ABCMdb

ABCC8 p.Val187Asp

Predicted by SNAP2:	A: N (78%), C: N (87%), D: D (63%), E: D (53%), F: N (82%), G: N (87%), H: N (53%), I: N (97%), K: D (59%), L: N (93%), M: N (82%), N: N (57%), P: D (53%), Q: N (53%), R: N (53%), S: N (82%), T: N (78%), W: N (61%), Y: N (61%),
Predicted by PROVEAN:	A: N, C: N, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: N, W: D, Y: N,

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[hide] Ubiquitin-mediated proteasomal degradation of ABC ... J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12. Nakagawa H, Toyoda Y, Wakabayashi-Nakao K, Tamaki H, Osumi M, Ishikawa T
Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts.
J Pharm Sci. 2011 Sep;100(9):3602-19. doi: 10.1002/jps.22615. Epub 2011 May 12., [PMID:21567408]

Abstract [show]
The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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155 Effect of Mutations and Nonsynonymous SNPs on Protein Trafficking, Maturation, or ERAD of ABC Transporters Protein AA Mutation/SNP Effect on Protein Reference ABCA1 W590S Mutation Functional defect 115 R587W Mutation Impaired glycol processing 115 Q597R Mutation Impaired glycol processing, ERAD 115,116 Y1532C Mutation Altered protein trafficking 117 R1925Q Mutation Altered protein trafficking 118 ABCA3 R43L Mutation Altered protein trafficking 119 L101P Mutation Altered protein trafficking 119 R280C Mutation Altered protein trafficking 119 ABCA4 L541P Mutation Mislocalization 120 R602W Mutation Mislocalization 120 A1038V Mutation Mislocalization 120 C1490Y Mutation Mislocalization 120 ABCB1a G268V Mutation ERAD 121 G341C Mutation ERAD 121 I1196S Mutation Reduced glycosylation 122 ABCB4 I541F Mutation Accumulation in ER 123 ABCB11a E135K Mutation Reduced level of mature protein 124 L198P Mutation Reduced level of mature protein 124 E297G Mutation Reduced level of mature protein 124 L413W Mutation Reduced level of mature protein 124 R432T Mutation Reduced level of mature protein 124 D482G Mutation Immature protein in ER 124,125 N490D Mutation Reduced level of mature protein 124 A570T Mutation Reduced level of mature protein 124 T655I Mutation Reduced level of mature protein 124 Y818F SNP Moderate reduction of protein 124 G982R Mutation Retention in ER 125 R1153C Mutation ERAD 125 R1286Q Mutation Retention in ER 125 ABCC2a R768W Mutation Impaired protein trafficking 126 I1173F Mutation Impaired protein maturation 127 R1392 Mutation Impaired protein maturation 128 M1393 Mutation Impaired protein maturation 129 ABCC4a E757K SNP Altered protein trafficking 23 ABCC7 F508 Mutation Misfolding, ERAD 36-39,130 G85E Mutation Impaired protein maturation 130-132 G91R Mutation Impaired protein maturation 130-132 N1303K Mutation Impaired protein maturation 130-132 ABCC8 WT Wild type Ubiquitin-proteasome degradation 133 A116P Mutation Ubiquitin-proteasome degradation 133 V187D Mutation Ubiquitin-proteasome degradation 133 F1388 Mutation Impaired protein trafficking 134 L1544P Mutation Impaired protein trafficking 135,136 ABCC11a G180R SNP Ubiquitin-proteasome degradation 50 27 Mutation Ubiquitin-proteasome degradation 50 ABCG2a V12M SNP Altered protein localization 96 Q141K SNP Ubiquitin-proteasome degradation 102 F208S SNP Ubiquitin-proteasome degradation 78,99 S441N SNP Ubiquitin-proteasome degradation 78,99 Mutations of ABCA1, ABCA3, ABCA4, ABCB4, ABCB11, ABCC2, ABCC7 (CFTR), and ABCC8 are associated with Tangier disease, fatal surfactant deficiency, Stargardt disease, progressive familial intrahepatic cholestasis type 3 (PFIC-3), progressive familial intrahepatic cholestasis type 2 (PFIC-2), Dubin-Johnson syndrome, cystic fibrosis, and familial hyperinsulinism, respectively. Login to comment

[hide] Role of ubiquitin-proteasome degradation pathway i... Am J Physiol Cell Physiol. 2005 Nov;289(5):C1351-9. Epub 2005 Jun 29. Yan FF, Lin CW, Cartier EA, Shyng SL
Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels.
Am J Physiol Cell Physiol. 2005 Nov;289(5):C1351-9. Epub 2005 Jun 29., [PMID:15987767]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.

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228 Three SUR1 mutations, A116P, V187D, and ⌬F1388, which we have previously shown to result in ER retention and surface expression defects of KATP channels, were tested. Login to comment
231 To further test this hypothesis, we examined the combined effect of glibenclamide and MG132 on surface expression of the A116P and V187D mutants. Login to comment
232 Sulfonylureas such as glibenclamide have previously been shown to significantly increase surface expression of the A116P and V187D mutants, presumably by acting as pharmacological chaperones to help mutant SUR1 fold more efficiently (42). Login to comment
233 We found that pretreatment of COS cells expressing the A116P or the V187D mutant with 5 ␮M glibenclamide led to a significant increase in mutant channel surface expression (P Ͻ 0.01) on subsequent exposure to the proteasome inhibitor MG132 (Fig. 7B). Login to comment
245 A: COSm6 cells transiently coexpressing Kir6.2 and wild-type (WT) fSUR1 or fSUR1 bearing the A116P, V187D, or ⌬F1388 mutation were treated with or without 10 ␮M MG132 for 6 h and processed for chemiluminescence assays to quantify channel expression level at the cell surface. Login to comment
248 B: cells expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated for 24 h with (Glib) or without (Control) 5 ␮M glibenclamide. Login to comment
260 Among them, ⌬F1388 has been proposed to cause severe folding defects, whereas A116P and V187D appear to have milder defects that can be partially overcome by sulfonylurea treatment. Login to comment
262 However, after pretreatment with glibenclamide, the A116P and V187D mutant channels responded to proteasome inhibitors with a statistically significant increase in surface expression (Fig. 7B). Login to comment
229 Three SUR1 mutations, A116P, V187D, and èc;F1388, which we have previously shown to result in ER retention and surface expression defects of KATP channels, were tested. Login to comment
234 We found that pretreatment of COS cells expressing the A116P or the V187D mutant with 5 òe;M glibenclamide led to a significant increase in mutant channel surface expression (P b0d; 0.01) on subsequent exposure to the proteasome inhibitor MG132 (Fig. 7B). Login to comment
246 A: COSm6 cells transiently coexpressing Kir6.2 and wild-type (WT) fSUR1 or fSUR1 bearing the A116P, V187D, or èc;F1388 mutation were treated with or without 10 òe;M MG132 for 6 h and processed for chemiluminescence assays to quantify channel expression level at the cell surface. Login to comment
249 B: cells expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated for 24 h with (Glib) or without (Control) 5 òe;M glibenclamide. Login to comment
264 However, after pretreatment with glibenclamide, the A116P and V187D mutant channels responded to proteasome inhibitors with a statistically significant increase in surface expression (Fig. 7B). Login to comment

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

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

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107 These mutations are all in the TMD0 of SUR1 (amino acids 1-196) and include G7R, N24K, F27S, R74W, A116P, E128K, and V187D. Login to comment
108 The functional properties of rescued A116P and V187D mutant channels had been characterized in detail and shown to be normal (13). Login to comment
272 If R74W and E128K cause functional uncoupling between TMD0-SUR1 and Kir6.2, one might ask if the mutations also result in reduced physical association between the two subunits. Login to comment
273 Several SUR1-TMD0 mutations have been reported to reduce physical association between TMD0 and Kir6.2 in co-immunoprecipitation experiments, including CHI-causing A116P and V187D mutations and PNDM-causing F132L mutation (10, 30). Login to comment

[hide] Genetics of congenital hyperinsulinism. Endocr Pathol. 2004 Fall;15(3):233-40. Fournet JC, Junien C
Genetics of congenital hyperinsulinism.
Endocr Pathol. 2004 Fall;15(3):233-40., [PMID:15640549]

Abstract [show]
Congenital hyperinsulinism (CHI) is a clinically and genetically heterogeneous entity and causes severe hypoglycemia in neonates and infants. The clinical heterogeneity is manifested by severity ranging from extremely severe, life-threatening disease to very mild clinical symptoms, which may even be difficult to identify. Furthermore, clinical responsiveness to medical and surgical management is extremely variable. Recent discoveries have begun to clarify the molecular etiology of this disease in about 50% of cases. Mutations in five different genes have been identified in patients with this clinical syndrome. Most cases are caused by mutations in the genes ABCC8 and KCNJ11 coding for either of the two subunits of the beta-cell KATP channel (SUR1 and Kir6.2). Recessive mutations of the beta-cell K(ATP) channel genes cause diffuse HI, whereas loss of heterozygosity together with inheritance of a paternal mutation causes focal adenomatous HI. In other cases, CHI is caused by mutations in genes coding for the beta-cell enzymes glucokinase (GK), glutamate dehydrogenase (GDH), and SCHAD. However, for as many as 50% of the cases, no genetic etiology has yet been determined. The study of the genetics of this disease has provided important new information regarding beta-cell physiology.

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73 Morerecently,afoundermutation(V187D) was found in Finnish patients with HI (29%) [12]. Login to comment

[hide] Noninvasive diagnosis of focal hyperinsulinism of ... Diabetes. 2006 Jan;55(1):13-8. Otonkoski T, Nanto-Salonen K, Seppanen M, Veijola R, Huopio H, Hussain K, Tapanainen P, Eskola O, Parkkola R, Ekstrom K, Guiot Y, Rahier J, Laakso M, Rintala R, Nuutila P, Minn H
Noninvasive diagnosis of focal hyperinsulinism of infancy with [18F]-DOPA positron emission tomography.
Diabetes. 2006 Jan;55(1):13-8., [PMID:16380471]

Abstract [show]
Congenital hyperinsulinism of infancy (CHI) is characterized by severe hypoglycemia due to dysregulated insulin secretion, associated with either focal or diffuse pathology of the endocrine pancreas. The focal condition is caused by a paternally inherited mutation in one of the genes encoding the subunits of the beta-cell ATP-sensitive potassium channel (SUR1/ABCC8 or Kir6.2/KCNJ11) and somatic loss of maternal 11p15 alleles within the affected area. Until now, preoperative diagnostics have relied on technically demanding and invasive catheterization techniques. We evaluated the utility of fluorine-18 l-3,4-dihydroxyphenylalanine ([(18)F]-DOPA) positron emission tomography (PET) to identify focal pancreatic lesions in 14 CHI patients, 11 of which carried mutations in the ABCC8 gene (age 1-42 months). To reduce bias in PET image interpretation, quantitative means for evaluation of pancreatic [(18)F]-DOPA uptake were established. Five patients had a visually apparent focal accumulation of [(18)F]-DOPA and standardized uptake value (SUV) >50% higher (mean 1.8-fold) than the maximum SUV of the unaffected part of the pancreas. When these patients were operated on, a focus of 4-5 x 5-8 mm matching with the PET scan was found, and all were normoglycemic after resection of the focus. The remaining nine patients had diffuse accumulation of [(18)F]-DOPA in the pancreas (SUV ratio <1.5). Diffuse histology was verified in four of these, and pancreatic catheterization was consistent with diffuse pathology in four cases. In conclusion, [(18)F]-DOPA PET is a promising noninvasive method for the identification and localization of the focal form of CHI.

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83 The two previously detected founder mutations SUR1-V187D (5) and SUR1-E1506K (24) were screened by direct sequencing in all Finnish patients. Login to comment
104 Genetic analysis showed that five of the patients were paternal heterozygotes for the Finnish major founder ABCC8 mutation V187D (Table 1). Login to comment
118 Representative illustrations of a patient with focal CHI who carries the paternally inherited SUR1-V187D mutation (patient 1, Table 1). Login to comment
100 In one of these (case no. 7), a pancreatic biopsy with diffuse-type pathol- TABLE 1 Clinical and genetic characteristics of the patients, together with the findings in PET scan, pancreatic catheterization, surgery, and histology Patient Age at diagnosis/PET ABCC8 mutation Response to medication Glucose need (mg/kg/min) PET PVS/PACS Surgery/histology 1 Neonatal/6 months V187D (561Tb0e;A) (Paternal) Dzxafa;, Octr af9; 12.3 focal/head focal/head focal resection/posterior neck PAD: focus 6.8 afb; 4 mm 2 4 months/13 months V187D (561Tb0e;A) (Paternal) Dzxafa;, Octr af9; 9.5 focal/body focal/body focal resection/body PAD: focus 5 afb; 4 mm 3 Neonatal/6 months G1469V (4408Gb0e;T) (Paternal) Dzxafa;, Octr af9; 12.7 focal/head focal/head focal resection/head PAD: focus 8 afb; 5 mm 4 Neonatal/3.5 years V187D (561Tb0e;A) (Paternal) Dzxafa;, Octr af9; 12.7 diffuse diffuse* ND 5 Neonatal/6 months A113V (338Cb0e;T) (Paternal) Dzxafa;, Octr af9; 12.7 diffuse diffuse Near-total pancreatectomy PAD: diffuse histology 6 Neonatal/5 years No mutations Dzx af9; 6.5 diffuse diffuse* ND 7 3 months/4 years No mutations Dzx af9; 6.5 diffuse ND Pancreas biopsy PAD: diffuse histology 8 Neonatal/9 months G92D (275Gb0e;A) Dzxafa;, Octr af9; 10.3 diffuse ND Near-total pancreatectomy PAD: diffuse histology 9 Neonatal/1 month V187D (561Tb0e;A) (Paternal) Dzxafa;, Octrafa; 20 focal/head ND focal resection/uncinate process PAD: focus 8 afb; 4 mm 10 Neonatal/2 months No mutations Dzx partial, Octr af9; 6.2 diffuse ND ND 11 5 months/13 months V187D (561Tb0e;A) (Paternal) Dzxafa;, Octr af9; 6.4 diffuse diffuse ND 12 Neonatal/1.5 months G474A (de novo) Dzxafa;, Octr af9; 15.9 diffuse ND Near-total pancreatectomy PAD diffuse; 13 Neonatal/3 months C418R (1252Tb0e;C) (Maternal) Dzxafa;, Octr af9; 26 focal/body ND focal resection/body PAD: focus 10 afb; 6 mm 14 Neonatal/6 months A113V (338Cb0e;T) (Paternal) Dzxafa;, Octr af9; 13 diffuse ND ND The glucose need refers to the glucose infusion rate that was required to maintain normoglycemia at the time of the PET scan. Login to comment
105 Genetic analysis showed that five of the patients were paternal heterozygotes for the Finnish major founder ABCC8 mutation V187D (Table 1). Login to comment
119 Representative illustrations of a patient with focal CHI who carries the paternally inherited SUR1-V187D mutation (patient 1, Table 1). Login to comment

[hide] Congenital hyperinsulinism associated ABCC8 mutati... Diabetes. 2007 Sep;56(9):2339-48. Epub 2007 Jun 15. Yan FF, Lin YW, MacMullen C, Ganguly A, Stanley CA, Shyng SL
Congenital hyperinsulinism associated ABCC8 mutations that cause defective trafficking of ATP-sensitive K+ channels: identification and rescue.
Diabetes. 2007 Sep;56(9):2339-48. Epub 2007 Jun 15., [PMID:17575084]

Abstract [show]
Congenital hyperinsulinism (CHI) is a disease characterized by persistent insulin secretion despite severe hypoglycemia. Mutations in the pancreatic ATP-sensitive K(+) (K(ATP)) channel proteins sulfonylurea receptor 1 (SUR1) and Kir6.2, encoded by ABCC8 and KCNJ11, respectively, is the most common cause of the disease. Many mutations in SUR1 render the channel unable to traffic to the cell surface, thereby reducing channel function. Previous studies have shown that for some SUR1 trafficking mutants, the defects could be corrected by treating cells with sulfonylureas or diazoxide. The purpose of this study is to identify additional mutations that cause channel biogenesis/trafficking defects and those that are amenable to rescue by pharmacological chaperones. Fifteen previously uncharacterized CHI-associated missense SUR1 mutations were examined for their biogenesis/trafficking defects and responses to pharmacological chaperones, using a combination of immunological and functional assays. Twelve of the 15 mutations analyzed cause reduction in cell surface expression of K(ATP) channels by >50%. Sulfonylureas rescued a subset of the trafficking mutants. By contrast, diazoxide failed to rescue any of the mutants. Strikingly, the mutations rescued by sulfonylureas are all located in the first transmembrane domain of SUR1, designated as TMD0. All TMD0 mutants rescued to the cell surface by the sulfonylurea tolbutamide could be subsequently activated by metabolic inhibition on tolbutamide removal. Our study identifies a group of CHI-causing SUR1 mutations for which the resulting K(ATP) channel trafficking and expression defects may be corrected pharmacologically to restore channel function.

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129 We first examined the effects of sulfonylureas, which were shown previously to improve surface expression of the A116P- and V187D-SUR1 mutants (16), on the trafficking mutants identified in this study (those that had surface expression Ͻ50% of wild type based on chemiluminescence assays shown in Fig. 3A). Login to comment
178 The first two trafficking mutations that we reported to be rescued by sulfonylurea drugs are A116P and V187D, both located in TMD0 of SUR1 (16). Login to comment
184 First, a truncated SUR1 of TMD0 alone containing the A116P or V187D trafficking mutations failed to respond to sulfonylurea rescue. Login to comment
177 The first two trafficking mutations that we reported to be rescued by sulfonylurea drugs are A116P and V187D, both located in TMD0 of SUR1 (16). Login to comment
183 First, a truncated SUR1 of TMD0 alone containing the A116P or V187D trafficking mutations failed to respond to sulfonylurea rescue. Login to comment

[hide] Sulfonylurea receptor 1 and Kir6.2 expression in t... Diabetes. 2000 Jun;49(6):953-60. Macfarlane WM, O'Brien RE, Barnes PD, Shepherd RM, Cosgrove KE, Lindley KJ, Aynsley-Green A, James RF, Docherty K, Dunne MJ
Sulfonylurea receptor 1 and Kir6.2 expression in the novel human insulin-secreting cell line NES2Y.
Diabetes. 2000 Jun;49(6):953-60., [PMID:10866047]

Abstract [show]
NES2Y is a proliferating human insulin-secreting cell line that we have derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy. This disease is characterized by unregulated insulin release despite profound hypoglycemia. NES2Y cells, like beta-cells isolated from the patient of origin, lack functional ATP-sensitive potassium channels (KATP) and also carry a defect in the insulin gene-regulatory transcription factor PDX1. Here, we report that the NES2Y beta-cells that are transfected with the genes encoding the components of KATP channels in beta-cells, sulfonylurea receptor (SUR) 1 and Kir6.2, have operational KATP channels and show normal intracellular Ca2+ and secretory responses to glucose. However, these cells, designated NESK beta-cells, have impaired insulin gene transcription responses to glucose. NES2Y beta-cells that are transfected with either Kir6.2 or SUR1 alone do not express functional KATP channels and have impaired intracellular free Ca2+ concentration-signaling responses to depolarization-dependent beta-cell agonists. These findings document that in NES2Y beta-cells, coexpression of both subunits is critically required for fully operational KATP channels and KATP channel-dependent signaling events. This article further characterizes the properties of the novel human beta-cell line, NES2Y, and documents the usefulness of these cells in diabetes-related research.

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188 Indeed, recent studies have demonstrated that even as V187D-SUR1 mutations in PHHI beta-cells led to the appearance of KATP channels in patient tissue, expression of the same mutation in Xenopus oocytes failed to reconstitute the channels (8). Login to comment
189 Indeed, recent studies have demonstrated that even as V187D-SUR1 mutations in PHHI b-cells led to the appearance of KATP channels in patient tissue, expression of the same mutation in Xenopus oocytes failed to reconstitute the channels (8). Login to comment

[hide] Function and distribution of the SUR isoforms and ... J Mol Cell Cardiol. 2005 Jul;39(1):51-60. Epub 2005 Feb 5. Shi NQ, Ye B, Makielski JC
Function and distribution of the SUR isoforms and splice variants.
J Mol Cell Cardiol. 2005 Jul;39(1):51-60. Epub 2005 Feb 5., [PMID:15978902]

Abstract [show]
Alternative splicing allows multiple mRNAs to be generated from a single gene, which in turn can be translated into a group of diverse proteins with different roles and structures. The outcome of alternative splicing leads to the co-existence of multiple splice variants of a gene at different concentrations in different tissues. The pore-forming subunit of the K(ATP) channel (K(IR)6.x) and the regulatory sulfonylurea receptor (SUR(x)) subunits exist in a 4:4 stoichiometry to form hetero-octameric ATP-sensitive potassium channel (K(ATP)) channels, which are widely distributed in various types of tissues at either the plasma membrane (cellK(ATP)) or mitochondrial inner membrane (the mitochondrial form of K(ATP) channel, mitoK(ATP)). They perform important physiological functions in regulating insulin secretion in pancreatic beta-cells, providing ischemic protection in heart and brain, and regulating vascular tone in smooth muscles. Two separate genes, the regulatory subunit protein I (SUR1) and the regulatory subunit protein II (SUR2) encode the high- and low-affinity SUR, respectively. This review summarizes the current studies on the function and distribution of the SUR isoforms and alternative splice variants, and to a lesser extent the K(IR)6.x subunits. The different isoforms and splice variants allow for many K(ATP) channel combinations, and therefore, increases the channel diversity and the possibility of complexity in function.

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69 When the RKR motif in a DF1388 mutant of SUR1 (a PHHI mutation) was mutated to AAA, the re-constituted DF1388SUR1AAA/KIR6.2 channel was somewhat active and could be expressed at the cell surface partially [22,23].A most recent study in trafficking issue of other mutant KATP channels revealed that the A116P and V187D mutants (mutations cause congenital hyperinsulinism) of SUR1 could be rescued to the cell membrane surface by sulfonylureas [24]. 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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134 The recessively inherited missense mutation V187D, located in a transmembrane domain of SUR1, leads to severe early-onset HI (46). Login to comment
136 Interestingly, the disease phenotype is almost as severe in patients homozygous or heterozygous for the mutation; even a single copy of the V187D mutation seems to lead to a severe drug-unresponsive form of HI in compound heterozygotes. Login to comment
138 Functional studies (intact cell recordings, cell-free inside-out patches) of beta-cells isolated from an HI patient homozygous for the V187D mutation, as well as the results of recombinant KATP channel experiments, are consistent with the phenotype and show that mutation SUR1[V187D] leads to a loss of functional KATP channels that are not activated by diazoxide or somatostatin. Login to comment
164 Birthplaces of parents of HI patients with founder mutations SUR1(E1506K) (E) and SUR1(V187D) (F) are indicated (8, 40). Login to comment
123 The recessively inherited missense mutation V187D, located in a transmembrane domain of SUR1, leads to severe early-onset HI (46). Login to comment
125 Interestingly, the disease phenotype is almost as severe in patients homozygous or heterozygous for the mutation; even a single copy of the V187D mutation seems to lead to a severe drug-unresponsive form of HI in compound heterozygotes. Login to comment
127 Functional studies (intact cell recordings, cell-free inside-out patches) of beta-cells isolated from an HI patient homozygous for the V187D mutation, as well as the results of recombinant KATP channel experiments, are consistent with the phenotype and show that mutation SUR1[V187D] leads to a loss of functional KATP channels that are not activated by diazoxide or somatostatin. Login to comment
153 Birthplaces of parents of HI patients with founder mutations SUR1(E1506K) (E) and SUR1(V187D) (F) are indicated (8, 40). Login to comment

[hide] Characterization of ABCC8 and KCNJ11 gene mutation... Eur J Endocrinol. 2011 Jun;164(6):919-26. Epub 2011 Mar 21. Park SE, Flanagan SE, Hussain K, Ellard S, Shin CH, Yang SW
Characterization of ABCC8 and KCNJ11 gene mutations and phenotypes in Korean patients with congenital hyperinsulinism.
Eur J Endocrinol. 2011 Jun;164(6):919-26. Epub 2011 Mar 21., [PMID:21422196]

Abstract [show]
OBJECTIVE: Congenital hyperinsulinism (CHI) is characterized by persistent hypoglycemia due to the inappropriate insulin secretion. Inactivating mutations in the ABCC8 and KCNJ11 genes, which encode the sulfonylurea receptor 1 and Kir6.2 subunits of the ATP-sensitive K(+) (K(ATP)) channel in pancreatic beta-cell, are the most common cause of CHI. We studied the genetic etiology and phenotypes of CHI in Korean patients. METHODS: ABCC8 and KCNJ11 mutational analysis was performed in 17 patients with CHI. Medical records were retrospectively reviewed to identify phenotypes. RESULTS: Mutations (12 ABCC8 and three KCNJ11) were identified in 82% (14/17) of patients. Of these, nine ABCC8 mutations (E100X, W430X, c.1630+1G>C, D813N, Q923X, E1087_A1094delinsDKSDT, Q1134H, H1135W, and E1209Rfs) and one KCNJ11 mutation (W91X) were novel. Of the 14 patients, four had confirming recessively inherited CHI. The remaining ten patients had single heterozygous mutations. The majority (12/17) of patients were medically responsive. Of the five diazoxide-responsive patients, four had an ABCC8 mutation. The five patients unresponsive to medical management and one diazoxide-responsive patient underwent pancreatectomy and had diffuse histology. Of the operated six patients, two had recessively inherited mutations; three patients had a single heterozygous mutation (one maternally and two paternally inherited); and one patient had no identifiable K(ATP) channel mutation. CONCLUSIONS: This is the first study to report genotype and phenotype correlations among Korean patients with CHI. Mutations in ABCC8 and KCNJ11 are the most common causes of CHI in Korean patients. Similar to other studies, there is marked genetic heterogeneity and no clear genotype-phenotype correlation.

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28 Founder mutations are reported in certain populations, such as certain ABCC8 gene mutations (c.3992-9GOA and F1388del) in Ashkenazi Jews and V187D and E1507K mutations among the Finnish population. Login to comment

[hide] A heterozygous activating mutation in the sulphony... Hum Mol Genet. 2006 Jun 1;15(11):1793-800. Epub 2006 Apr 13. Proks P, Arnold AL, Bruining J, Girard C, Flanagan SE, Larkin B, Colclough K, Hattersley AT, Ashcroft FM, Ellard S
A heterozygous activating mutation in the sulphonylurea receptor SUR1 (ABCC8) causes neonatal diabetes.
Hum Mol Genet. 2006 Jun 1;15(11):1793-800. Epub 2006 Apr 13., [PMID:16613899]

Abstract [show]
Neonatal diabetes is a genetically heterogeneous disorder with nine different genetic aetiologies reported to date. Heterozygous activating mutations in the KCNJ11 gene encoding Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, are the most common cause of permanent neonatal diabetes. The sulphonylurea receptor (SUR) SUR1 serves as the regulatory subunit of the K(ATP) channel in pancreatic beta cells. We therefore hypothesized that activating mutations in the ABCC8 gene, which encodes SUR1, might cause neonatal diabetes. We identified a novel heterozygous mutation, F132L, in the ABCC8 gene of a patient with severe developmental delay, epilepsy and neonatal diabetes (DEND syndrome). This mutation had arisen de novo and was not present in 150 control chromosomes. Residue F132 shows evolutionary conservation across species and is located in the first set of transmembrane helices (TMD0) of SUR1, which is proposed to interact with Kir6.2. Functional studies of recombinant K(ATP) channels demonstrated that F132L markedly reduces the sensitivity of the K(ATP) channel to inhibition by MgATP and this increases the whole-cell K(ATP) current. The functional consequence of this ABCC8 mutation mirrors that of KCNJ11 mutations causing neonatal diabetes and provides new insights into the interaction of Kir6.2 and SUR1. As SUR1 is expressed in neurones as well as in beta cells, this mutation can account for both neonatal diabetes and the neurological phenotype. Our results demonstrate that SUR1 mutations constitute a new genetic aetiology for neonatal diabetes and that they act by reducing the K(ATP) channel's ATP sensitivity.

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140 Two mutations in TMD0 (A116P and V187D), which cause congenital hyperinsulinism, abrogate the association of SUR1 and Kir6.2 and lead to loss of KATP channel function (27,35). Login to comment

[hide] Carriers of an inactivating beta-cell ATP-sensitiv... Diabetes Care. 2002 Jan;25(1):101-6. Huopio H, Vauhkonen I, Komulainen J, Niskanen L, Otonkoski T, Laakso M
Carriers of an inactivating beta-cell ATP-sensitive K(+) channel mutation have normal glucose tolerance and insulin sensitivity and appropriate insulin secretion.
Diabetes Care. 2002 Jan;25(1):101-6., [PMID:11772909]

Abstract [show]
OBJECTIVE: Insulin release from the pancreatic beta-cells is controlled by ATP-sensitive K(+) (K(ATP)) channels, which consist of a hetero-octameric complex of four sulfonylurea receptor 1 (SUR1) and four Kir6.2 proteins. Mutations in the SUR1 gene are the major cause of congenital hyperinsulinemia (CHI). Despite the hereditary nature of CHI, studies of glucose homeostasis in heterozygous relatives of CHI patients are lacking. Theoretically, in the heterozygous state of the SUR1 gene mutation, only 1 of 16 K(ATP) channels consists of entirely normal subunits. The aim of our study was to investigate in vivo the glucose homeostasis of heterozygous SUR1 mutation carriers. RESEARCH DESIGN AND METHODS: We studied 8 parents of CHI patients, all 8 of whom were heterozygous for the inactivating SUR1 mutation V187D, and 10 matched control subjects. We evaluated glucose tolerance and insulin secretory capacity with oral and intravenous glucose tests, rates of whole-body glucose uptake with hyperinsulinemic-euglycemic clamps, C-peptide response to hypoglycemia during hyperinsulinemic-hypoglycemic clamp, and function of the K(ATP) channels with intravenous tolbutamide test. RESULTS: Carriers of the V187D substitution had normal glucose tolerance, normal tissue sensitivity to insulin, and no signs of inappropriate insulin secretion. The normal insulin response to tolbutamide indicated that heterozygosity for the V187D mutation did not impair K(ATP) channel function. CONCLUSIONS: We conclude that the heterozygous carriers of the SUR1 mutation had normal glucose metabolism and insulin secretion, indicating that carriers of recessive K(ATP) channel mutations are unlikely to be at an increased risk of hypoglycemia or other disturbances in glucose metabolism.

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5 RESEARCH DESIGN AND METHODS - We studied 8 parents of CHI patients, all 8 of whom were heterozygous for the inactivating SUR1 mutation V187D, and 10 matched control subjects. Login to comment
7 RESULTS - Carriers of the V187D substitution had normal glucose tolerance, normal tissue sensitivity to insulin, and no signs of inappropriate insulin secretion. Login to comment
8 The normal insulin response to tolbutamide indicated that heterozygosity for the V187D mutation did not impair KATP channel function. Login to comment
31 We have identified a missense mutation, V187D, in the SUR1 gene that is responsible for the majority of severe CHI cases in Finland. Login to comment
35 To determine in vivo the effects of the heterozygous state of this mutation on the regulation of beta-cell secretion, whole-body glucose metabolism, and glucose homeostasis, including the counterregulatory system against hypoglycemia, we studied eight parents of CHI patients, all eight of whom are carriers of the V187D substitution. Login to comment
37 The subjects for the present study were the parents of five patients with diffuse CHI caused by the homozygous SUR1 mutation V187D. Login to comment
39 Genetic analysis confirmed that all eight parents were heterozygous carriers of the V187D mutation. Login to comment
49 Several weeks after the other tests, four carriers of the V187D mutation and six control subjects participated in the tolbutamide test. Login to comment
73 The tolbutamide test was performed in four V187D heterozygotes and six control subjects to investigate the beta-cell response to the KATP channel antagonist tolbutamide. Login to comment
75 It was thus hypothesized that the increment in insulin secretion would be lower in the V187D carriers if their KATP channels are defective. Login to comment
98 Altogether, 7 of 8 V187D carriers and 4 of 10 control subjects had suffered from hypoglycemic symptoms. Login to comment
99 OGTT All individuals in both study groups had normal glucose tolerance according to World Health Organization criteria (16), as determined by OGTT (fasting blood glucose: V187D heterozygotes 4.2-5.5 mmol/l, control subjects 2.8-6.2 mmol/l; 2-h blood glucose: V187D heterozygotes 2.8-6.2 mmol/l, control subjects 3.46.4 mmol/l). Login to comment
101 The blood glucose levels were similar in both groups (V187D heterozygotes and control subjects) at all time points measured after the oral glucose load. Login to comment
104 Moreover, the plasma insulin response, expressed as the incremental insulin area under the curve, was similar in the two groups (V187D heterozygotes 519 Ϯ 98 pmol/l ⅐ h, control subjects 553 Ϯ 89 pmol/l ⅐ h). Login to comment
106 Similarly, the incremental C-peptide area under the curve was comparable between the study groups (V187D hetrozygotes 3,125 Ϯ Figure 1-Blood glucose (A), plasma insulin (B), and plasma C-peptide (C) concentrations during the OGTT. Login to comment
107 E, Control subjects; f, SUR1 V187D heterozygotes. Login to comment
108 Table 1-Clinical and biochemical characteristics of the study groups Control subjects V187D carriers P n 10 8 Age (years) 35.6 Ϯ 1.1 35.3 Ϯ 0.9 NS Sex (M/F) 5/5 4/4 NS BMI (kg/m2 ) 23.2 Ϯ 0.6 24.0 Ϯ 1.2 NS Fasting blood glucose (mmol/l) 4.3 Ϯ 0.2 4.7 Ϯ 0.2 NS Fasting insulin (pmol/l) 49.2 Ϯ 6.6 42.0 Ϯ 5.4 NS Fasting C-peptide (pmol/l) 530 Ϯ 35 450 Ϯ 51 NS Systolic blood pressure (mmHg) 128 Ϯ 4 129 Ϯ 3 NS Diastolic blood pressure (mmHg) 84 Ϯ 2 76 Ϯ 2 NS HbA1c (%) 5.4 Ϯ 0.1 5.4 Ϯ 0.1 NS Fasting hepatic insulin extraction (pmol C-peptide/pmol P-insulin) 10.7 Ϯ 0.3 10.7 Ϯ 0.4 NS Data are means Ϯ SEM or n. Login to comment
113 Furthermore, the incremental glucose area under the curve was similar in both study groups (71.1 Ϯ 3.9 and 70.2 Ϯ 2.7 mmol/l ⅐ min for the V187D heterozygote group and control subjects, respectively). Login to comment
117 Finally, neither the counterregulatory hormone responses in normoglycemia (serum glucagon 84.3 Ϯ 5.5 vs. 81.0 Ϯ 9.9 pmol/l, serum epinephrine 0.14 Ϯ 0.03 vs. 0.24 Ϯ 0.05 nmol/l, serum norepinephrine 1.5 Ϯ 0.2 vs. 1.8 Ϯ 0.3 nmol/l, serum cortisol 213.3 Ϯ 37.4 vs. 253.8 Ϯ 30.7 nmol/l, and serum growth hormone 0.88 Ϯ 0.13 vs. 0.46 Ϯ 0.15 ␮g/l in the V187D heterozygote group and in the control group, respectively) and in hypoglycemia (serum glucagon 95.1 Ϯ 4.2 vs. 120.7 Ϯ 18.9 pmol/l, serum epinephrine 1.3 Ϯ 0.4 vs. 1.6 Ϯ 0.3 nmol/l, serum norepinephrine 1.9 Ϯ 0.3 vs. 2.1 Ϯ 0.3 nmol/l, serum cortisol 355.9 Ϯ 85.4 vs. 493.4 Ϯ 59.3 nmol/l, and serum growth hormone 11.4 Ϯ 2.9 vs. 14.7 Ϯ 3.9 ␮g/l in the V187D heterozygote group and control subjects, respectively) nor the symptoms of hypoglycemia evaluated during the hypoglycemic clamp differed significantly between the groups. Login to comment
118 Tolbutamide test Figure 4 shows that plasma insulin and C-peptide responses to the tolbutamide injection were similar in V187D heterozygotes and control subjects when expressed as the difference between the hormone levels measured at 0 and 3 min after the tolbutamide bolus. Login to comment
119 The incremental areas under the curve (0-10 min) did not differ, either (P-insulin 1,744 Ϯ 338 vs. 2,226 Ϯ 491 pmol/l ⅐ min and C-peptide 5,950 Ϯ 1,266 vs. 6,607 Ϯ 870 pmol/l ⅐ min for V187D carriers and control subjects, respectively). Login to comment
124 In this study, we investigated in detail the glucose metabolism of such individuals, who carried the previously described SUR1 loss of function mutation V187D (9). Login to comment
131 E, Control subjects; f, SUR1 V187D heterozygotes. Login to comment
133 E And Ⅺ, control subjects; f, SUR1 V187D heterozygotes. Login to comment
136 Our present findings, demonstrating normal insulin and C-peptide responses after tolbutamide injection, indicate that normal KATP channel function is maintained in the beta-cells of the SUR1 V187D heterozygotes. Login to comment
144 In our study, only one of the mothers of V187D homozygous patients had transiently elevated blood glucose levels during pregnancy. Login to comment
145 All V187D mutation carriers had normal glucose tolerance and normal rates of whole-body glucose uptake. Login to comment
146 In addition, first-phase insulin secretion, which is known to be impaired in individuals at high risk for type 1 (24) and type 2 (25,26) diabetes, was not impaired in subjects with the V187D substitution. Login to comment
147 Our results indicate that the carriers of the V187D substitution do not have any features of type 2 diabetes. Login to comment
150 In our study, 7 of the 8 V187D carriers but only 4 of the 10 control individuals had suffered from such symptoms. Login to comment
151 Therefore, we determined whether insulin levels of the V187D carriers were higher after overnight fasting and during hypoglycemia. Login to comment
160 In the present study, carriers of the V187D substitution had quite normal counterregulatory system function. Login to comment
163 Some mutations impair the function of KATP channels only slightly, whereas the V187D mutation leads to total inactivation of pancreatic beta-cell KATP channels. Login to comment

[hide] Low temperature completely rescues the function of... FEBS Lett. 2005 Aug 1;579(19):4113-8. Yang K, Fang K, Fromondi L, Chan KW
Low temperature completely rescues the function of two misfolded K ATP channel disease-mutants.
FEBS Lett. 2005 Aug 1;579(19):4113-8., [PMID:16023110]

Abstract [show]
The pancreatic ATP-sensitive potassium channels comprise two subunits: SUR1 and Kir6.2. Two SUR1 mutations, A116P and V187D, reduce channel activity causing persistent hyperinsulinemic hypoglycemia of infancy. We investigated whether these mutations cause temperature sensitive misfolding. We show that the processing defect of these mutants is temperature sensitive and these two mutations disrupt the association between SUR1 and Kir6.2 by causing misfolding in SUR1 at 37 degrees C but can be rescued at 18 degrees C. Extensive electrophysiological characterization of these mutants indicated that low temperature largely, if not completely, corrects the folding defect of these two SUR1 mutants observed at 37 degrees C.

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1 Two SUR1 mutations, A116P and V187D, reduce channel activity causing persistent hyperinsulinemic hypoglycemia of infancy. Login to comment
17 Two SUR1 mutations, A116P and V187D, have been reported to cause PHHI (Fig. 1) [7,8]. Login to comment
60 Maturation of A116P and V187D SUR1 is temperature sensitive and requires Kir6.2 We tested whether the maturation of A116P and V187D SUR1 was temperature sensitive by co-expressing them with Kir6.2 at three different temperatures. Login to comment
65 Only the lower band was found for either A116P or V187D, indicating that they were retained in the ER. Login to comment
67 At 30 °C, both mutants could form the mature upper band but the percentage of the upper band normalized to that of the WT was only 33% for A116P and 86% for V187D (Fig. 2A and B). Login to comment
68 Interestingly, at 18 °C, similar proportions of WT and mutants matured to form the upper bands (relative to WT, 96% for A116P and 99% for V187D). Login to comment
70 The reduced rescue in the processing of A116P compared to V187D at Fig. 1. Login to comment
73 SUR1 is made up of TMD0 and the core domain connected together through the cytoplasmic linker L0. A116P and V187D are two mutations that cause PHHI. Login to comment
76 Processing and misfolding of A116P and V187D mutants are temperature sensitive. Login to comment
81 However, the upper band was not detected for A116P-AAA and V187D-AAA. Login to comment
86 When expressed at 37 °C in the absence of Kir6.2, WT SUR1 but neither A116P nor V187D have been shown to be mature glycosylated when their RKR motifs were mutated to AAA (three alanines) [9]. Login to comment
87 We found that even at 18 °C, A116P and V187D SUR1-AAA could not mature to form the upper band (Fig. 2B) in the absence of Kir6.2. Login to comment
90 A116P and V187D disrupt heteromeric subunits interaction at 37 °C, but not at 18 °C, by causing misfolding One explanation for the temperature sensitive processing of these mutants is that the mutations cause misfolding in SUR1 that is temperature sensitive. Login to comment
102 We have previously shown that A116P or V187D mutation abolished the association between TMD0 and Kir6.2 at 18 °C [5], which seems to contradict with our current result obtained at the same temperature. Login to comment
106 Mutant and the WT channels show indistinguishable channel properties at 18 °C suggesting folding defect is completely corrected It is possible that A116P and V187D mutations still cause small conformational changes in SUR1 that are not detected by immunoprecipitation when expressed at 18 °C. Login to comment
115 t1/2 for azide activation is the time required to reach half of the maximally azide activated current (t1/2 for WT = 264 ± 10 s; A116P = 246 ± 10 s; and V187D = 277 ± 10 s; n = 6). Login to comment
116 Azide activated current is the maximum current obtained in the presence of azide minus the background current remained in the presence of glibenclamide (Iaz for WT = À14.53 ± 2.66 lA; A116P = À15.96 ± 3.98 lA; V187D = À14.18 ± 2.80 lA). Login to comment
117 Diazoxide/azide activation is the ratio of the current activated by diazoxide divided by the current activated by azide (Idzx/Iaz for WT = 0.95 ± 0.08; A116P = 0.92 ± 0.13; V187D = 1.12 ± 0.10). Login to comment
127 Lastly, we investigated the effect of A116P and V187D mutations on the single channel characteristics of the SUR1/Kir6.2 channels. Login to comment
134 (A) Representative current traces obtained at À100 mV from patches excised from oocytes expressing WT or A116P SUR1/Kir6.2 channels (trace for V187D mutant channels were not shown). Login to comment
139 The obtained values for k (lM) and n are: 16.71 ± 0.88 (k) and 1.43 ± 0.09 (n) for WT; 12.17 ± 0.25 and 1.17 ± 0.03 for A116P; 11.21 ± 0.22 and 1.42 ± 0.04 for V187D. Login to comment
146 Table 1 Single channel parameters for WT, A116P and V187D SUR1/Kir6.2 channelsa SUR1 + Kir6.2 A116P + Kir6.2 V187D + Kir6.2 sc1 (ms) 0.36 ± 0.002 0.43 ± 0.007 0.38 ± 0.007 sc2 (ms) 9.36 ± 2.238 7.28 ± 1.711 10.37 ± 2.895 sc3 (ms) 63.12 ± 14.543 42.92 ± 7.702 71.31 ± 37.949 ac1 0.969 ± 0.006 0.961 ± 0.008 0.975 ± 0.0006 ac2 0.025 ± 0.007 0.025 ± 0.005 0.016 ± 0.003 ac3 0.006 ± 0.002 0.014 ± 0.003 0.009 ± 0.003 so (ms) 1.31 ± 0.042 1.47 ± 0.027 1.46 ± 0.056 sb (ms) 67.66 ± 11.93 68.92 ± 17.77 77.96 ± 4.06 Po 0.595 ± 0.034 0.554 ± 0.047 0.583 ± 0.022 i (pA) 6.05 ± 0.43 6.07 ± 0.06 6.26 ± 0.17 a Explanations of the symbols can be found in the legends of Figs. 5 and 6. Login to comment
148 Conclusion Our data prove that A116P and V187D disrupt the association between the two KATP channel subunits by causing misfolding in SUR1 at physiological temperature (37 °C). Login to comment
82 However, the upper band was not detected for A116P-AAA and V187D-AAA. Login to comment
88 When expressed at 37 C in the absence of Kir6.2, WT SUR1 but neither A116P nor V187D have been shown to be mature glycosylated when their RKR motifs were mutated to AAA (three alanines) [9]. Login to comment
89 We found that even at 18 C, A116P and V187D SUR1-AAA could not mature to form the upper band (Fig. 2B) in the absence of Kir6.2. Login to comment
92 A116P and V187D disrupt heteromeric subunits interaction at 37 C, but not at 18 C, by causing misfolding One explanation for the temperature sensitive processing of these mutants is that the mutations cause misfolding in SUR1 that is temperature sensitive. Login to comment
110 Mutant and the WT channels show indistinguishable channel properties at 18 C suggesting folding defect is completely corrected It is possible that A116P and V187D mutations still cause small conformational changes in SUR1 that are not detected by immunoprecipitation when expressed at 18 C. Login to comment
119 t1/2 for azide activation is the time required to reach half of the maximally azide activated current (t1/2 for WT = 264 &#b1; 10 s; A116P = 246 &#b1; 10 s; and V187D = 277 &#b1; 10 s; n = 6). Login to comment
120 Azide activated current is the maximum current obtained in the presence of azide minus the background current remained in the presence of glibenclamide (Iaz for WT = 14.53 &#b1; 2.66 lA; A116P = 15.96 &#b1; 3.98 lA; V187D = 14.18 &#b1; 2.80 lA). Login to comment
121 Diazoxide/azide activation is the ratio of the current activated by diazoxide divided by the current activated by azide (Idzx/Iaz for WT = 0.95 &#b1; 0.08; A116P = 0.92 &#b1; 0.13; V187D = 1.12 &#b1; 0.10). Login to comment
131 Lastly, we investigated the effect of A116P and V187D mutations on the single channel characteristics of the SUR1/Kir6.2 channels. Login to comment
144 The obtained values for k (lM) and n are: 16.71 &#b1; 0.88 (k) and 1.43 &#b1; 0.09 (n) for WT; 12.17 &#b1; 0.25 and 1.17 &#b1; 0.03 for A116P; 11.21 &#b1; 0.22 and 1.42 &#b1; 0.04 for V187D. Login to comment
151 Table 1 Single channel parameters for WT, A116P and V187D SUR1/Kir6.2 channelsa SUR1 + Kir6.2 A116P + Kir6.2 V187D + Kir6.2 sc1 (ms) 0.36 &#b1; 0.002 0.43 &#b1; 0.007 0.38 &#b1; 0.007 sc2 (ms) 9.36 &#b1; 2.238 7.28 &#b1; 1.711 10.37 &#b1; 2.895 sc3 (ms) 63.12 &#b1; 14.543 42.92 &#b1; 7.702 71.31 &#b1; 37.949 ac1 0.969 &#b1; 0.006 0.961 &#b1; 0.008 0.975 &#b1; 0.0006 ac2 0.025 &#b1; 0.007 0.025 &#b1; 0.005 0.016 &#b1; 0.003 ac3 0.006 &#b1; 0.002 0.014 &#b1; 0.003 0.009 &#b1; 0.003 so (ms) 1.31 &#b1; 0.042 1.47 &#b1; 0.027 1.46 &#b1; 0.056 sb (ms) 67.66 &#b1; 11.93 68.92 &#b1; 17.77 77.96 &#b1; 4.06 Po 0.595 &#b1; 0.034 0.554 &#b1; 0.047 0.583 &#b1; 0.022 i (pA) 6.05 &#b1; 0.43 6.07 &#b1; 0.06 6.26 &#b1; 0.17 a Explanations of the symbols can be found in the legends of Figs. 5 and 6. Login to comment
153 Conclusion Our data prove that A116P and V187D disrupt the association between the two KATP channel subunits by causing misfolding in SUR1 at physiological temperature (37 C). Login to comment

[hide] Genotype-phenotype correlations in children with c... J Clin Endocrinol Metab. 2005 Feb;90(2):789-94. Epub 2004 Nov 23. Henwood MJ, Kelly A, Macmullen C, Bhatia P, Ganguly A, Thornton PS, Stanley CA
Genotype-phenotype correlations in children with congenital hyperinsulinism due to recessive mutations of the adenosine triphosphate-sensitive potassium channel genes.
J Clin Endocrinol Metab. 2005 Feb;90(2):789-94. Epub 2004 Nov 23., [PMID:15562009]

Abstract [show]
Congenital hyperinsulinism (HI) is most commonly caused by recessive mutations of the pancreatic beta-cell ATP-sensitive potassium channel (K(ATP)), encoded by two genes on chromosome 11p, SUR1 and Kir6.2. The two mutations that have been best studied, SUR1 g3992-9a and SUR1 delF1388, are null mutations yielding nonfunctional channels and are characterized by nonresponsiveness to diazoxide, a channel agonist, and absence of acute insulin responses (AIRs) to tolbutamide, a channel antagonist, or leucine. To examine phenotypes of other K(ATP) mutations, we measured AIRs to calcium, leucine, glucose, and tolbutamide in infants with recessive SUR1 or Kir6.2 mutations expressed as diffuse HI (n = 8) or focal HI (n = 14). Of the 24 total mutations, at least seven showed evidence of residual K(ATP) channel function. This included positive AIR to both tolbutamide and leucine in diffuse HI cases or positive AIR to leucine in focal HI cases. One patient with partial K(ATP) function also responded to treatment with the channel agonist, diazoxide. Six of the seven patients with partial defects had amino acid substitutions or insertions; whereas, the other patient was compound heterozygous for two premature stop codons. These results indicate that some K(ATP) mutations can yield partially functioning channels, including cases of hyperinsulinism that are fully responsive to diazoxide therapy.

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116 Whereas one patient who was homozygous for the common Finnish SUR1 V187D mutation had a negative AIR to tolbutamide (0.14 ␮U/ml), a second patient with the same mutations had a modest response (11.7 ␮U/ml); the third patient with compound heterozygosity for Kir6.2 (c 1-54 t)/K67N mutations had an AIR to tolbutamide of 68 ␮U/ml, implying considerable residual channel function. Login to comment

[hide] Mutations in the genes encoding the pancreatic bet... Hum Mutat. 2006 Mar;27(3):220-31. Gloyn AL, Siddiqui J, Ellard S
Mutations in the genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) in diabetes mellitus and hyperinsulinism.
Hum Mutat. 2006 Mar;27(3):220-31., [PMID:16416420]

Abstract [show]
The beta-cell ATP-sensitive potassium channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events, bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis, it is not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1. It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinemia (HI) of infancy; however, heterozygous activating mutations in KCNJ11 that result in the opposite phenotype of diabetes have recently been described. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment when patients are diagnosed with mutations in these genes.

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240 population two founder mutations have been reported (V187D and E1507 K) [Huopio et al., 2000; Otonkoski et al., 1999]. Login to comment
241 The V187D mutation is associated with 50% of HI in this population [Otonkoski et al., 1999]. Login to comment

[hide] Sulfonylureas correct trafficking defects of ATP-s... J Biol Chem. 2004 Mar 19;279(12):11096-105. Epub 2004 Jan 5. Yan F, Lin CW, Weisiger E, Cartier EA, Taschenberger G, Shyng SL
Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor.
J Biol Chem. 2004 Mar 19;279(12):11096-105. Epub 2004 Jan 5., [PMID:14707124]

Abstract [show]
The pancreatic ATP-sensitive potassium (K(ATP)) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to beta-cell membrane potential. Sulfonylureas inhibit K(ATP) channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue K(ATP) channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional K(ATP) channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain K(ATP) channel trafficking mutations.

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2 We report here that sulfonylureas also function as chemical chaperones to rescue KATP channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Login to comment
3 Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. Login to comment
5 Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional KATP channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain KATP channel trafficking mutations. Login to comment
43 Here, we report that two PHHI-associated SUR1 mutations, A116P and V187D (2, 21, 29, 37), located in the first transmembrane domain (TM0), prevent trafficking of KATP channels from the ER to the plasma membrane. Login to comment
93 RESULTS Both the A116P and V187D Mutations in SUR1 Prevent Normal Cell Surface Expression of KATP Channels-Several recent studies have shown that defective KATP channel trafficking is an underlying mechanism of congenital hyperinsulinism. Login to comment
95 To examine whether mutations located in other parts of the molecule also affect channel trafficking, we focused our attention to two mutations, A116P and V187D, that are located in the first transmembrane domain, or TM0, of SUR1 (Fig. 1), and that have previously been reported to not form functional channels when co-expressed with Kir6.2 (6, 21, 37). Login to comment
96 To investigate how the A116P and V187D mutations lead to loss of functional KATP channels, we first performed Western blot analysis. Login to comment
101 Fig. 2A shows that, although both the immature and mature forms were seen with cells co-expressing WT-fSUR1 and Kir6.2, only the immature form was evident in cells co-expressing Kir6.2 and the A116P- or the V187D-fSUR1 mutants. Login to comment
104 In contrast to the abundant surface staining observed in cells transfected with Kir6.2 and WT-fSUR1, surface staining in cells transfected with Kir6.2 and A116P-fSUR1 or V187D-fSUR1 was barely detectable (Fig. 2B, top panels). Login to comment
106 These results led us to conclude that the A116P and V187D mutations cause loss of functional KATP channels by preventing channels from trafficking to the cell surface. Login to comment
107 The Trafficking Defects of the A116P and V187D Mutants Are Intrinsic to SUR1-One potential explanation for the trafficking defects seen with the A116P- and V187D-fSUR1 mutations is that the SUR1 mutants are unable to associate with Kir6.2. Login to comment
109 To address this possibility, we used a heterotandem dimer construct in which the C terminus of the mutant fSUR1 has been fused to the N terminus of Kir6.2 (referred to as A116P- or V187D-fSUR1/Kir6.2 fusion) to achieve obligatory physical association between the two subunits; similar SUR1/ Kir6.2 fusion constructs have been used previously by a number of groups for structure-function and trafficking studies (7, 9, 10, 28, 34). Login to comment
111 Although WT fSUR1/Kir6.2 fusion protein was expressed at a level comparable with that observed in cells transfected with WT-fSUR1 and Kir6.2 as individual subunits, fusion proteins carrying the A116P- or V187D-SUR1 mutation had poor surface expression, ϳ10 and 20% that of WT fSUR1/ Kir6.2 fusion, respectively (Fig. 3A). Login to comment
114 Analysis of the A116- and V187D-SUR1 mutants by immunoblotting and immunofluorescent staining experiments. Login to comment
117 In contrast, only the immature band is observed in cells expressing Kir6.2 and A116P- or V187D-fSUR1. Login to comment
118 The total steady-state protein level of A116P- and V187D-fSUR1 also appears less than that of WT-fSUR1. Login to comment
120 B, top panels, surface staining of COSm6 cells transiently transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1 using the M2 anti-FLAG mouse monoclonal antibodies followed by Cy-3-conjugated anti-mouse secondary antibody. Login to comment
122 Whereas cells expressing WT-fSUR1 channels have abundant surface staining, those expressing A116P- or V187D-fSUR1 channels have barely detectable staining. Login to comment
125 Both A116P- and V187D-fSUR1 were detected inside the cell, with a perinuclear staining pattern. Login to comment
126 fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
128 The A116P and V187D mutations in SUR1. Login to comment
129 The locations of the A116P and V187D mutations in SUR1 are shown. Login to comment
133 The surface expression levels of A116P-fSUR1AAA and V187D-fSUR1AAA are 10 and 20% that of WT-fSUR1AAA, respectively (Fig. 3B). Login to comment
135 In addition, although the surface expression levels of the A116P and V187D mutant channels were very low (7 and 19% of WT; see Figs. Login to comment
139 Taken together, the results led us to propose that the A116P and V187D mutations cause trafficking defects in SUR1, possibly by promoting protein misfolding, but that the mutant proteins retain the ability to associate with Kir6.2 to form functional channels. Login to comment
140 Another potential mechanism for the deficient surface expression of KATP channels is that the A116P and V187D mutations interfere with proper shielding of the RKR signals in the channel complex. Login to comment
142 We found that inactivation of the RKR signal in SUR1 slightly increased surface expression of the A116P mutant channels (from 6 to 18% of normal expression level) but not the V187D mutant, and removal of the RKR signal in Kir6.2 (Kir6.2⌬C25) also had very little effect on the surface expression of either mutant (Fig. 3C). Login to comment
144 Glibenclamide Corrects the Channel Trafficking Defects Caused by the A116P and V187D Mutations in SUR1-The data presented so far suggest that the two mutations likely cause defective channel trafficking by promoting misfolding of SUR1. Login to comment
151 We found that treating cells with 5% glycerol for 24 h slightly improved surface expression of both the A116P and V187D mutants as well as the WT channel (Fig. 4A). Login to comment
152 Treating cells with 5 ␮M glibenclamide, however, dramatically increased surface expression of A116P-fSUR1, from 5 to 55%, and of V187D-fSUR1, from 19% to 70% of normal WT channel expression level (Fig. 4A). Login to comment
154 The effect of glibenclamide on the A116P and V187D mu- FIG. 3. Login to comment
155 The trafficking defects of the A116P- and V187D-fSUR1 mutants are intrinsic to SUR1. Login to comment
156 A, obligatory association between SUR1 and Kir6.2 does not overcome trafficking defects caused by A116P or V187D. Login to comment
157 Fusion fSUR1/Kir6.2 constructs containing either A116P or V187D mutation still exhibit poor surface expression compared with the WT fusion construct. Login to comment
162 B, the A116P and V187D mutations cause trafficking defects in SUR1. Login to comment
164 However, introducing A116P or V187D to fSUR1AAA (fA116PAAA and fV187DAAA) abolishes the ability of the proteins to traffic to the cell surface. Login to comment
165 C, trafficking defects caused by A116P and V187D do not involve improper shielding of RKR signals in the channel complex. Login to comment
166 Inactivation of the RKR signal in SUR1 (SUR1RKR/AAA) only slightly improved surface expression of A116P but not V187D, whereas removal of RKR in Kir6.2 (Kir6.2⌬C25) slightly improved surface expression of V187D but not A116P. Login to comment
167 fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
170 The response of the A116P- and the V187D-fSUR1 mutants to glibenclamide was specific; another SUR1 ligand, FIG. 4. Login to comment
171 Glibenclamide rescues surface expression of the A116P and V187D mutant KATPchannels. A, cells co-expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were subjected to the different drug treatments indicated for 24 h, and surface expression of fSUR1 was quantified using the chemiluminescence assay as described in Fig. 3. Login to comment
172 Without any drug treatment, expression levels of the A116P and V187D mutants are 6.4 Ϯ 1.2 and 19.1 Ϯ 4.8% that of WT, respectively. Login to comment
174 Glibenclamide treatment at 5 ␮M for 24 h dramatically improved surface expression of both A116P- and V187D-fSUR1 (to 55.4 Ϯ 4.8 and 70.4 Ϯ 14.5% of WT, respectively) but only slightly increased WT expression (by 6.6 Ϯ 2.8%). Login to comment
176 B, Western blots showing that in cells expressing Kir6.2 and the A116P or V187D mutant fSUR1, treatment with 5 ␮M glibenclamide for 24 h led to appearance of the mature band, which was not detected in untreated cells (Fig. 2A). Login to comment
178 Top panels, surface staining of COSm6 cells transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1, as described for Fig. 2B. Login to comment
180 In contrast to the data shown in Fig. 2B, cells expressing A116P- or V187D-fSUR1 mutant channels had strong surface staining that is nearly comparable with cells expressing WT-fSUR1 channels. Login to comment
183 fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1; Glib, glibenclamide. Login to comment
185 In fact, diazoxide slightly decreased the surface expression of both A116P and V187D mutants. Login to comment
196 Cells expressing A116P-fSUR1 alone were pulse-labeled for 30 min and chased for up to 18 h in the presence or absence of 5 ␮M glibenclamide. Login to comment
199 Tolbutamide Also Rescues SUR1 A116P Mutant Channels to the Cell Surface, and the Expressed Channels Are Fully Functional after Tolbutamide Washout-Following our observation that glibenclamide significantly improves cell surface expression of the A116P and V187D mutants, the question arises as to whether the rescued channels are still glibenclamide-bound and whether they are physiologically functional. Login to comment
204 Concentration and time dependence of the effect of glibenclamide on surface expression of A116P- and V187D-fSUR1 mutant KATPchannels. A, cells transfected with Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated with different concentrations of glibenclamide for 24 h, and the surface expression of fSUR1 was quantified by the chemiluminescence assay. Login to comment
210 The cells expressing A116P- or V187D-fSUR1 mutant channels were treated with 5 ␮M for different periods of time as indicated, and surface expression of the mutant channel was quantified by chemiluminescence assays. Login to comment
212 Each data point represents the average from 2-3 experiments, and the error bar is the deviation from the average or the S.E. fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
216 Fig. 7A shows that in chemiluminescence assays, tolbutamide also increases surface expression of both A116P- and V187D-fSUR1 mutant channels, at concentrations of 100 and 300 ␮M that we tested (only 300 ␮M is shown). Login to comment
217 Consistently, in inside-out patch clamp recording experiments, tolbutamide treatment led to parallel increases in the current size of both mutant channels; the average patch current amplitudes in K-INT for A116P- and V187D-fSUR1 channels are 3.24 Ϯ 0.75 nA (n ϭ 12) and 5.40 Ϯ 1.19 nA (n ϭ 13), respectively, compared with 7.05 Ϯ 1.02 nA (n ϭ 17) for control WT-fSUR1 channels. Login to comment
231 In this study, we show that two SUR1 point mutations, A116P and V187D, identified in patients with congenital hyperinsulinism (2, 37) cause defective trafficking and a lack of cell surface expression of KATP channels. Login to comment
233 Mechanisms of Trafficking Defects Caused by the A116P and V187D Mutations-Multiple steps are involved in the proper expression of KATP channels on the cell surface. Login to comment
252 fWT, WT-fSUR1; fA116P, A116P-fSUR1. Login to comment
253 ported by our metabolic pulse-chase labeling experiments, which showed that glibenclamide slowed the degradation rate of the A116P mutant SUR1 and, in the presence of Kir6.2, promoted maturation of the mutant protein. Login to comment
255 This conclusion differs somewhat from that reached by Chan et al. (29), who proposed that the A116P and V187D mutations cause PHHI by preventing association between SUR1 and FIG. 7. Login to comment
256 Tolbutamide rescues functional A116P and V187D mutant channels to the cell surface. Login to comment
257 A, cells transfected with Kir6.2 and A116P- or V187D-fSUR1 were treated with 300 ␮M tolbutamide for 24 h, and surface expression of channels was measured by chemiluminescence assays. Login to comment
258 At 300 ␮M, tolbutamide was nearly as effective as 5 ␮M glibenclamide and restored surface expression of A116P- and V187D-fSUR1 channels from 6.4 Ϯ 1.2 to 49.9 Ϯ 7.6% and from 19.1 Ϯ 4.8 to 48.9 Ϯ 6.0% of normal levels, respectively. Login to comment
267 C, representative KATP current traces recorded from inside-out membrane patches containing WT-fSUR1, A116P-fSUR1, or V187D-fSUR1 channels 2 h after tolbutamide removal. Login to comment
272 fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
284 However, the first transmembrane domain (TM0) where the A116P and V187D mutations are located has not been implicated in glibenclamide binding. Login to comment
292 By contrast, diazoxide, which too binds SUR1 but is structurally quite different from sulfonylureas and results in channel stimulation, does not correct the trafficking defect of either A116P- or V187D-fSUR1. Login to comment
293 Comparison with Other Trafficking Mutants-A number of missense or point deletion mutations in SUR1 have been reported to reduce or prevent cell surface expression of KATP channels, including ⌬F1388, R1394H, L1544P, A1457T, V1550D, and L1551V (34-36, 50). Login to comment
296 Although like A116P and V187D they all result in a lack of surface channel expression phenotype, the mechanisms leading to this phenotype differ, as revealed by their responses to the different rescuing strategies. Login to comment
300 Although sulfonylureas rescue the surface expression of mutant channels bearing the A116P or the V187D mutation, they do not rescue surface expression of either ⌬F1388 or L1544P mutant channels (36). Login to comment
307 Although genetic and clinical data on the A116P mutation have not been published, the V187D mutation has been shown to account for the majority of PHHI cases in Finland (37). Login to comment
309 The results presented in this study show that sulfonylureas have rapid, potent, and long lasting (for at least 12 h after drug removal) effects on rescuing the A116P and V187D mutant channels to the cell surface. Login to comment
310 Most importantly, both A116P- and V187D-SUR1 mutant channels rescued to the cell surface by tolbutamide are fully functional upon drug removal and respond to MgADP and diazoxide stimulation like WT channels. Thus, mutant channels rescued to the surface will be able to respond to metabolic signals and to diazoxide treatment. Login to comment
42 Here, we report that two PHHI-associated SUR1 mutations, A116P and V187D (2, 21, 29, 37), located in the first transmembrane domain (TM0), prevent trafficking of KATP channels from the ER to the plasma membrane. Login to comment
90 The data were presented as the means afe; S.E. RESULTS Both the A116P and V187D Mutations in SUR1 Prevent Normal Cell Surface Expression of KATP Channels-Several recent studies have shown that defective KATP channel trafficking is an underlying mechanism of congenital hyperinsulinism. Login to comment
92 To examine whether mutations located in other parts of the molecule also affect channel trafficking, we focused our attention to two mutations, A116P and V187D, that are located in the first transmembrane domain, or TM0, of SUR1 (Fig. 1), and that have previously been reported to not form functional channels when co-expressed with Kir6.2 (6, 21, 37). Login to comment
98 Fig. 2A shows that, although both the immature and mature forms were seen with cells co-expressing WT-fSUR1 and Kir6.2, only the immature form was evident in cells co-expressing Kir6.2 and the A116P- or the V187D-fSUR1 mutants. Login to comment
103 These results led us to conclude that the A116P and V187D mutations cause loss of functional KATP channels by preventing channels from trafficking to the cell surface. Login to comment
108 Although WT fSUR1/Kir6.2 fusion protein was expressed at a level comparable with that observed in cells transfected with WT-fSUR1 and Kir6.2 as individual subunits, fusion proteins carrying the A116P- or V187D-SUR1 mutation had poor surface expression, b03;10 and 20% that of WT fSUR1/ Kir6.2 fusion, respectively (Fig. 3A). Login to comment
115 The total steady-state protein level of A116P- and V187D-fSUR1 also appears less than that of WT-fSUR1. Login to comment
119 Whereas cells expressing WT-fSUR1 channels have abundant surface staining, those expressing A116P- or V187D-fSUR1 channels have barely detectable staining. Login to comment
123 fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
130 The surface expression levels of A116P-fSUR1AAA and V187D-fSUR1AAA are 10 and 20% that of WT-fSUR1AAA, respectively (Fig. 3B). Login to comment
132 In addition, although the surface expression levels of the A116P and V187D mutant channels were very low (7 and 19% of WT; see Figs. Login to comment
136 Taken together, the results led us to propose that the A116P and V187D mutations cause trafficking defects in SUR1, possibly by promoting protein misfolding, but that the mutant proteins retain the ability to associate with Kir6.2 to form functional channels. Login to comment
137 Another potential mechanism for the deficient surface expression of KATP channels is that the A116P and V187D mutations interfere with proper shielding of the RKR signals in the channel complex. Login to comment
141 Glibenclamide Corrects the Channel Trafficking Defects Caused by the A116P and V187D Mutations in SUR1-The data presented so far suggest that the two mutations likely cause defective channel trafficking by promoting misfolding of SUR1. Login to comment
148 We found that treating cells with 5% glycerol for 24 h slightly improved surface expression of both the A116P and V187D mutants as well as the WT channel (Fig. 4A). Login to comment
149 Treating cells with 5 òe;M glibenclamide, however, dramatically increased surface expression of A116P-fSUR1, from 5 to 55%, and of V187D-fSUR1, from 19% to 70% of normal WT channel expression level (Fig. 4A). Login to comment
153 A, obligatory association between SUR1 and Kir6.2 does not overcome trafficking defects caused by A116P or V187D. Login to comment
159 B, the A116P and V187D mutations cause trafficking defects in SUR1. Login to comment
161 However, introducing A116P or V187D to fSUR1AAA (fA116PAAA and fV187DAAA) abolishes the ability of the proteins to traffic to the cell surface. Login to comment
163 Inactivation of the RKR signal in SUR1 (SUR1RKR/AAA) only slightly improved surface expression of A116P but not V187D, whereas removal of RKR in Kir6.2 (Kir6.2èc;C25) slightly improved surface expression of V187D but not A116P. Login to comment
168 Glibenclamide rescues surface expression of the A116P and V187D mutant KATPchannels. A, cells co-expressing Kir6.2 and WT-, A116P-, or V187D-fSUR1 were subjected to the different drug treatments indicated for 24 h, and surface expression of fSUR1 was quantified using the chemiluminescence assay as described in Fig. 3. Login to comment
169 Without any drug treatment, expression levels of the A116P and V187D mutants are 6.4 afe; 1.2 and 19.1 afe; 4.8% that of WT, respectively. Login to comment
173 B, Western blots showing that in cells expressing Kir6.2 and the A116P or V187D mutant fSUR1, treatment with 5 òe;M glibenclamide for 24 h led to appearance of the mature band, which was not detected in untreated cells (Fig. 2A). Login to comment
175 Top panels, surface staining of COSm6 cells transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1, as described for Fig. 2B. Login to comment
177 In contrast to the data shown in Fig. 2B, cells expressing A116P- or V187D-fSUR1 mutant channels had strong surface staining that is nearly comparable with cells expressing WT-fSUR1 channels. Login to comment
182 In fact, diazoxide slightly decreased the surface expression of both A116P and V187D mutants. Login to comment
201 Concentration and time dependence of the effect of glibenclamide on surface expression of A116P- and V187D-fSUR1 mutant KATPchannels. A, cells transfected with Kir6.2 and WT-, A116P-, or V187D-fSUR1 were treated with different concentrations of glibenclamide for 24 h, and the surface expression of fSUR1 was quantified by the chemiluminescence assay. Login to comment
207 The cells expressing A116P- or V187D-fSUR1 mutant channels were treated with 5 òe;M for different periods of time as indicated, and surface expression of the mutant channel was quantified by chemiluminescence assays. Login to comment
209 Each data point represents the average from 2-3 experiments, and the error bar is the deviation from the average or the S.E. fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
213 Fig. 7A shows that in chemiluminescence assays, tolbutamide also increases surface expression of both A116P- and V187D-fSUR1 mutant channels, at concentrations of 100 and 300 òe;M that we tested (only 300 òe;M is shown). Login to comment
214 Consistently, in inside-out patch clamp recording experiments, tolbutamide treatment led to parallel increases in the current size of both mutant channels; the average patch current amplitudes in K-INT for A116P- and V187D-fSUR1 channels are 3.24 afe; 0.75 nA (n afd; 12) and 5.40 afe; 1.19 nA (n afd; 13), respectively, compared with 7.05 afe; 1.02 nA (n afd; 17) for control WT-fSUR1 channels. Login to comment
228 In this study, we show that two SUR1 point mutations, A116P and V187D, identified in patients with congenital hyperinsulinism (2, 37) cause defective trafficking and a lack of cell surface expression of KATP channels. Login to comment
230 Mechanisms of Trafficking Defects Caused by the A116P and V187D Mutations-Multiple steps are involved in the proper expression of KATP channels on the cell surface. Login to comment
254 A, cells transfected with Kir6.2 and A116P- or V187D-fSUR1 were treated with 300 òe;M tolbutamide for 24 h, and surface expression of channels was measured by chemiluminescence assays. Login to comment
264 C, representative KATP current traces recorded from inside-out membrane patches containing WT-fSUR1, A116P-fSUR1, or V187D-fSUR1 channels 2 h after tolbutamide removal. Login to comment
269 fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1. Login to comment
281 However, the first transmembrane domain (TM0) where the A116P and V187D mutations are located has not been implicated in glibenclamide binding. Login to comment
289 By contrast, diazoxide, which too binds SUR1 but is structurally quite different from sulfonylureas and results in channel stimulation, does not correct the trafficking defect of either A116P- or V187D-fSUR1. Login to comment
297 Although sulfonylureas rescue the surface expression of mutant channels bearing the A116P or the V187D mutation, they do not rescue surface expression of either èc;F1388 or L1544P mutant channels (36). Login to comment
304 Although genetic and clinical data on the A116P mutation have not been published, the V187D mutation has been shown to account for the majority of PHHI cases in Finland (37). Login to comment
306 The results presented in this study show that sulfonylureas have rapid, potent, and long lasting (for at least 12 h after drug removal) effects on rescuing the A116P and V187D mutant channels to the cell surface. Login to comment

[hide] N-terminal transmembrane domain of the SUR control... EMBO J. 2003 Aug 1;22(15):3833-43. Chan KW, Zhang H, Logothetis DE
N-terminal transmembrane domain of the SUR controls trafficking and gating of Kir6 channel subunits.
EMBO J. 2003 Aug 1;22(15):3833-43., [PMID:12881418]

Abstract [show]
The sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, assembles with a potassium channel subunit (Kir6) to form the ATP-sensitive potassium channel (K(ATP)) complex. Although SUR is an important regulator of Kir6, the specific SUR domain that associates with Kir6 is still unknown. All functional ABC proteins contain two transmembrane domains but some, including SUR and MRP1 (multidrug resistance protein 1), contain an extra N-terminal transmembrane domain called TMD0. The functions of any TMD0s are largely unclear. Using Xenopus oocytes to coexpress truncated SUR constructs with Kir6, we demonstrated by immunoprecipitation, single-oocyte chemiluminescence and electrophysiological measurements that the TMD0 of SUR1 strongly associated with Kir6.2 and modulated its trafficking and gating. Two TMD0 mutations, A116P and V187D, previously correlated with persistent hyperinsulinemic hypoglycemia of infancy, were found to disrupt the association between TMD0 and Kir6.2. These results underscore the importance of TMD0 in K(ATP) channel function, explaining how specific mutations within this domain result in disease, and suggest how an ABC protein has evolved to regulate a potassium channel.

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No. Sentence Comment
4 Using Xenopus oocytes to coexpress truncated SUR constructs with Kir6, we demonstrated by immunoprecipitation, single-oocyte chemiluminescence and electrophysiological measurements that the TMD0 of SUR1 strongly associated with Kir6.2 and modulated its traf®cking and gating. Two TMD0 mutations, A116P and V187D, previously correlated with persistent hyperinsulinemic hypoglycemia of infancy, were found to disrupt the association between TMD0 and Kir6.2. Login to comment
147 Two PHHI mutations located in TMD0 abolish its association with 6.2 Two TMD0 mutations, A116P and V187D, have been reported to cause PHHI (Aguilar-Bryan and Bryan, 1999; Otonkoski et al., 1999). Login to comment
215 Two PHHI mutations located in TMD0, A116P and V187D disrupt the association between TMD0 and 6.2. Login to comment
216 (A) A116P and V187D mutations completely abolish the total current expressed from TMD0+6.2HA. Login to comment
218 (B) A116P and V187D mutations completely abolish the ability of TMD0 to enhance the surface expression of 6.2D26. Login to comment
220 (C) A116P and V187D mutations completely abolish the ability of TMD0 to traf®c to the cell surface. Login to comment
222 (D) A116P and V187D mutations disrupt the association between TMD0 and 6.2. Login to comment
232 PHHI mutations affect KATP channels by disrupting the function of TMD0 Two mutations, A116P and V187D, have been reported to correlate with PHHI (Aguilar-Bryan and Bryan, 1999; Otonkoski et al., 1999). Login to comment
234 While detailed analysis of A116P has not been reported, V187D has been shown to abolish the function of the pancreatic KATP channels both in native b cells and when expressed in Xenopus oocytes (Otonkoski et al., 1999). Login to comment
249 Four nanograms of SUR1, 1 ng of TMD0 [including F195, F195(A116P), F195(V187D), TMD0*, 1±195, S2-TMD0 and MRP1-TMD0*], 3 ng of F196-917, 3 ng of 918M and 2 ng of Kir6 (including various 6.2 and 6.1 constructs) RNAs were used in independent or coexpression experiments for TEVC, macropatch recording, western blotting and immunoprecipitation. Login to comment

[hide] Sulfonylureas correct trafficking defects of disea... J Biol Chem. 2006 Nov 3;281(44):33403-13. Epub 2006 Sep 6. Yan FF, Casey J, Shyng SL
Sulfonylureas correct trafficking defects of disease-causing ATP-sensitive potassium channels by binding to the channel complex.
J Biol Chem. 2006 Nov 3;281(44):33403-13. Epub 2006 Sep 6., [PMID:16956886]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels mediate glucose-induced insulin secretion by coupling metabolic signals to beta-cell membrane potential and the secretory machinery. Reduced K(ATP) channel expression caused by mutations in the channel proteins: sulfonylurea receptor 1 (SUR1) and Kir6.2, results in loss of channel function as seen in congenital hyperinsulinism. Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. In this study, we investigated the mechanism by which sulfonylureas rescue these mutants. We found that glinides, another class of SUR-binding hypoglycemic drugs, also markedly increased surface expression of the trafficking mutants. Attenuating or abolishing the ability of mutant SUR1 to bind sulfonylureas or glinides by the following mutations: Y230A, S1238Y, or both, accordingly diminished the rescuing effects of the drugs. Interestingly, rescue of the trafficking defects requires mutant SUR1 to be co-expressed with Kir6.2, suggesting that the channel complex, rather than SUR1 alone, is the drug target. Observations that sulfonylureas also reverse trafficking defects caused by neonatal diabetes-associated Kir6.2 mutations in a way that is dependent on intact sulfonylurea binding sites in SUR1 further support this notion. Our results provide insight into the mechanistic and structural basis on which sulfonylureas rescue K(ATP) channel surface expression defects caused by channel mutations.

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No. Sentence Comment
2 Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. Login to comment
40 We have previously reported that sulfonylureas rescue surface expression defects of KATP channels caused by two CHI-associated SUR1 mutations, A116P and V187D (23). Login to comment
42 In this work, we investigated the mechanism by which sulfonylureas correct the channel surface expression defects caused by the A116P or V187D mutations and by the PNDM-associated Kir6.2 mutations. Login to comment
44 Interestingly and somewhat unexpectedly, we found that Kir6.2 is required for sulfonylureas to rescue the A116P and V187D mutant SUR1 at the cell surface. Login to comment
59 A, topology model of SUR1 showing the three transmembrane domains, the location of the A116P and V187D trafficking mutations, the location of the -RKR-ER retention motif, and the location of the S1238Y and Y230A mutations that have been proposed to disrupt the Aand B- sulfonyl- urea binding sites (as shown in B), respectively. Login to comment
89 Of these, two mutations, A116P and V187D, were rescued by the pharmacological agent sulfonylurea (23). Login to comment
91 Because both A116P and V187D are located in TMD0, we first tested if sulfonylureas bind directly to TMD0 to facilitate mutant protein biogenesis and trafficking, even though TMD0 has not been implicated in sulfonylurea binding in prior studies (21). Login to comment
92 We compared surface expression of recombinant SUR1-TMD0 (amino acids 1-197) containing either the A116P or the V187D mutation in the presence or absence of 5 ␮M glibenclamide. Login to comment
95 By con- FIGURE2.ThefirsttransmembranedomainofSUR1(TMD0)doesnotconfer the sulfonylurea rescue effect on the A116P or V187D mutations. Login to comment
96 A, schematic showing the fSUR1-TMD0 (amino acids 1-197) and the Kir6.2⌬C25 constructs used in experiments shown in B. B, surface expression of fSUR1-TMD0 harboring mutations A116P or V187D in cells treated with or without glibenclamide (5 ␮M for 24 h). Login to comment
99 Each bar represents the mean Ϯ S.E. of three to four independent experiments. Sulfonylureas and KATP Channel Trafficking trast, when the A116P or V187D mutations were introduced into fSUR1-TMD0, surface expression was greatly reduced (by Ͼ70%), even though the total mutant recombinant proteins were abundantly expressed as assessed by Western blots and immunofluorescent staining of permeabilized cells (not shown). Login to comment
102 Restoration of A116P- and V187D-mutant Channel Expression by Sulfonylureas Is Dependent on Intact Sulfonylurea Binding Sites in SUR1-Several studies indicate that the high affinity tolbutamide binding site in SUR1 resides in transmembrane segments 13-16 (19, 20). Login to comment
105 If sulfonylureas rescue the A116P and V187D trafficking mutants by binding to the channel protein, then introducing the S1237Y mutation should also reduce or abolish the ability of sulfonylureas to correct the trafficking defect. Login to comment
106 We made the equivalent sulfonylurea binding site mutation (S1238Y) in hamster SUR1 (16) and examined how it affects the response of the A116P- or V187D-mutant channels to sulfonylureas. Login to comment
107 Initial assessment by immunofluorescent staining indicates that the S1238Y mutation by itself does not affect fSUR1 surface expression when coexpressed with Kir6.2; however, when combined with the A116P or V187D mutation, it indeed reduced or prevented the ability of glibenclamide to rescue the surface expression defect caused by the A116P and V187D mutations (Fig. 3). Login to comment
116 However, when combined with the A116P- or V187D-SUR1 trafficking mutations, S1238Y completely abolished the rescue effect of tolbutamide at 300 (Fig. 4B) and 600 ␮M (not shown). Login to comment
118 In the WT background (no sulfonylurea binding mutation), 24-h treatment with 1 ␮M glibenclamide increased surface expression of the A116P mutant from 3.1 Ϯ 0.7 to 34.5 Ϯ 7.2% and the V187D mutant from 12.8 Ϯ 1.6 to 49.2 Ϯ 6.8% of WT channels. Login to comment
119 But in the S1238Y background, the same treatment only slightly increased surface expression of the A116P and V187D mutants, from 0.7 Ϯ 1.0 to 6.6 Ϯ 2.1% and 9.8 Ϯ 1.4 to 15.6 Ϯ 2.3% of WT, respectively. Login to comment
120 Increasing the concentration of glibenclamide to 5 ␮M led to a much greater effect on surface expression of the mutants (to 21.8 and 23.9% of WT for A116P and V187D, respectively; Fig. 4C). Login to comment
125 Bryan et al. (14) have shown that mutation of Tyr230 located in the intracellular loop between TMD0 and TMD1 to alanine (Y230A) results in loss of [125 I]azidoglibenclamide photoaffinity labeling, suggesting that the benzamido group lies in close proximity to Tyr230 during binding, although it is possible that Y230A abolishes binding indirectly by affecting a distant site. Login to comment
126 We therefore tested whether this mutation also interferes with the ability of sulfonylureas to rescue the A116P and V187D trafficking mutants. Login to comment
136 The Y230A or S1238Y mutations reduced the ability of glibenclamide to restore surface expression of the A116P mutant and simultaneous mutation of Y230A and S1238Y completely abolished the ability of glibenclamide to rescue A116P to the cell surface. Login to comment
137 C, same as B except that the V187D trafficking mutation was introduced into WT or sulfonylurea binding mutation fSUR1 background. Login to comment
138 Note as previously reported, the V187D mutation showed slightly higher surface expression compared with the A116P mutation, with faint surface staining barely visible (23). Login to comment
140 Accordingly, we examined how repaglinide, a glinide that contains the benzamido moiety but not the sulfonylurea moiety, affects surface expression of A116P and V187D channels in the presence or absence of the Y230A mutation. Login to comment
141 Fig. 5B shows that repaglinide at 10 ␮M effectively rescued surface expression of the A116P and V187D mutants, whereas the Tyr230 mutation abolished this rescue effect; in contrast, the S1238Y mutation had little effect on the ability of repaglinide to rescue the trafficking mutants (not shown). Login to comment
142 Surprisingly, we found that the Y230A mutation also renders tolbutamide unable to rescue the A116P and V187D trafficking mutants (Fig. 5C), suggesting a role of Tyr230 in tolbutamide binding or in conferring tolbutamide sensitivity toward trafficking rescue through an allosteric effect. Login to comment
153 Surface expression of tolbutamide-treated cells was significantly higher than untreated for A116P and V187D (p Ͻ 0.001) but not A116P/S1238Y and V187D/S1238Y. Login to comment
155 Without the S1238Y mutation, both 1 and 5 ␮M glibenclamide significantly increased surface expression of the A116P and V187D trafficking mutants (p Ͻ 0.001). Login to comment
156 However, for the A116P/ S1238Y and V187D/S1238Y mutants, 1 ␮M glibenclamide did not lead to a statistically significant increase in surface expression, whereas 5 ␮M glibenclamide did (p Ͻ 0.01). Login to comment
158 FIGURE 5. Impact of the Y230A mutation on the effectiveness of sulfonylureas and glinides to rescue KATP channel trafficking defects in the presence of Kir6.2. Login to comment
159 The A116P or V187D trafficking mutation was introduced onto the WTor Y230A-fSUR1 background. Login to comment
165 Each bar is the mean Ϯ S.E. of three to five independent experiments. Sulfonylureas and KATP Channel Trafficking 33408 the trafficking defects of the A116P and V187D mutants via direct interactions with the mutant channel proteins. Login to comment
175 The Role of Kir6.2 in Sulfonylurea Rescue of Channel Trafficking Defects-The data we presented so far demonstrate that intact sulfonylurea binding sites in SUR1 are necessary for effective rescue of the A116P- or V187D-SUR1 trafficking mutants by sulfonylureas. Login to comment
177 To investigate the role of Kir6.2 in sulfonylurea rescue of the A116P- or V187D-SUR1 trafficking mutants, we took advantage of the fact that inactivation of the -RKR-ER retention/retrieval motif by mutation to AAA (referred to as WTAAA in Fig. 8A) in SUR1 allows SUR1 to traffic to the cell surface without co-expression of Kir6.2 (29). Login to comment
178 If sulfonylurea binding to SUR1 is sufficient to correct the defect caused by the A116P or V187D mutations, we expect sulfonylureas to improve the surface expression of A116P- or V187D-SUR1 in which the -RKR- motif has been mutated to -AAA- (referred to as A116PAAA and V187DAAA) in the absence of Kir6.2. Login to comment
180 These results are consistent with the A116P and V187D mutations causing defects in the SUR1 protein itself. Login to comment
185 Effect of the Y230A/S1238Y double mutation on sulfonylurea and glinide rescue of KATP channel trafficking mutants in the presence of Kir6.2. Login to comment
186 The experiments were as described in the legend to Fig. 5 except that A116P and V187D were each introduced onto the WTor Y230A/S1238Y-fSUR1 backgrounds. Login to comment
212 Our results demonstrate that mutations in SUR1 previously reported to abolish or reduce sulfonylurea binding also abolish or reduce the ability of sulfonylureas to rescue channel trafficking defects caused by the A116P or V187D SUR1 mutations and that both the sulfonylurea and benzoamido moieties of glibenclamide contribute to the rescue effect. Login to comment
218 A reasonable hypothesis for how sulfonylureas improve surface expression of the A116P and V187D mutants is that A116P or V187D cause SUR1 misfolding and sulfonylureas, upon binding to the mutant SUR1, help it to adopt the correct conformation. Login to comment
219 Our results that the A116P or V187D mutations are sufficient to prevent trafficking of TMD0 as well as SUR1AAA to the cell surface support the idea that the A116P or V187D mutations have an effect on the folding/processing of SUR1 protein itself. Login to comment
227 The A116P or V187D trafficking mutation was engineered into WT-fSUR1 or fSUR1 in which the -RKR-ER retention motif has been inactivated by mutation to AAA. Login to comment
238 It is also worth pointing out that TMD0 harboring the A116P and V187D mutations have been shown to not coimmunoprecipitate with Kir6.2 (27), suggesting weakened association between mutant SUR1 and Kir6.2. Login to comment
254 Although we used the A116P and V187D-SUR1 mutations as two examples for probing the mechanism by which sulfonylureas rescue channel trafficking defect, we have found many more CHI-causing KATP mutants with trafficking defects to respond to sulfonylurea rescue.3 Our findings are therefore applicable to a growing number of naturally occurring channel mutations whose trafficking defects could be targeted for therapy. Login to comment
58 A, topology model of SUR1 showing the three transmembrane domains, the location of the A116P and V187D trafficking mutations, the location of the -RKR-ER retention motif, and the location of the S1238Y and Y230A mutations that have been proposed to disrupt the A- and B- sulfonyl- urea binding sites (as shown in B), respectively. Login to comment
88 Of these, two mutations, A116P and V187D, were rescued by the pharmacological agent sulfonylurea (23). Login to comment
90 Because both A116P and V187D are located in TMD0, we first tested if sulfonylureas bind directly to TMD0 to facilitate mutant protein biogenesis and trafficking, even though TMD0 has not been implicated in sulfonylurea binding in prior studies (21). Login to comment
94 By con- FIGURE2.ThefirsttransmembranedomainofSUR1(TMD0)doesnotconfer the sulfonylurea rescue effect on the A116P or V187D mutations. Login to comment
115 However, when combined with the A116P- or V187D-SUR1 trafficking mutations, S1238Y completely abolished the rescue effect of tolbutamide at 300 (Fig. 4B) and 600 òe;M (not shown). Login to comment
117 In the WT background (no sulfonylurea binding mutation), 24-h treatment with 1 òe;M glibenclamide increased surface expression of the A116P mutant from 3.1 afe; 0.7 to 34.5 afe; 7.2% and the V187D mutant from 12.8 afe; 1.6 to 49.2 afe; 6.8% of WT channels. Login to comment
139 Accordingly, we examined how repaglinide, a glinide that contains the benzamido moiety but not the sulfonylurea moiety, affects surface expression of A116P and V187D channels in the presence or absence of the Y230A mutation. Login to comment
152 Surface expression of tolbutamide-treated cells was significantly higher than untreated for A116P and V187D (p b0d; 0.001) but not A116P/S1238Y and V187D/S1238Y. Login to comment
154 Without the S1238Y mutation, both 1 and 5 òe;M glibenclamide significantly increased surface expression of the A116P and V187D trafficking mutants (p b0d; 0.001). Login to comment
164 Each bar is the mean afe; S.E. of three to five independent experiments. Sulfonylureas and KATP Channel Trafficking 33408 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281ߦNUMBER 44ߦNOVEMBER 3, 2006 the trafficking defects of the A116P and V187D mutants via direct interactions with the mutant channel proteins. Login to comment
174 The Role of Kir6.2 in Sulfonylurea Rescue of Channel Trafficking Defects-The data we presented so far demonstrate that intact sulfonylurea binding sites in SUR1 are necessary for effective rescue of the A116P- or V187D-SUR1 trafficking mutants by sulfonylureas. Login to comment
176 To investigate the role of Kir6.2 in sulfonylurea rescue of the A116P- or V187D-SUR1 trafficking mutants, we took advantage of the fact that inactivation of the -RKR-ER retention/retrieval motif by mutation to AAA (referred to as WTAAA in Fig. 8A) in SUR1 allows SUR1 to traffic to the cell surface without co-expression of Kir6.2 (29). Login to comment
179 These results are consistent with the A116P and V187D mutations causing defects in the SUR1 protein itself. Login to comment
211 Our results demonstrate that mutations in SUR1 previously reported to abolish or reduce sulfonylurea binding also abolish or reduce the ability of sulfonylureas to rescue channel trafficking defects caused by the A116P or V187D SUR1 mutations and that both the sulfonylurea and benzoamido moieties of glibenclamide contribute to the rescue effect. Login to comment
217 A reasonable hypothesis for how sulfonylureas improve surface expression of the A116P and V187D mutants is that A116P or V187D cause SUR1 misfolding and sulfonylureas, upon binding to the mutant SUR1, help it to adopt the correct conformation. Login to comment
226 The A116P or V187D trafficking mutation was engineered into WT-fSUR1 or fSUR1 in which the -RKR-ER retention motif has been inactivated by mutation to AAA. Login to comment
236 It is also worth pointing out that TMD0 harboring the A116P and V187D mutations have been shown to not coimmunoprecipitate with Kir6.2 (27), suggesting weakened association between mutant SUR1 and Kir6.2. Login to comment
252 Although we used the A116P and V187D-SUR1 mutations as two examples for probing the mechanism by which sulfonylureas rescue channel trafficking defect, we have found many more CHI-causing KATP mutants with trafficking defects to respond to sulfonylurea rescue.3 Our findings are therefore applicable to a growing number of naturally occurring channel mutations whose trafficking defects could be targeted for therapy. Login to comment

[hide] Regulation of KATP channel expression and activity... Channels (Austin). 2007 Jul-Aug;1(4):315-23. Epub 2007 Sep 25. Masia R, Caputa G, Nichols CG
Regulation of KATP channel expression and activity by the SUR1 nucleotide binding fold 1.
Channels (Austin). 2007 Jul-Aug;1(4):315-23. Epub 2007 Sep 25., [PMID:18708750]

Abstract [show]
ATP-sensitive K(+) (K(ATP)) channels are oligomeric complexes of pore-forming Kir6 subunits and regulatory Sulfonylurea Receptor (SUR) subunits. SUR, an ATP-Binding Cassette (ABC) transporter, confers Mg-nucleotide stimulation to the channel via nucleotide interactions with its two cytoplasmic domains (Nucleotide Binding Folds 1 and 2; NBF1 and NBF2). Regulation of K(ATP) channel expression is a complex process involving subunit assembly in the ER, SUR glycosylation in the Golgi, and trafficking to the plasma membrane. Dysregulation can occur at different steps of the pathway, as revealed by disease-causing mutations. Here, we have addressed the role of SUR1 NBF1 in gating and expression of reconstituted channels. Deletion of NBF1 severely impairs channel expression and abolishes MgADP stimulation. Total SUR1 protein levels are decreased, suggestive of increased protein degradation, but they are not rescued by treatment with sulfonylureas or the proteasomal inhibitor MG-132. Similar effects of NBF1 deletion are observed in recombinant K(ATP) channels obtained by "splitting" SUR1 into two separate polypeptides (a N-terminal "half" and a C-terminal "half"). Interestingly, the location of the "splitting point" in the vicinity of NBF1 has marked effects on the MgADP stimulation of resulting channels. Finally, ablation of the ER retention motif upstream of NBF1 (in either "split" or full-length SUR1) does not rescue expression of channels lacking NBF1. These results indicate that, in addition to NBF1 being required for MgADP stimulation of the channel, it plays an important role in the regulation of channel expression that is independent of the ER retention checkpoint and the proteasomal degradation pathway.

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142 The ubiquitination‑proteasomal pathway of degradation may be involved: this is the mechanism that underlies the effects of the PHHI mutants A116P and V187D.26 However, the proteasomal inhibitor MG‑132 failed to rescue total DNBF1 protein levels, and incubation with sulfonylureas, which facilitates SUR1 folding and prevents its degradation, was also without effect. Login to comment

[hide] Update of mutations in the genes encoding the panc... Hum Mutat. 2009 Feb;30(2):170-80. Flanagan SE, Clauin S, Bellanne-Chantelot C, de Lonlay P, Harries LW, Gloyn AL, Ellard S
Update of mutations in the genes encoding the pancreatic beta-cell K(ATP) channel subunits Kir6.2 (KCNJ11) and sulfonylurea receptor 1 (ABCC8) in diabetes mellitus and hyperinsulinism.
Hum Mutat. 2009 Feb;30(2):170-80., [PMID:18767144]

Abstract [show]
The beta-cell ATP-sensitive potassium (K(ATP)) channel is a key component of stimulus-secretion coupling in the pancreatic beta-cell. The channel couples metabolism to membrane electrical events bringing about insulin secretion. Given the critical role of this channel in glucose homeostasis it is therefore not surprising that mutations in the genes encoding for the two essential subunits of the channel can result in both hypo- and hyperglycemia. The channel consists of four subunits of the inwardly rectifying potassium channel Kir6.2 and four subunits of the sulfonylurea receptor 1 (SUR1). It has been known for some time that loss of function mutations in KCNJ11, which encodes for Kir6.2, and ABCC8, which encodes for SUR1, can cause oversecretion of insulin and result in hyperinsulinism of infancy, while activating mutations in KCNJ11 and ABCC8 have recently been described that result in the opposite phenotype of diabetes. This review focuses on reported mutations in both genes, the spectrum of phenotypes, and the implications for treatment on diagnosing patients with mutations in these genes.

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139 In the Finnish population, two founder mutations have been reported (V187D and E1507 K) [Otonkoski et al., 1999; Huopio et al., 2000]; the V187D mutation is associated with 50% of HI in this population [Otonkoski et al., 1999]. Login to comment

[hide] Acute insulin response tests for the differential ... J Clin Endocrinol Metab. 2002 Oct;87(10):4502-7. Huopio H, Jaaskelainen J, Komulainen J, Miettinen R, Karkkainen P, Laakso M, Tapanainen P, Voutilainen R, Otonkoski T
Acute insulin response tests for the differential diagnosis of congenital hyperinsulinism.
J Clin Endocrinol Metab. 2002 Oct;87(10):4502-7., [PMID:12364426]

Abstract [show]
Mutations in genes encoding the two subunits of the beta-cell ATP-sensitive potassium channel (K(ATP)) channel (SUR1 and Kir6.2) are the major cause of congenital hyperinsulinism (CHI). In this study, the K(ATP) channel genes were screened in a population-based study that included all verified Finnish CHI patients (n = 43) in a 27-yr period. Seven different mutations were identified, which accounted for 60% of all cases. The functional consequences of the major missense mutations were studied in vivo by determining acute (1-3 min) plasma insulin and C-peptide responses to calcium (n = 18), glucose (n = 12), and tolbutamide (n = 11) in those CHI patients who were able to take part in these studies. C-peptide and insulin responses to calcium were significantly higher in the patients with SUR1-E1506K mutation, compared with patients without K(ATP) channel mutations. The patients with SUR1-V187D mutation showed a reduced response to tolbutamide but unexpectedly did not show any response to calcium stimulation. A compound heterozygous patient with Kir6.2-(-54)/K67N mutations responded to calcium but also to tolbutamide. In conclusion, our results show that a positive response in the calcium test is indicative of a K(ATP) channel mutation, but all mutations cannot be identified with this method. The insulin response to tolbutamide in patients with SUR1 mutations is impaired to different extents, depending on the genotype. The combination of calcium and tolbutamide tests is a useful tool for the detection of CHI patients with K(ATP) channel dysfunction. Our results, however, also demonstrate the complexity of these responses and the difficulties in their interpretation.

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5 The patients with SUR1-V187D mutation showed a reduced response to tolbutamide but unexpectedly did not show any response to calcium stimulation. Login to comment
20 Indeed, the previously reported SUR1 mutations V187D (3) and E1506K (14) are the cause of most genetically characterized CHI cases. Login to comment
21 The mutation SUR1-V187D leads to total loss of function of KATP channels and severe drug-resistant phenotype. Login to comment
39 The fourth group was composed of one homozygous and five compound heterozygote patients with the mutation SUR1-V187D (aged 1-14 yr). Login to comment
41 The patients with paternal SUR1-V187D and maternal SUR1-A1457T (n ϭ 1) or SUR1-V1550D (n ϭ 1) were excluded from AIR tests because of the requirement of insulin more than 0.5 U/kg per day. Login to comment
43 All pancreatectomized patients who were included in AIR tests had the diffuse form of CHI as judged by histopathological examination (no KATP channel mutation, n ϭ 1; Kir6.2-(-54)/K67N, n ϭ 1; SUR1-E1506K, n ϭ 1; SUR1-V187D, n ϭ 5). Login to comment
58 Clinical characteristics of the patients Case Sex Age Cause of hyperinsulinism Previous treatment of hyperinsulinism No KATP channel mutation 1 M 2 Unknown Diazoxide 2 F 3 Unknown Octreotide 3 M 5 Unknown Diazoxide 4 M 20 Unknown Diazoxide, subtotal pancreatectomy 5 F 26 Unknown Diazoxide Kir6.2-(-54)/K67N 6 M 8 Paternal Kir6.2-K67N, maternal Kir6.2-(-54) Octreotide, subtotal pancreatectomy SUR1-E1506K 7 F 6 Dominant maternal SUR1-E1506K Diazoxide 8 F 9 Dominant maternal SUR1-E1506K Diazoxide 9 F 15 Dominant maternal SUR1-E1506K Frequent feeds 10 F 16 Dominant maternal SUR1-E1506K Diazoxide 11 F 19 Dominant maternal SUR1-E1506K Frequent feeds 12 M 27 Dominant maternal SUR1-E1506K Diazoxide, subtotal pancreatectomy SUR1-V187D 13 F 1 Paternal SUR1-V187D, maternal genotype pending Octreotide 14 F 6 Maternal SUR1-V187D, paternal genotype pending Subtotal pancreatectomy 15 M 8 Paternal SUR1-V187D, maternal genotype pending Subtotal pancreatectomy 16 F 8 Homozygous SUR1-V187D Subtotal pancreatectomy 17 F 9 Maternal SUR1-V187D, paternal genotype pending Subtotal pancreatectomy 18 F 14 Maternal SUR1-V187D, paternal genotype pending Subtotal pancreatectomy 19 M 11 Paternal SUR1-V187D, maternal SUR1-A1457T Subtotal pancreatectomy 20 F 13 Paternal SUR1-V187D, maternal SUR1-V1550D Subtotal pancreatectomy SUR1-L1551V 21 M 2 Paternal SUR1-L1551V, maternal genotype pending Diazoxide 22 F 0.2 Paternal SUR1-L1551V, maternal genotype pending Diazoxide Diabetic patients are shown in italics. Login to comment
82 The mutation A1457T in exon 36 was found to be maternally inherited in one compound heterozygote patient with the paternally inherited mutation SUR1-V187D (case 19). Login to comment
83 The mutation V1550D in exon 39 of SUR1 was maternally inherited in one individual who also had paternally inherited SUR1-V187D (case 20). Login to comment
87 Despite two separate screening processes of the KATP channel genes, we were not able to identify another SUR1 mutation in the five SUR1-V187D compound heterozygote patients who were tested with the AIR tests. Login to comment
90 First, three of the patients (cases 14, 17, and 18) had inherited the V187D mutation from their mother, which is inconsistent with focal disease. Login to comment
95 in all study groups: 0.19 mmol/liter in CHI patients without KATP mutations, 0.23 mmol/liter in the patient with both Kir6.2 mutations, 0.17 mmol/liter in SUR1-E1506K patients, and 0.23 mmol/liter in SUR1-V187D patients. Login to comment
96 The acute plasma C-peptide response to calcium was significantly increased in patients with SUR1-E1506K (159 Ϯ 28 pmol/liter), compared with either patients without KATP channel mutations (33 Ϯ 25 pmol/liter) (P Ͻ 0.05) or SUR1-V187D carriers (41 Ϯ 15 pmol/liter) (P Ͻ 0.05). Login to comment
97 The response to calcium was not significantly different between the SUR1-V187D carriers and patients without KATP channel mutations. Login to comment
98 It is obvious that the subjects with SUR1-V187D have very little remaining beta-cell function after the subtotal pancreatectomy and that this is maximally stimulated even under basal conditions. Login to comment
101 The plasma insulin and C-peptide responses to tolbutamide appeared to be lower in subjects with SUR-V187D and SUR-E1506K channel mutations, compared with the subjects without KATP channel mutations, but the differences were not statistically significant because of the small number of observations. Login to comment
105 It was clearly subnormal in the prepubertal SUR1-V187D homozygous patient (case 16) and in the postpubertal SUR1-E1506K heterozygotes. Login to comment
108 The two previously reported founder SUR1 mutations, V187D (3) and E1506K (14), account for 88% of the genetically characterized cases. Login to comment
110 Correlation between genotype and phenotype The verified compound heterozygote subjects (A1457T/ V187D and V1550D/V187D) show a very severe and drug-resistant disease phenotype. Login to comment
111 This is likely to be due to the total loss of channel activity by both of the novel mutations because we have shown that the mutation V187D alone does not cause any impairment of insulin secretion in heterozygous carriers (22). Login to comment
118 AIRs in nondiabetic patients shown as the means of the increments at 1 and 3 min Case Insulin response to calcium C-peptide response to calcium Insulin response to tolbutamide C-peptide response to tolbutamide Insulin response to glucose No KATP channel mutation 1 37 62 175 727 299 2 0 0 392 1099 607 3 20 49 139 5 38 101 906 1413 1438 Median 29 55 450 1132 453 Kir6.2-(-54)/K67N 6 284 652 491 1136 1083 SUR1-E1506K 7 62 147 127 306 197 8 55 171 476 1148 1001 9 36 77 34 36 105 10 83 265 34 93 75 11 42 200 26 33 74 Median 55 171 34 93 105 SUR1-V187D 13 10 80 166 550 216 16 5 30 1 8 42 Median 8 55 84 279 129 Reference values 1 Ϯ 4a 318 Ϯ 72b 252 Ϯ 54b (-12-25) (158-478) The individual results and median values are shown for each group, expressed as picomoles per liter. Login to comment
147 Unexpectedly, subjects with SUR1-V187D did not respond to calcium stimulation. Login to comment
154 The C-peptide response to tolbutamide was severely decreased in SUR1-V187D subjects. Login to comment
155 This finding is in agreement with the previous results of ion channel recordings of beta-cells isolated from a SUR1-V187D homozygous patient. Login to comment
156 Unlike control cells that were activated by diazoxide and inhibited by tolbutamide, no actions of KATP channel agonists diazoxide or octreotide were seen in the cells with SUR1-V187D mutation (3). Login to comment
163 The results show, however, that a negative response to calcium stimulation does not exclude the possibility that a subject has a KATP channel mutation, as clearly demonstrated by the major Finnish SUR1 mutation V187D. 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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38 Review J Med Genet 2009;46:289-299. doi:10.1136/jmg.2008.064337 Homozygous, compound heterozygous and heterozygous recessive inactivating mutations (missense, frameshift, nonsense, insertions/deletions (macrodeletion), splice site and regulatory mutations) have been reported in ABCC8 and KCNJ11.10-17 67 68 So far, more than 150 mutations have been reported in ABCC8 and 25 in KCNJ11.69 In the Ashkenazi Jewish population two common mutations (F1388del and c.3992-9G4A) account for 90% of all cases of CHI9 10 whereas in the Finnish population, two founder mutations have been reported (V187D and E1507 K).14 22 Recessive inactivating mutations in ABCC8 and KCNJ11 usually cause severe CHI which in the vast majority of patients is unresponsive to medical treatment with diazoxide. Login to comment

[hide] A point mutation inactivating the sulfonylurea rec... Diabetes. 1999 Feb;48(2):408-15. Otonkoski T, Ammala C, Huopio H, Cote GJ, Chapman J, Cosgrove K, Ashfield R, Huang E, Komulainen J, Ashcroft FM, Dunne MJ, Kere J, Thomas PM
A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland.
Diabetes. 1999 Feb;48(2):408-15., [PMID:10334322]

Abstract [show]
Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.

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4 Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Login to comment
5 Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No KATP channel activity was observed in -cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Login to comment
57 To detect the V187D mutation, PCR amplification of genomic DNA was performed using the primers 5 -GTGAGTGTACACATGATG and 5 -CAGAGCCA GAGCCTCTGCTT. Login to comment
85 Oocytes were then co-injected with a mixture of mRNAs encoding Kir6.2 (~0.04 ng) and either wild-type SUR1 or V187D SUR1 (~2 ng) at dilutions yielding a final injection volume of about ~50 nl/oocyte. Login to comment
89 Open circles represent carriers of the V187D mutation of SUR1, detected with the Tth111I restriction endonuclease test. Login to comment
123 Even in this TABLE 1 Major clinical findings in Finnish PHHI patients Gestational Birth weight Blood glucose (mmol/l); Age at Mutation Patient Sex age (weeks) (g) plasma insulin (pmol/l) pancreatectomy (days) V187D 1 M 34 3,920 (+4.6) 0.5; 534 23 +/- 2 M 33 2,350 (+0.2) 5.0; 282 29 - 3 F 35 3,120 (+1.1) 5.0; 342 22 +/- 4 M 36 3,940 (+2.4) 3.0; 564 16 +/+ 5 M 39 5,080 (+3.3) 1.8; 96 57 +/- 6 M 39 3,970 (+0.9) 1.2; 78 - - 7 F 37 3,420 (+0.7) 1.4; 236 38 +/- 8 F 39 4,020 (+1.3) 1.5; 341 161 +/- 9 F 29 2,070 (+4.6) 2.2; 440 31 +/+ 10 M 40 3,710 (0.0) 1.5; 190 38 +/- 11 F 39 3,900 (+1.1) 1.6; 654 11 +/- 12 F 33 3,710 (+5.0) 1.4; 1040 12 +/+ 13 F 37 4,100 (+2.4) 1.7; 149 59 +/+ 14 F 34 3,700 (+4.0) 2.2; 396 - - 15 M 39 3,845 (+0.7) 2.2; 1200 11 +/- 16 M 36 3,150 (+0.4) 1.2; 186 - - 17 M 36 5,250 (+5.6) 2.2; 874 21/196 +/+ 18 M 35 2,910 (+0.3) 2.8; 215 82 +/- 19 M 35 4,565 (+5.2) 4.1; 850 11 - 20 F 38 4,330 (+0.6) 1.5; 138 - - 21 M 28 1,770 (+3.8) 1.8; 615 15 (+/+)* 22 M 39 2,900 (-1.5) 2.1; 294 - - 23 F 37 5,360 (+5.7) 2.4; 984 - - 24 F 37 4,490 (+3.3) 1.2; 257 - - Data are n or n (SD). Login to comment
136 The geographicaldistribution of birthplaces for parents carrying the marker haplotype and V187D supports a single origin for the mutation (Fig. 1). Login to comment
144 This point mutation is predicted to substitute a valine for an aspartic acid residue at site 187 of the SUR1 protein (V187D). Login to comment
150 An additional 20 Finnish PHHI families were analyzed for the presence of the V187D mutation. Login to comment
153 Presence of the V187D mutation was associated with a severeclinical phenotype. Login to comment
155 None of the 23 additional individuals who came from outside Finland, were of diverse ethnic origin, and were affected with classic PHHI possessed the V187D mutation as assessed by the Tth111I restriction assay. Login to comment
156 This demonstrated that the V187D mutation is rare in the PHHI population at large and that its prevalence within the Finnish population may be attributed to a founder effect. Login to comment
160 of control Haplotype with V187D without V187D chromosomes 2-10-5-7-8-8 13 0 0 3-10-5-7-8-8 1 0 1 2-10-5-7-8-7 1 0 0 2-10-5-7-10-7 1 0 0 2-10-5-7-2-9 1 0 0 2-10-5-7-10-10 1 0 0 All other haplotypes 0 20 32 Total 18 24 33 The PHHI-associated chromosomes were divided in two groups based on the occurrence of the V187D mutation. Login to comment
161 All haplotypes associated with V187D were considered derivatives of a single ancestral chromosome with historical recombinations. Login to comment
166 We therefore screened 100 chromosomes from normal individuals living in this area and found the V187D mutation in heterozygous form in one of them. Login to comment
168 These results are consistent with the idea that V187D is not a common polymorphism. Login to comment
170 The functional properties of KATP channels in -cells isolated from a PHHI patient homozygous for the V187D mutation (case 4) were studied using both intact cell recordings and cell-free inside-out patches. Login to comment
180 The relationship between the novel SUR1 gene defect and loss of channel function was therefore investigated in recombinant experiments with SUR1 containing the V187D mutation identified in our PHHI patients. Login to comment
182 Whole-cell currents were recorded from Xenopus oocytes injected with mRNA encoding Kir6.2 and with either wild-type SUR1 (wt-SUR1) or SUR1 engineered to carry the mutation found in the Finnish population of PHHI patients (SUR1-V187D). Login to comment
183 Under basal conditions, currents recorded from oocytes injected with Kir6.2/wt-SUR1 (n = 5) or Kir6.2/SUR1-V187D (n = 5) were no different from those measured in oocytes injected with water (n = 4), entirely consistent with previous observations (24). Login to comment
185 No such increase in current was observed in response to sodium azide in either control oocytes or oocytes injectedwith Kir6.2/SUR1-V187D (Fig. 4A and B). Login to comment
186 This lack of response observed in Kir6.2/SUR1-V187D-injected oocytes may have arisen because the mutant channel is insensitive to metabolic regulation or because it does not form a functional channel in the plasma membrane. Login to comment
187 To determine which of these possibilities is correct, we recordedrecombinant KATP channel activityingiant inside-out membranepatches.Figure4C shows currents elicitedbya voltage-ramp from -100 to +100 mV in the cell-attached condition (c/a) and after excision (i/o) into the ATP-free solution recorded from oocytes injected with wt-SUR1/Kir6.2 or Kir6.2/SUR1-V187D. Login to comment
189 By contrast, there was no increase in KATP channel activity (Fig. 4C [right panel] and D) in patches excised fromoocytes injected with Kir6.2/SUR1-V187D. Login to comment
190 These findings were consistent with those obtained from acutely isolated PHHI -cells (Fig. 3B) and suggested that the presence of the V187D mutation renders the channel completely nonfunctional. Login to comment
192 We have reported here that a single mutation in the SUR1 gene, V187D, detected in 18of42disease-associated chromosomes,revealed FIG. 2. Login to comment
193 Demonstration of the V187D mutation. Login to comment
205 The V187D mutationwas not detected in 9 families, although in 5 of these families, one or both of the disease-associated chromosomes carried partial haplotypes observedin otherpatients heterozygous for V187D. Login to comment
210 The in vitro studies described here have demonstrated clearly that the V187D point mutation leads to loss of functional KATP channelexpression, even in excised patches, and thus falls into the former group. Login to comment
214 The V187D mutation appears to be more severe than N188S, as we could not observe any KATP channel activity in excised patches. Login to comment
222 In contrast, in Finnish patients the V187D mutation was strongly associated with a severe form of the disease. Login to comment
223 It is interesting that the clinical phenotype of PHHI was equally severe, whether the patients were homo- or heterozygous for the V187D mutation. Login to comment
234 presence ofeven a single copy of the V187D mutation will prevent the generation of functionally operating KATP channels with high diazoxide sensitivity, if another, possibly less severe mutation is also present. Login to comment
236 The presence of the V187D mutation can be easily detected. Login to comment
238 Identification of the V187D mutation is prognostically valuable because we observed that this mutation was invariably associated with a severe disease that responded poorly to medication in all cases. Login to comment

[hide] Characterisation of new KATP-channel mutations ass... Diabetologia. 2003 Feb;46(2):241-9. Epub 2003 Jan 9. Reimann F, Huopio H, Dabrowski M, Proks P, Gribble FM, Laakso M, Otonkoski T, Ashcroft FM
Characterisation of new KATP-channel mutations associated with congenital hyperinsulinism in the Finnish population.
Diabetologia. 2003 Feb;46(2):241-9. Epub 2003 Jan 9., [PMID:12627323]

Abstract [show]
AIMS/HYPOTHESIS: ATP-sensitive potassium (K(ATP)) channels are crucial for the regulation of insulin secretion from pancreatic beta cells and mutations in either the Kir6.2 or SUR1 subunit of this channel can cause congenital hyperinsulinism (CHI). The aim of this study was to analyse the functional consequences of four CHI mutations (A1457T, V1550D and L1551V in SUR1, and K67N in Kir6.2) recently identified in the Finnish population. METHODS: Wild type or mutant Kir6.2 and SUR1 subunits were coexpressed in Xenopus oocytes. The functional properties of the channels were examined by measuring currents in intact oocytes or giant inside-out membrane patches. Surface expression was measured by enzyme-linked immunosorbance assay, using HA-epitope-tagged subunits. RESULTS: Two mutations (A1457T and V1550D) prevented trafficking of the channel to the plasma membrane. The L1551V mutation reduced surface expression 40-fold, and caused loss of MgADP and diazoxide activation. Both these factors will contribute to the lack of K(ATP) current activation observed in response to metabolic inhibition in intact oocytes. The L1551V mutation also increased the channel open probability, thereby producing a reduction in ATP-sensitivity (from 10 micro mol/l to 120 micro mol/l). The fourth mutation (K67N mutation in Kir6.2) did not affect surface expression nor alter the properties of K(ATP) channels in excised patches, but resulted in a reduced K(ATP) current amplitude in intact cells on metabolic inhibition, through an unidentified mechanism. CONCLUSION/INTERPRETATION: The four CHI mutations disrupted K(ATP) channel activity by different mechanisms. Our results are discussed in relation to the CHI phenotype observed in patients with these mutations.

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64 The mutations V187D and E1506K have been described previously [3, 5]. Login to comment
186 In both cases they occurred as part of a complex heterozygous genotype, with the SUR1 mutation V187D on the second allele [3]. Login to comment
187 As SUR1-V187D also abolished KATP channel activity [3], these two subjects would be predicted to have no functional KATP channels, consistent with the observed severe, drug-resistant CHI phenotype [23]. Login to comment

[hide] The spectrum of ABCC8 mutations in Norwegian patie... Clin Genet. 2009 May;75(5):440-8. Sandal T, Laborie LB, Brusgaard K, Eide SA, Christesen HB, Sovik O, Njolstad PR, Molven A
The spectrum of ABCC8 mutations in Norwegian patients with congenital hyperinsulinism of infancy.
Clin Genet. 2009 May;75(5):440-8., [PMID:19475716]

Abstract [show]
Potassium channels in the plasma membrane of the pancreatic beta cells are critical in maintaining glucose homeostasis by responding to ATP and coupling metabolic changes to insulin secretion. These channels consist of subunits denoted the sulfonylurea receptor SUR1 and the inwardly rectifying ion channel KIR6.2, which are encoded by the genes ABCC8 and KCNJ11, respectively. Activating mutations in the subunit genes can result in monogenic diabetes, whereas inactivating mutations are the most common cause of congenital hyperinsulinism of infancy (CHI). Twenty-six Norwegian probands with CHI were analyzed for alterations in ABCC8 and KCNJ11. Fifteen probands (58%) had mutations in the ABCC8 gene. Nine patients were homozygous or compound heterozygous for the mutations, indicating diffuse pancreatic disease. In five patients, heterozygous and paternally inherited mutations were found, suggesting focal disease. One patient had a de novo mutation likely to cause a milder, dominant form of CHI. Altogether, 16 different ABCC8 mutations (including the novel alterations W231R, C267X, IVS6-3C>G, I462V, Q917X and T1531A) were identified. The mutations IVS10+1G>T, R1493W and V21D occurred in five, three and two families, respectively. KCNJ11 mutations were not found in any patients. Based on our mutation screening, we estimate the minimum birth prevalence of ABCC8-CHI in Norway to 1:70,000 during the past decade. Our results considerably extend the knowledge of the molecular genetics behind CHI in Scandinavia.

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109 Clinical characteristics of Norwegian CHI patients carrying mutations in ABCC8 Proband Sex Birth weight (g)/gestation length (weeks)a Treatment Mutationsd Medicalb Surgeryc Maternal chromosome Paternal chromosome Hypo-N3 F 6190/38 Deceased Yes (S) R1493W R1493W Hypo-N6 M 5340/38 Somatostatin, diet (FM, PEG) No V21D V21D Hypo-N8 F 5740/37 Insulin Yes (S) G1400R R1493W Hypo-N9 F 5130/40 Diet (FM) Yes (S) - IVS1011G.T Hypo-N11 M 4000/38 None No - G1478Re Hypo-N14 M 5000/40 Somatostatin, diet (FM, PEG) No - IVS1011G.T Hypo-N16 F 3780/38 Diet (FM) No - C267X Hypo-N19 F 5240/40 Somatostatin, diet (FM, PEG) No IVS1011G.T T1531Af Hypo-N22 M 4500/39 Diazoxide Yes (S) IVS6-3C.G, I462V Q917X Hypo-N23 F 4860/38 Insulin Yes (S) P1413Lg IVS1011G.Tg Hypo-N25 M 3910/34 Insulin Yes (S) V21Dg E490Xg Hypo-N26 M 3790/35 Diet (FM, PEG) Yes (H) V187D R248X Hypo-N29 F 3350/37 None Yes (P) - IVS1011G.T Hypo-N30 F 3800/37 Diazoxide No W231R L503P Hypo-N31 M 4340/40 None Yes (P) - R1493W a All cases had birth weights 12 standard deviation scores except for Hypo-N29 whose score was 11. b Current therapy is given. Login to comment
122 We classified the mutations as either MnMn Hypo-N3 R1493W MMMM Mn Mn Hypo-N6 V21D MM MnMn Hypo-N8 G1400R / R1493W MM nnMn Hypo-N9 IVS10 Mn Hypo-N11 G1478R Mn nnMn Hypo-N16 C267X Mn Mn Hypo-N19 IVS10 / T1531A MM Mn nn Hypo-N29 IVS10 Mn Mn Hypo-N30 W231R / L503P MM MM x Hypo-N23 IVS10 / P1413L MM x Hypo-N14 IVS10 Mn Hypo-N22 IVS6 (I462V) / Q917X MM Hypo-N25 V21D / E490X MM xx Hypo-N26 V187D / R248 X MM x Hypo-N31 R1493W nnMnMnMn nnnnMn MnMn MM MnMn Mn nnnn Fig. 1. Login to comment
133 ABCC8 mutations found in Norwegian CHI patientsa Nucleotide change Location Amino acid change Mutation type PSIC score PD Number of families Reference c.62 T.A Exon 1 V21D Mis 1.96 PoD 2 (24) c.560 T.A Exon 4 V187D Mis 2.01 PrD 1 (2) c.691 T.C Exon 5 W231R Mis 4.03 PrD 1 NR c.742 C.T Exon 5 R248X Non - - 1 (34, 42) c.801 C.A Exon 5 C267X Non - - 1 NR IVS6-3C.G Intron 6 - AS - - 1 NR c.1384 A.G Exon 9 I462V Mis 0.62 PrB 1 NR c.1468 G.T Exon 10 E490X Non - - 1 (43) c.1508 T.C Exon 10 L503P Mis 2.36 PrD 1 (24) IVS1011G.T Intron 10 - AS - - 5 (44) c.2749 C.T Exon 23 Q917X Non - - 1 NR c.4198 G.A Exon 35 G1400R Mis 2.37 PrD 1 (42) c.4238 C.T Exon 35 P1413L Mis 2.76 PrD 1 (25) c.4432 G.A Exon 37 G1478R Mis 2.37 PrD 1 (14, 31) c.4477 C.T Exon 37 R1493W Mis 2.79 PrD 3 (26) c.4591 A.G Exon 38 T1531A Mis 1.93 PoD 1 NR AS, aberrant splicing; Mis, missense; NR, not previously reported; Non, nonsense; PD, pathogenic description; PoD, possibly damaging; PrB, predicted to be benign; PrD, probably damaging; PSIC, position-specific independent counts. Login to comment
168 A largenumber (.150)ofABCC8 alterations have been reported to cause CHI (19) including 10 of the mutations observed in this study (V21D, V187D, R248X, E490X, L503P, IVS1011G.T, G1400R, P1413L, G1478R, and R1493W). Login to comment
187 One example is V187D that is located in TMD0 and was found in family Hypo-N26. Login to comment
189 V187D is a founder mutation in Finland (2), and further investigation revealed that the mother of the proband was of Finnish ancestry. Login to comment

[hide] Compounds that correct F508del-CFTR trafficking ca... Orphanet J Rare Dis. 2013 Jan 14;8:11. doi: 10.1186/1750-1172-8-11. Sampson HM, Lam H, Chen PC, Zhang D, Mottillo C, Mirza M, Qasim K, Shrier A, Shyng SL, Hanrahan JW, Thomas DY
Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines.
Orphanet J Rare Dis. 2013 Jan 14;8:11. doi: 10.1186/1750-1172-8-11., [PMID:23316740]

Abstract [show]
BACKGROUND: Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. METHODS: To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also known as hERG), and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1). We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). RESULTS: Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested. CONCLUSIONS: Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases.

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24 We investigated mutants from a diverse set of well-studied protein trafficking diseases including the nephrogenic diabetes insipidus mutations V206D and L292P in the arginine-vasopressin receptor 2 (AVPR2, also known as V2R) [24,25], the LQTS2 mutations G601S and F805C in the human ether-a-go-go-related gene (KCNH2, also known as hERG) [26,27], and finally the persistent hyperinsulinemic hypoglycemia of infancy (PHHI, also known as congenital hyperinsulinism) mutations A116P and V187D in the sulfonylurea receptor 1 (ABCC8, also known as SUR1) [10,28,29], a component of the KATP channel. Login to comment
34 FLAG-tagged hamster SUR1 WT, A116P and V187D mutants and rat Kir6.2 plasmids have been described previously [32]. Login to comment
120 6 F508del-CFTR correctors correct the trafficking of SUR1 mutants V187D and A116P. Login to comment
121 Representative immunoblots are shown for SUR1 V187D (A-C) and SUR1 A116P (D-F). Login to comment
122 Representative immunoblot for cells expressing SUR1 V187D treated with decreasing concentrations of ouabain (ouab), KM57, and RDR1 (A), KM60, ABT-888, and latonduine (latond) (B), glafenine and carbamazepine (carbam) (C). Login to comment
125 Lanes where Kir6.2 and SUR1 V187D or SUR1 A116P are expressed are indicated by a line above the corresponding lanes. Login to comment
131 We next tested the SUR1 mutants V187D and A116P for correction with F508del-CFTR correctors. Login to comment
158 Interestingly, sulfonylureas correct the trafficking of A116P and V187D mutants by binding to sites outside the affected domain in SUR1, and possibly also with a weak affinity site in Kir6.2 [36]. Login to comment
167 Table 1 F508del-CFTR corrector compounds show distinct profiles of correction for other ER-retained proteins Corrector CFTR F508del hERG G601S hERG F805C SUR1 A116P SUR1 V187D V2R L292P V2R V206D VRT-325 + + - - ND - ND Glycerol + ND ND + ND ND +/- 29&#b0;C ++ + + + + ++ ++ KM60 + + - + + - + KM57 +/- ++ - +/- - - +/- ABT-888 + - ND + + - + Glafenine + + - + - - - RDR1 + - - + + - - Ouabain + +/-* +* + + + + Carbamazepine + - ND - +/- - ND Latonduine + +/- - + ++ +/- + Astemizole ND + - ND ND ND ND Glibenclamide ND ND ND ++ ++ ND ND A qualitative assessment of correction as determined by glycosylation status in immunoblotting is shown for each mutation following treatment with a corrector compound, where "-" indicates no correction observed, "+/-" indicates slight correction, "+" and "++" indicate more and best correction observed, respectively, and "ND" indicates not determined. Login to comment

[hide] Carbamazepine as a novel small molecule corrector ... J Biol Chem. 2013 Jul 19;288(29):20942-54. doi: 10.1074/jbc.M113.470948. Epub 2013 Jun 6. Chen PC, Olson EM, Zhou Q, Kryukova Y, Sampson HM, Thomas DY, Shyng SL
Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.
J Biol Chem. 2013 Jul 19;288(29):20942-54. doi: 10.1074/jbc.M113.470948. Epub 2013 Jun 6., [PMID:23744072]

Abstract [show]
ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to beta-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human beta-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

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125 At 10 òe;M, the F27S and E128K mutations exhibited the greatest improvement to nearly the level seen with 5 òe;M glibenclamide; R74W, A116P, and V187D showed moderate responses; whereas G7R and N24K, which have less severe processing defects (31), had weak responses (Fig. 1C). Login to comment
126 Dose-response relationships were further determined for F27S, A116P, and V187D. Login to comment
136 In surface protein biotinylation experiments, there was a significant increase in biotinylated F27S, A116P, or V187D SUR1 in cells treated with carbamazepine or glibenclamide as compared with cells treated with vehicle alone (Fig. 3A). Login to comment
138 Surface staining of FLAG-tagged (N terminus) SUR1 showed a clear increase in surface expression of the F27S mutant upon Carbamazepine as a Novel KATP Channel Corrector JULY 19, 2013ߦVOLUME 288ߦNUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20945 carbamazepine treatment, resembling that seen in cells treated with the sulfonylurea drug tolbutamide (Fig. 3B). Login to comment
127 At 10 òe;M, the F27S and E128K mutations exhibited the greatest improvement to nearly the level seen with 5 òe;M glibenclamide; R74W, A116P, and V187D showed moderate responses; whereas G7R and N24K, which have less severe processing defects (31), had weak responses (Fig. 1C). Login to comment
128 Dose-response relationships were further determined for F27S, A116P, and V187D. Login to comment

[hide] 5'-adenosine monophosphate mediated cooling treatm... J Biomed Sci. 2015 Sep 4;22:72. doi: 10.1186/s12929-015-0178-3. Zhang Y, O'Brien WG 3rd, Zhao Z, Lee CC
5'-adenosine monophosphate mediated cooling treatment enhances DeltaF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo.
J Biomed Sci. 2015 Sep 4;22:72. doi: 10.1186/s12929-015-0178-3., [PMID:26335336]

Abstract [show]
BACKGROUND: Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5'-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (DeltaF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of DeltaF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport. RESULTS: Here, we report that whole body cooling enhanced the retention of DeltaF508-CFTR in intestinal epithelial cells. Functional analysis based on beta-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice. CONCLUSIONS: Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.

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20 Two mutations in the pancreatic ATP-sensitive potassium channels A116P and V187D, located in the SUR1 subunit, reduce channel activity leading to persistent infancy hyperinsulinemic hypoglycemia. Login to comment

[hide] KATP channel mutations in congenital hyperinsulini... Semin Pediatr Surg. 2011 Feb;20(1):18-22. doi: 10.1053/j.sempedsurg.2010.10.012. Saint-Martin C, Arnoux JB, de Lonlay P, Bellanne-Chantelot C
KATP channel mutations in congenital hyperinsulinism.
Semin Pediatr Surg. 2011 Feb;20(1):18-22. doi: 10.1053/j.sempedsurg.2010.10.012., [PMID:21185999]

Abstract [show]
Adenosine triphosphate (ATP)-sensitive potassium channels (K(ATP) channels) have a central role in the regulation of insulin secretion in pancreatic beta cells. They are octameric complexes organized around the central core constituted by the Kir6.2 subunits. The regulation of the channel itself takes place on the sulfonylurea receptor-1 subunit. The channel opens and closes according to the balance between adenine nucleotide ATP and adenosine diphosphate. Hyperinsulinemic hypoglycemia (also named congenital hyperinsulinism, or CHI) is associated with loss-of-function K(ATP) channel mutations. Their frequency depends on the histopathological form and the responsiveness of CHI patients to diazoxide. ABCC8/KCNJ11 defects are identified in approximately 80% of patients with CHI refractory to diazoxide. Within this group, focal forms are related to a paternally inherited KCNJ11 or ABCC8 mutation and the loss of the corresponding maternal allele in some pancreatic beta cells leading to a focal lesion. Diffuse forms are mostly associated with recessively inherited mutations. Some patients with diffuse forms also carried a single K(ATP) channel mutation. In contrast, K(ATP) mutations are involved in 15% of diazoxide-responsive CHI cases that are either sporadic or dominantly inherited.

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73 Missense, frameshift, nonsense, insertions/deletions, splice-site mutations, and rare exonic deletions have been reported in CHI.7,15 No "hot spots" for mutations are present; most mutations are unique to the respective family except for 2 mutations common (F1388del and c.3992-9G b0e; A) in the Ashkenazi Jewish population18 and 2 founder mutations (V187D and E1507K) in the Finnish population.19,20 The frequency of KATP mutations identified is different according to the histopathological form and the responsiveness of CHI patients to diazoxide. Login to comment

[hide] Clinical and molecular characterisation of 300 pat... Eur J Endocrinol. 2013 Mar 15;168(4):557-64. doi: 10.1530/EJE-12-0673. Print 2013 Apr. Kapoor RR, Flanagan SE, Arya VB, Shield JP, Ellard S, Hussain K
Clinical and molecular characterisation of 300 patients with congenital hyperinsulinism.
Eur J Endocrinol. 2013 Mar 15;168(4):557-64. doi: 10.1530/EJE-12-0673. Print 2013 Apr., [PMID:23345197]

Abstract [show]
BACKGROUND: Congenital hyperinsulinism (CHI) is a clinically heterogeneous condition. Mutations in eight genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A and HNF1A) are known to cause CHI. AIM: To characterise the clinical and molecular aspects of a large cohort of patients with CHI. METHODOLOGY: Three hundred patients were recruited and clinical information was collected before genotyping. ABCC8 and KCNJ11 genes were analysed in all patients. Mutations in GLUD1, HADH, GCK and HNF4A genes were sought in patients with diazoxide-responsive CHI with hyperammonaemia (GLUD1), raised 3-hydroxybutyrylcarnitine and/or consanguinity (HADH), positive family history (GCK) or when CHI was diagnosed within the first week of life (HNF4A). RESULTS: Mutations were identified in 136/300 patients (45.3%). Mutations in ABCC8/KCNJ11 were the commonest genetic cause identified (n=109, 36.3%). Among diazoxide-unresponsive patients (n=105), mutations in ABCC8/KCNJ11 were identified in 92 (87.6%) patients, of whom 63 patients had recessively inherited mutations while four patients had dominantly inherited mutations. A paternal mutation in the ABCC8/KCNJ11 genes was identified in 23 diazoxide-unresponsive patients, of whom six had diffuse disease. Among the diazoxide-responsive patients (n=183), mutations were identified in 41 patients (22.4%). These include mutations in ABCC8/KCNJ11 (n=15), HNF4A (n=7), GLUD1 (n=16) and HADH (n=3). CONCLUSIONS: A genetic diagnosis was made for 45.3% of patients in this large series. Mutations in the ABCC8 gene were the commonest identifiable cause. The vast majority of patients with diazoxide-responsive CHI (77.6%) had no identifiable mutations, suggesting other genetic and/or environmental mechanisms.

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168 Two ABCC8 mutations, c.3992-9GOA and p.F1388del, are associated with CHI in the Ashkenazi Jewish population (11) and the p.V187D mutation has been associated with the Finnish population (46). Login to comment

[hide] Pharmacological rescue of trafficking-impaired ATP... Front Physiol. 2013 Dec 24;4:386. doi: 10.3389/fphys.2013.00386. Martin GM, Chen PC, Devaraneni P, Shyng SL
Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels.
Front Physiol. 2013 Dec 24;4:386. doi: 10.3389/fphys.2013.00386., [PMID:24399968]

Abstract [show]
ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic beta-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The beta-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed.

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214 Among the mutations documented, A116P- and V187D-SUR1, both located in TMD0, exhibited reduced association with Kir6.2 in co-immunoprecipitation experiments (Chan et al., 2003), supporting a role of TMD0 in subunit-subunit interactions. Login to comment
218 Mutation Domain Rescue Rescue Gating References by SU by CBZ property SUR1 G7R TMD0 Yes Yes Normal Yan et al., 2007 N24K TMD0 Yes Yes Normal Yan et al., 2007 F27S TMD0 Yes Yes Normal Yan et al., 2007 R74W TMD0 Yes Yes ATP-insensitive Yan et al., 2007 A116P TMD0 Yes Yes Normal Yan et al., 2004 E128K TMD0 Yes Yes ATP-insensitive Yan et al., 2007 V187D TMD0 Yes Yes Normal Yan et al., 2004 R495Q TMD1 Yes Yes Unknown Yan et al., 2007 E501K TMD1 Yes Yes Unknown Yan et al., 2007 L503P TMD1 No No Unknown Yan et al., 2007 F686S NBD1 No No Unknown Yan et al., 2007 G716V NBD1 No No Unknown Yan et al., 2007 E1324K TMD2 N.D.3 N.D. Login to comment
248 Subsequent work identified additional SUR1 mutations in CHI patients that impair the proper trafficking of KATP channels, including L1544P, A116P, and V187D (Taschenberger et al., 2002; Yan et al., 2004). Login to comment
250 Yan et al. (2004) demonstrated that two CHI mutations, A116P and V187D, both located in the first transmembrane domain TMD0 of SUR1, could be rescued by sulfonylureas in vitro. Login to comment
254 Previously, Chan et al. showed that TMD0 domain of SUR1 harboring the A116P or V187D mutation, had reduced association with Kir6.2 in co-immunoprecipitation experiments (Chan et al., 2003). Login to comment
256 Yan et al. showed, however, that the trafficking defect in A116P and V187D is intrinsic to SUR1. Login to comment
257 This is based on the observation that in the absence of Kir6.2, A116P and V187D also prevented Kir6.2-independent surface expression of a SUR1 protein in which the RKR ER retention signal is inactivated by mutation to AAA (SUR1AAA). Login to comment
259 Yet mutation of these signals in both subunits also failed to improve surface expression of the A116P or V187D mutants. Login to comment
261 Consistent with this notion, channel trafficking defects caused by A116P and V187D could be overcome by culturing cells at lower temperature (Yang et al., 2005), a condition known to facilitate protein folding. Login to comment
266 Accordingly, mutation of S1238 to tyrosine abolished tolbutamide rescue of SUR1 mutants A116P and V187D. Login to comment
305 A more recent study by Sampson et al. tested the effects of multiple CFTR correctors identified in a chemical library screen (Carlile et al., 2007) on the processing efficiency of two SUR1 trafficking mutants, A116P and V187D (Sampson et al., 2013). Login to comment

[hide] Structurally distinct ligands rescue biogenesis de... J Biol Chem. 2015 Mar 20;290(12):7980-91. doi: 10.1074/jbc.M114.634576. Epub 2015 Jan 30. Devaraneni PK, Martin GM, Olson EM, Zhou Q, Shyng SL
Structurally distinct ligands rescue biogenesis defects of the KATP channel complex via a converging mechanism.
J Biol Chem. 2015 Mar 20;290(12):7980-91. doi: 10.1074/jbc.M114.634576. Epub 2015 Jan 30., [PMID:25637631]

Abstract [show]
Small molecules that correct protein misfolding and misprocessing defects offer a potential therapy for numerous human diseases. However, mechanisms underlying pharmacological correction of such defects, especially in heteromeric complexes with structurally diverse constituent proteins, are not well understood. Here we investigate how two chemically distinct compounds, glibenclamide and carbamazepine, correct biogenesis defects in ATP-sensitive potassium (KATP) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2. We present evidence that despite structural differences, carbamazepine and glibenclamide compete for binding to KATP channels, and both drugs share a binding pocket in SUR1 to exert their effects. Moreover, both compounds engage Kir6.2, in particular the distal N terminus of Kir6.2, which is involved in normal channel biogenesis, for their chaperoning effects on SUR1 mutants. Conversely, both drugs can correct channel biogenesis defects caused by Kir6.2 mutations in a SUR1-dependent manner. Using an unnatural, photocross-linkable amino acid, azidophenylalanine, genetically encoded in Kir6.2, we demonstrate in living cells that both drugs promote interactions between the distal N terminus of Kir6.2 and SUR1. These findings reveal a converging pharmacological chaperoning mechanism wherein glibenclamide and carbamazepine stabilize the heteromeric subunit interface critical for channel biogenesis to overcome defective biogenesis caused by mutations in individual subunits.

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148 TMD0 trafficking mutations F27S, A116P, and V187D used in the study as well as the ER retention motif RKR are also shown. Login to comment
163 For this set of experiments, SUR1-TMD0 trafficking mutations A116P and V187D, which we have shown previously to respond to GBC and CBZ rescue (20, 22, 23, 41, 44), were used as examples. Login to comment
164 In SUR1RKR3AAA bearing A116P or V187D expressed without Kir6.2, both exhibited only the core-glycosylated lower band, in contrast to WT-SUR1RKR3AAA, which showed both lower and upper bands; treatment with CBZ failed to correct the mutant SUR1 processing defects (Fig. 3A). Login to comment
167 Interestingly, we noted that CBZ treatment significantly enhanced the core-glycosylated A116P- and V187D- SUR1RKR3AAA band intensity even in the absence of Kir6.2. Login to comment