ABCMdb

ABCC8 p.Arg1420Cys

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Publications

PMID: 11226335 [PubMed] Cartier EA et al: "Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy."

No. Sentence Comment

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

PMID: 15910875 [PubMed] Matsuo M et al: "KATP channel interaction with adenine nucleotides."

No. Sentence Comment

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

PMID: 10993895 [PubMed] Matsuo M et al: "Functional analysis of a mutant sulfonylurea receptor, SUR1-R1420C, that is responsible for persistent hyperinsulinemic hypoglycemia of infancy."

No. Sentence Comment

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

PMID: 12565699 [PubMed] Seino S et al: "Physiological and pathophysiological roles of ATP-sensitive K+ channels."

No. Sentence Comment

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

PMID: 12110524 [PubMed] Huopio H et al: "K(ATP) channels and insulin secretion disorders."

No. Sentence Comment

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

PMID: 15807877 [PubMed] Ohkubo K et al: "Genotypes of the pancreatic beta-cell K-ATP channel and clinical phenotypes of Japanese patients with persistent hyperinsulinaemic hypoglycaemia of infancy."

No. Sentence Comment

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

PMID: 10615958 [PubMed] Tanizawa Y et al: "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."

No. Sentence Comment

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

PMID: 14593442 [PubMed] Campbell JD et al: "Potassium channel regulation."

No. Sentence Comment

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

PMID: 19254908 [PubMed] James C et al: "The genetic basis of congenital hyperinsulinism."

No. Sentence Comment

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

PMID: 10893240 [PubMed] Matsuo M et al: "Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B."

No. Sentence Comment

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

PMID: 20460728 [PubMed] Matsuo M et al: "ATP-binding cassette proteins involved in glucose and lipid homeostasis."

No. Sentence Comment

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

PMID: 23926410 [PubMed] Saito-Hakoda A et al: "Nateglinide is Effective for Diabetes Mellitus with Reactive Hypoglycemia in a Child with a Compound Heterozygous ABCC8 Mutation."

No. Sentence Comment

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

PMID: 25733449 [PubMed] Rahman SA et al: "Molecular mechanisms of congenital hyperinsulinism."

No. Sentence Comment

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

PMID: 26246406 [PubMed] Baier LJ et al: "ABCC8 R1420H Loss-of-Function Variant in a Southwest American Indian Community: Association With Increased Birth Weight and Doubled Risk of Type 2 Diabetes."

No. Sentence Comment

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