hyperglycemia due to poor early insulin response and subsequent hypoglycemia due to delayed prolonged insulin secretion. Hypoglycemia was improved by the combination of nateglinide, which stimulates early insulin secretion, and an alpha-glucosidase inhibitor, voglibose. Sequencing of the ABCC8 identified a compound heterozygous mutation (R1420H/F591fs604X), suggesting that this mutation may alter regulation of insulin secretion with advancing age, leading to diabetes mellitus with reactive hypoglycemia from hyperinsulinism. Therefore, long-term follow-up and periodic OGTTs are important for early detection of insulin dysregulation in congenital hyperinsulinism patients carrying the ABCC8 mutation, even though hypoglycemia resolves spontaneously during infancy. Furthermore, nateglinide may be useful therapeutically in the treatment of not only diabetes mellitus but also reactive hypoglycemia.

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79 (NBF2) of SUR1, and homozygous missense mutation R1420C is reported in Japanese CHI siblings(22).Althoughtheywerebornmacrosomic and developed hypoglycemia soon after birth, hypoglycemia resolve spontaneously after 3 mo ofage.FunctionalanalysisofthemutantR1420C SUR1 showed lower expression of the mutant channelandreducedaffinityofNFB2forMgADP that might leadtotheenhancedinsulinsecretion (23). Login to comment

[hide] Molecular mechanisms of congenital hyperinsulinism... J Mol Endocrinol. 2015 Apr;54(2):R119-29. doi: 10.1530/JME-15-0016. Epub 2015 Mar 2. Rahman SA, Nessa A, Hussain K
Molecular mechanisms of congenital hyperinsulinism.
J Mol Endocrinol. 2015 Apr;54(2):R119-29. doi: 10.1530/JME-15-0016. Epub 2015 Mar 2., [PMID:25733449]

Abstract [show]
Congenital hyperinsulinism (CHI) is a complex heterogeneous condition in which insulin secretion from pancreatic beta-cells is unregulated and inappropriate for the level of blood glucose. The inappropriate insulin secretion drives glucose into the insulin-sensitive tissues, such as the muscle, liver and adipose tissue, leading to severe hyperinsulinaemic hypoglycaemia (HH). At a molecular level, genetic abnormalities in nine different genes (ABCC8, KCNJ11, GLUD1, GCK, HNF4A, HNF1A, SLC16A1, UCP2 and HADH) have been identified which cause CHI. Autosomal recessive and dominant mutations in ABCC8/KCNJ11 are the commonest cause of medically unresponsive CHI. Mutations in GLUD1 and HADH lead to leucine-induced HH, and these two genes encode the key enzymes glutamate dehydrogenase and short chain 3-hydroxyacyl-CoA dehydrogenase which play a key role in amino acid and fatty acid regulation of insulin secretion respectively. Genetic abnormalities in HNF4A and HNF1A lead to a dual phenotype of HH in the newborn period and maturity onset-diabetes later in life. This state of the art review provides an update on the molecular basis of CHI.

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64 The R1420C mutation is located in SUR1-NBD2, and results of mutational studies have indicated that it reduces the affinity of NBD2 for ATP and ADP (Matsuo et al. 2000). Login to comment

[hide] ABCC8 R1420H Loss-of-Function Variant in a Southwe... Diabetes. 2015 Dec;64(12):4322-32. doi: 10.2337/db15-0459. Epub 2015 Aug 5. Baier LJ, Muller YL, Remedi MS, Traurig M, Piaggi P, Wiessner G, Huang K, Stacy A, Kobes S, Krakoff J, Bennett PH, Nelson RG, Knowler WC, Hanson RL, Nichols CG, Bogardus C
ABCC8 R1420H Loss-of-Function Variant in a Southwest American Indian Community: Association With Increased Birth Weight and Doubled Risk of Type 2 Diabetes.
Diabetes. 2015 Dec;64(12):4322-32. doi: 10.2337/db15-0459. Epub 2015 Aug 5., [PMID:26246406]

Abstract [show]
Missense variants in KCNJ11 and ABCC8, which encode the KIR6.2 and SUR1 subunits of the beta-cell KATP channel, have previously been implicated in type 2 diabetes, neonatal diabetes, and hyperinsulinemic hypoglycemia of infancy (HHI). To determine whether variation in these genes affects risk for type 2 diabetes or increased birth weight as a consequence of fetal hyperinsulinemia in Pima Indians, missense and common noncoding variants were analyzed in individuals living in the Gila River Indian Community. A R1420H variant in SUR1 (ABCC8) was identified in 3.3% of the population (N = 7,710). R1420H carriers had higher mean birth weights and a twofold increased risk for type 2 diabetes with a 7-year earlier onset age despite being leaner than noncarriers. One individual homozygous for R1420H was identified; retrospective review of his medical records was consistent with HHI and a diagnosis of diabetes at age 3.5 years. In vitro studies showed that the R1420H substitution decreases KATP channel activity. Identification of this loss-of-function variant in ABCC8 with a carrier frequency of 3.3% affects clinical care as homozygous inheritance and potential HHI will occur in 1/3,600 births in this American Indian population.

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62 Functional Analysis of the ABCC8 R1420H and R1420C Missense Mutations Site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) of hamster ABCC8 cDNA cloned into pCMV6B was used to generate the R1420H and R1420C missense mutations. Login to comment
104 In addition to R1420H, a different substitution at the same amino acid (R1420C, rs28938469), which has previously been shown to decrease KATP channel (32,33), was also Table 1-Subjects from the Gila River Indian Community genotyped and analyzed for all missense variants N Males/females Age (years) Adult BMI (kg/m2 ) or birth weight (g) Type 2 diabetes With type 2 diabetes 2,549 982/1,567 46.5 6 14.6 38.7 6 8.6 Full-heritage Pima Indian 1,684 623/1,061 49.1 6 14.0 38.7 6 8.6 Non-full-heritage Pima Indian 865 359/506 41.2 6 14.1 38.7 6 8.7 Without type 2 diabetes 5,161 2,444/2,717 27.6 6 13.5 34.6 6 8.6 Full-heritage Pima Indian 1,941 930/1,011 32.1 6 14.6 36.0 6 8.5 Non-full-heritage Pima Indian 3,220 1,514/1,706 24.9 6 11.9 33.5 6 8.4 BMI Full-heritage Pima Indian 2,862 1,239/1,623 32.2 6 11.8 36.3 6 8.3 Non-full-heritage Pima Indian 3,056 1,367/1,689 27.3 6 10.5 34.0 6 8.4 Birth weight Full-heritage Pima Indian 988 439/549 - 3,468 6 523 Non-full-heritage Pima Indian 1,389 619/770 - 3,483 6 521 Metabolic studies (full-heritage Pima Indian) 298 207/119 26.3 6 6.2 33.4 6 7.4 Data are shown as n or mean 6 SD. Login to comment
107 However, when KATP-specific fluxes were activated by metabolic inhibition or diazoxide administration, 86 Rb+ effluxes were lower for both R1420H and R1420C KATP channels (Fig. 6). Login to comment
109 These data indicate that the R1420H amino acid change acts in a similar manner to the previously characterized R1420C substitution (32,33) to reduce KATP channel activity in intact cells, predicting that in vivo insulin hypersecretion will result in individuals carrying the R1420H variant. Login to comment
172 R1420H is positioned in the second nucleotide binding fold (NBF2) of the SUR1 protein where other amino acid substitutions have been identified including R1420C, which was previously shown to be associated with a sporadic case of persistent HHI (15,63) (note: R1420C is referred to as R1421C in [63]). Login to comment
173 In our study, a similar decrease in KATP channel activity was also seen for R1420C, which is consistent with earlier reports showing diminished activity for this variant (32,33). Login to comment
174 R1420C may reduce KATP channel activity by lowering NBF2 binding affinities for ATP and ADP and impairing the cooperative ATP binding between NBF2 and NBF1 (32,33). Login to comment
177 In addition to the small sample size, these data may be difficult to interpret because this Figure 6-Decreased KATP activity for SUR1 (ABCC8) channels harboring R1420H or R1420C mutations. Login to comment
178 Relative 86 Rb+ efflux is shown as a function of time under basal conditions, in the presence of metabolic inhibition (MI), in the presence of KATP channel opener diazoxide, and for both control cells expressing green fluorescent protein and cells expressing R1420R, R1420H, or R1420C channels. Login to comment