ABCMdb

ABCC8 p.Ala116Pro

Predicted by SNAP2:	C: N (61%), D: N (53%), E: N (57%), F: N (57%), G: N (78%), H: N (61%), I: N (53%), K: N (57%), L: N (72%), M: D (59%), N: N (72%), P: N (61%), Q: N (57%), R: D (53%), S: N (87%), T: N (87%), V: N (72%), W: D (71%), Y: D (59%),
Predicted by PROVEAN:	C: N, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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438 Two TMD0 mutations, A116P and V187P, abrogated the association of TMD0 and Kir6.2. Login to comment

[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] Role of Hsp90 in biogenesis of the beta-cell ATP-s... Mol Biol Cell. 2010 Jun 15;21(12):1945-54. Epub 2010 Apr 28. Yan FF, Pratt EB, Chen PC, Wang F, Skach WR, David LL, Shyng SL
Role of Hsp90 in biogenesis of the beta-cell ATP-sensitive potassium channel complex.
Mol Biol Cell. 2010 Jun 15;21(12):1945-54. Epub 2010 Apr 28., [PMID:20427569]

Abstract [show]
The pancreatic beta-cell ATP-sensitive potassium (K(ATP)) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K(ATP) channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K(ATP) channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K(ATP) channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with beta-cell K(ATP) channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K(ATP) channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K(ATP) channels via SUR1, thereby affecting functional expression of the channel in beta-cell membrane.

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177 The five mutants examined harbor mutation N24K, A116P, D310N, ⌬F1388, or D1472N in the SUR1 subunit. Login to comment
179 However, the A116P and ⌬F1388 mutants did not show improved surface expression (Figure 5). Login to comment
180 Interestingly, the N24K, D310N and D1472N mutants have relatively milder processing/trafficking defects in that they do express at the cell surface to some extent even under control conditions, in contrast to A116P and ⌬F1388 that show virtually no surface expression. Login to comment
236 Although Hsp90beta improved surface expression of the N24K, D310N, and D1472N mutant (p ϭ 0.01, 0.01, 0.05, and 0.03 for WT, N24K, D310N, and D1472N, respectively), it did not significantly increase surface expression of the A116P or ⌬F1388 mutants. Login to comment
270 Because Hsp90 is thought to act on substrates at a late stage of folding, it is possible that the A116P and ⌬F1388-SUR1 mutations render folding difficulties at an early stage that cannot be overcome by upregulation of Hsp90 function. 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
140 To ensure that the trafficking defects of TMD0 mutations and their rescue by sulfonylureas are also seen in a cellular environment in which the channels normally reside, we examined whether A116P, a TMD0 trafficking mutant we documented previously, behaves the same in the insulin-secreting cell line INS-1 as in COS cells. Login to comment
142 As shown in Fig. 5D, A116P-SUR1 expressed in INS-1 cells failed to mature into the complex-glycosylated form; however, overnight treatment of cells with 1 ␮mol/l glibenclamide overcame this processing defect and led to the appearance of the complex-glycosylated form. Login to comment
143 These observations recapitulate what we have reported previously for A116P-SUR1 expressed in COS cells (16), FIG. 5. Login to comment
150 D: Western blot showing that the A116P-SUR1 expressed in INS-1 cells lacks the complex-glycosylated band and that treatment of cells with glibenclamide led to appearance of the complex-glycosylated band, as observed in COS cells. 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
214 Because there are many examples of cell-type-specific protein trafficking regulation (13,21,43), what we found in COS cells may not extrapolate directly to beta-cells. In this regard, our results that a previously published TMD0 trafficking mutant, A116P, exhibits the same trafficking defect and response to sulfonylurea rescue in INS-1 cells as in COS cells provide some assurance that the TMD0 mutants are likely to behave similarly in their native environment. 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
212 Because there are many examples of cell-type-specific protein trafficking regulation (13,21,43), what we found in COS cells may not extrapolate directly to beta-cells. In this regard, our results that a previously published TMD0 trafficking mutant, A116P, exhibits the same trafficking defect and response to sulfonylurea rescue in INS-1 cells as in COS cells provide some assurance that the TMD0 mutants are likely to behave similarly in their native environment. 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] 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] 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
85 30 °C indicates that A116P causes more severe perturbation in the processing of SUR1. 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] 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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No. Sentence Comment
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
188 Glibenclamide Rescues Surface Expression of A116P Mutant Channels by Slowing the Degradation of the Mutant SUR1 Protein-We presume that glibenclamide acts as a chemical chaperone to facilitate folding of the mutant fSUR1 in the ER, thereby increasing maturation and cell surface expression of the channel complex. Login to comment
189 To test this, we examined the effect of glibenclamide on metabolically labeled A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment
191 In the absence of glibenclamide, A116P-fSUR1 was detected as a core glycosylated immature band following a 30-min pulse-labeling period and remained as the immature form throughout the chase period of up to 24 h (Fig. 6A). Login to comment
193 We quantified the degradation rate of A116P-fSUR1 co-expressed with Kir6.2 in control or in glibenclamide-treated cells (the sum of both immature and mature forms) and compared it with that of WT-fSUR1 (coexpressed with Kir6.2) in control cells (the sum of both immature and mature forms). Login to comment
194 As shown in Fig. 6B, whereas the overall degradation rate of the A116P-fSUR1 mutant protein in glibenclamide-treated cells is similar to that of WT-fSUR1 in control cells, it is markedly slower than that of A116P-fSUR1 in control cells. Login to comment
195 To further determine whether glibenclamide stabilizes the A116P-fSUR1 mutant protein by stabilizing the mutant SUR1 itself, we measured the degradation rate of A116P-fSUR1 in the absence of Kir6.2. 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
197 We found that glibenclamide indeed slowed the degradation of A116P-fSUR1, although the rate is still faster than that of WT-fSUR1 in control cells (Fig. 6C). 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
223 The ability of the rescued mutant channels to respond to metabolic changes was further examined by 86 Rbϩ efflux experiments using the A116P mutant as an example. Login to comment
224 Without tolbutamide treatment, the cells transfected with A116P exhibited very low KATP channel activities upon metabolic inhibition (11% efflux in 40 min; compare with 9% in untransfected cells) in contrast to cells transfected with WT channels (81% efflux). Login to comment
225 Following tolbutamide treatment (300 ␮M for 24 h) and subsequent washout of tolbutamide (for 12 h), cells transfected with Kir6.2 and A116P exhibited a substantial increase in channel activities upon metabolic inhibition (ϳ40% efflux; compare with ϳ80% in cells transfected with Kir6.2 and WT-fSUR1 and 10% in untransfected cells). Login to comment
226 Thus, tolbutamide can be used as a pharmacological chaperone to recruit A116P mutant channels to the cell surface. 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
236 In our pulse-chase labeling experiments, the mutant A116P-fSUR1 never became complex-glycosylated, arguing that the lack of surface expression was a result of ER retention rather than increased degradation of surface channels. Login to comment
239 Glibenclamide slows the degradation of A116P-fSUR1. Login to comment
240 A, metabolic pulse-chase of A116P-fSUR1 co-expressed with Kir6.2 (labeled as fA116P-SUR1 in the figure). Login to comment
242 Although A116P-fSUR1 in cells not treated with glibenclamide appeared as a single immature band throughout the chase, A116P-fSUR1 in cells treated with 5 ␮M glibenclamide following the pulse label was converted to the mature form with time. Login to comment
244 B, degradation of A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment
245 In glibenclamide-treated cells expressing A116P-fSUR1 and Kir6.2 and in control cells expressing WT-fSUR1 and Kir6.2, both the mature band and immature band were included for the quantification of residual label. Login to comment
246 The overall degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is comparable to that of WT-fSUR1 in control cells (open circles) but obviously slower than that of A116P-fSUR1 in control (without glibenclamide treatment) cells (open squares). Login to comment
249 C, degradation of A116P-fSUR1 in the absence of Kir6.2. Login to comment
250 The degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is apparently slower than that in control untreated cells (open squares), but it is still faster than that of WT-fSUR1 in control cells (open circles). 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
282 It is expected that mutation of alanine 116 to a proline and valine 187 to a charged aspartate would be disruptive to the transmembrane ␣-helix structure and affect the normal protein folding process. 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
186 To test this, we examined the effect of glibenclamide on metabolically labeled A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment
190 We quantified the degradation rate of A116P-fSUR1 co-expressed with Kir6.2 in control or in glibenclamide-treated cells (the sum of both immature and mature forms) and compared it with that of WT-fSUR1 (coexpressed with Kir6.2) in control cells (the sum of both immature and mature forms). Login to comment
192 To further determine whether glibenclamide stabilizes the A116P-fSUR1 mutant protein by stabilizing the mutant SUR1 itself, we measured the degradation rate of A116P-fSUR1 in the absence of Kir6.2. 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
220 The ability of the rescued mutant channels to respond to metabolic changes was further examined by 86 Rbaf9; efflux experiments using the A116P mutant as an example. Login to comment
221 Without tolbutamide treatment, the cells transfected with A116P exhibited very low KATP channel activities upon metabolic inhibition (11% efflux in 40 min; compare with 9% in untransfected cells) in contrast to cells transfected with WT channels (81% efflux). Login to comment
222 Following tolbutamide treatment (300 òe;M for 24 h) and subsequent washout of tolbutamide (for 12 h), cells transfected with Kir6.2 and A116P exhibited a substantial increase in channel activities upon metabolic inhibition (b03;40% efflux; compare with b03;80% in cells transfected with Kir6.2 and WT-fSUR1 and 10% in untransfected cells). 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
237 A, metabolic pulse-chase of A116P-fSUR1 co-expressed with Kir6.2 (labeled as fA116P-SUR1 in the figure). Login to comment
241 B, degradation of A116P-fSUR1 in cells co-expressing Kir6.2. Login to comment
243 The overall degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is comparable to that of WT-fSUR1 in control cells (open circles) but obviously slower than that of A116P-fSUR1 in control (without glibenclamide treatment) cells (open squares). Login to comment
247 The degradation rate of A116P-fSUR1 in glibenclamide-treated cells (filled squares) is apparently slower than that in control untreated cells (open squares), but it is still faster than that of WT-fSUR1 in control cells (open circles). 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
279 It is expected that mutation of alanine 116 to a proline and valine 187 to a charged aspartate would be disruptive to the transmembrane ॷ-helix structure and affect the normal protein folding process. 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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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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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
109 Consistent results were obtained using Western blot analysis. A representative blot of A116P fSUR1 in the WT or S1238Y background from cells treated with or without 5 ␮M glibenclamide for 24 h is shown in Fig. 4A. 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
133 B, addition of the A116P mutation in WT or the various sulfonylurea binding mutation backgrounds abolished surface staining (top panel), although the mutant proteins were detected inside the cell (middle panel). Login to comment
135 Treatment of cells with 5 ␮M glibenclamide significantly increased surface expression of the A116P mutant as reported previously. 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
149 For the A116P-fSUR1, however, only the immature band was detected in untreated cells (A116P); upon treatment with 1 ␮M glibenclamide for 24 h (A116PϩGlib),theupperA116P-fSUR1bandbecameapparent,indicatingres- cue of the mutant protein out of the ER. Login to comment
150 When the S1238Y mutation was introduced into A116P-fSUR1 (A116P/S1238Y), the same glibenclamide treatment was less effective in promoting expression of the upper band (A116P/ S1238YϩGlib). 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
132 B, addition of the A116P mutation in WT or the various sulfonylurea binding mutation backgrounds abolished surface staining (top panel), although the mutant proteins were detected inside the cell (middle panel). Login to comment
134 Treatment of cells with 5 òe;M glibenclamide significantly increased surface expression of the A116P mutant as reported previously. 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
148 For the A116P-fSUR1, however, only the immature band was detected in untreated cells (A116P); upon treatment with 1 òe;M glibenclamide for 24 h (A116Paf9;Glib),theupperA116P-fSUR1bandbecameapparent,indicatingres- cue of the mutant protein out of the ER. 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] Role of Derlin-1 protein in proteostasis regulatio... J Biol Chem. 2012 Mar 23;287(13):10482-93. Epub 2012 Feb 6. Wang F, Olson EM, Shyng SL
Role of Derlin-1 protein in proteostasis regulation of ATP-sensitive potassium channels.
J Biol Chem. 2012 Mar 23;287(13):10482-93. Epub 2012 Feb 6., [PMID:22311976]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2 regulate insulin secretion by linking glucose metabolism with membrane potential. The number of K(ATP) channels in the plasma membrane affects the sensitivity of beta-cells to glucose. Aberrant surface channel expression leads to insulin secretion disease. Previously, we have shown that K(ATP) channel proteins undergo endoplasmic reticulum (ER)-associated degradation (ERAD) via the ubiquitin-proteasome pathway, and inhibition of proteasome function results in an increase in channel surface expression. Here, we investigated whether Derlin-1, a protein involved in retrotranslocation of misfolded or misassembled proteins across the ER membrane for degradation by cytosolic proteasomes, plays a role in ERAD and, in turn, biogenesis efficiency of K(ATP) channels. We show that both SUR1 and Kir6.2 form a complex with Derlin-1 and an associated AAA-ATPase, p97. Overexpression of Derlin-1 led to a decrease in the biogenesis efficiency and surface expression of K(ATP) channels. Conversely, knockdown of Derlin-1 by RNA interference resulted in increased processing of SUR1 and a corresponding increase in surface expression of K(ATP) channels. Importantly, knockdown of Derlin-1 increased the abundance of disease-causing misfolded SUR1 or Kir6.2 proteins and even partially rescued surface expression in a mutant channel. We conclude that Derlin-1, by being involved in ERAD of SUR1 and Kir6.2, has a role in modulating the biogenesis efficiency and surface expression of K(ATP) channels. The results suggest that physiological or pathological changes in Derlin-1 expression levels may affect glucose-stimulated insulin secretion by altering surface expression of K(ATP) channels.

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119 A116P- and F1388-SUR1 are mutations identified from patients with congenital hyperinsulinism (13,36). Login to comment
121 We infected INS-1 cells with adenoviruses carrying Kir6.2 and A116P or F1388 f-SUR1 and performed co-immunoprecipitation experiments using FLAG-antibody agarose beads. Login to comment
171 We therefore studied three SUR1 mutations, C26S, A116P and F1388, predicted to have folding defects in the extracellular (ER-luminal), transmembrane and cytosolic domains based on current topology model of SUR1 (Supplemental Figure 3) (43). Login to comment
175 In the A116P mutant, knockdown of Derlin-1 even led to appearance of the mature upper band. Login to comment
176 Surface biotinylation experiments further confirmed expression of the A116P mutant channel in the plasma membrane (Figure 6C, D). Login to comment
190 The amount of p97 detected in the immunoprecipitates was much higher for truncated SUR1 that had significantly reduced protein levels (a.a. 1-1022 and a.a. 1-607), reminiscent of that observed for the misfolding mutant A116P and F1388 (Figure 1D). Login to comment
228 Curiously, while a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite that A116P had overall lower protein levels than C26S and F1388. Login to comment
123 D, INS-1 cells infected with recombinant adenoviruses to express exogenous WT f-SUR1 alone (lane 2), WT f-SUR1 and Kir6.2 (lane 3), mutant A116P f-SUR1 and Kir6.2 (lane 4), or mutant èc;F1388 f-SUR1 and Kir6.2 (lane 5). Login to comment
136 A116P- and èc;F1388-SUR1 are mutations identified from patients with congenital hyperinsulinism (13, 36). Login to comment
138 We infected INS-1 cells with adenoviruses carrying Kir6.2 and A116P or èc;F1388 f-SUR1 and performed co-immunoprecipitation experiments using FLAG antibody-agarose beads. Login to comment
188 We therefore studied three SUR1 mutations, C26S, A116P, and èc;F1388, predicted to have folding defects in the extracellular (ER-luminal), transmembrane and cytosolic domains based on the current topology model of SUR1 (supplemental Fig. 3) (43). Login to comment
227 A, HEK293 cells were transfected with Kir6.2 and WT, C26S, A116P, or èc;F1388 SUR1 along with Derlin-1 shRNA or the scramble control. Login to comment
229 Knockdown of Derlin-1 increased the intensity of the core-glycosylated SUR1 in all cases and also the complex-glycosylated SUR1 in WT and A116P. Login to comment
231 C, surface biotinylation was performed in HEK293 cells transfected with A116P SUR1 and Kir6.2 together with Derlin-1 shRNA or the scramble plasmid. Login to comment
250 In the A116P mutant, knockdown of Derlin-1 even led to the appearance of the mature upper band. Login to comment
251 Surface biotinylation experiments further confirmed expression of the A116P mutant channel in the plasma membrane (Fig. 6, C and D). Login to comment
264 The amount of p97 detected in the immunoprecipitates was much higher for truncated SUR1 that had significantly reduced protein levels (aa 1-1022 and aa 1-607), reminiscent of that observed for the misfolding mutant A116P and èc;F1388 (Fig. 1D). Login to comment
317 Curiously, although a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite the fact that A116P had overall lower protein levels than C26S and èc;F1388. Login to comment

[hide] Sur domains that associate with and gate KATP pore... J Biol Chem. 2003 Oct 24;278(43):41577-80. Epub 2003 Aug 26. Babenko AP, Bryan J
Sur domains that associate with and gate KATP pores define a novel gatekeeper.
J Biol Chem. 2003 Oct 24;278(43):41577-80. Epub 2003 Aug 26., [PMID:12941953]

Abstract [show]
Structure-function analyses of K+ channels identify a common pore architecture whose gating depends on diverse signal sensing elements. The "gatekeepers" of the long, ATP-inhibited KIR6.0 pores of KATP channels are ABC proteins, SURs, receptors for channel opening and closing drugs. Several competing models for SUR/KIR coupling exist. We show that SUR TMD0, the N-terminal bundle of five transmembrane helices, specifically associates with KIR6.2, forcing nearly silent pores to burst like native KATP channels and enhancing surface expression. Inclusion of adjacent submembrane residues of L0, the linker between TMD0 and the stimulatory nucleotide- and drug-binding ABC core, generates constitutively active channels, whereas additional cytoplasmic residues counterbalance this activation establishing a relationship between the mean open and burst times of intact pores. SUR fragments, lacking TMD0, fail to modulate KIR. TMD0 is thus the domain that anchors SUR to the KIR pore. Consistent with data on chimeric ABCC/KIRs and a modeled channel structure, we propose that interactions of TMD0-L0 with the outer helix and N terminus of KIR bidirectionally modulate gating. The results explain and predict pathologies associated with alteration of the 5' ends of clustered ABCC8 (9)/KCNJ11 (8) genes.

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144 The model suggests how SUR1 mutations in the 5Ј portion of human ABCC8, e.g. A116P, cause familial hyperinsulinemia (30) and how a polymorphism, E23K, in the distal N terminus of KIR6.2 can increase the PO of KATP channels in insulin secreting beta-cells of pancreatic islets, increasing the risk of type 2 diabetes (31). Login to comment
148 The model suggests how SUR1 mutations in the 5b18; portion of human ABCC8, e.g. A116P, cause familial hyperinsulinemia (30) and how a polymorphism, E23K, in the distal N terminus of KIR6.2 can increase the PO of KATP channels in insulin secreting beta-cells of pancreatic islets, increasing the risk of type 2 diabetes (31). 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
123 Representative immunoblot for cells expressing SUR1 A116P treated with 10% glycerol (glycerol), decreasing concentrations of ouabain and glafenine (D), carbamazepine (carbam), KM57, and KM60 (E), latonduine, RDR1, ABT-888 (F). 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
134 In contrast to cells expressing wild type SUR1 protein, those expressing the SUR1 A116P mutant showed only low levels of channel activity due to loss of surface expression after treatment with the DMSO control. Login to comment
135 Exposing A116P-expressing cells to the reversible sulfonylurea drug tolbutamide for > 24 h followed by a washout 2 h prior to the assay led to almost complete recovery of channel activity, as reported previously [32]. Login to comment
136 Among the correctors Figure 7 The F508del-CFTR corrector RDR1 improves the function of the SUR1 A116P mutant. Login to comment
137 (A) Representative 86 Rb+ efflux profiles from COS cells transiently transfected with SUR1 A116P and wt Kir6.2 and treated with 0.1% DMSO, 300bc;M tolbutamide (a reversible sulfonylurea) or 10bc;M RDR1 as described in the METHODS. Login to comment
154 that enhanced the processing of A116P, KM57, KM60 and RDR1 were tested for their effects on functional recovery of the mutant channel. 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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68 Groups of islets (100 islet equivalents) in each well of a 12-well plate were infected with Ad-tTA (m.o.i., 500), Ad-Kir6.2 (m.o.i., 2000), and either Ad-f-SUR1 (m.o.i., 1000) or mutant Ad-A116P f-SUR1 (m.o.i., 1000) or Ad-F27S f-SUR1 (m.o.i., 500) for 16 h in 0.5 ml of Opti-MEM (Invitrogen) at 37 &#b0;C. The islets were then incubated for an additional 24 h in RPMI 1640 medium with 10% FBS containing either DMSO, glibenclamide, or carbamazepine before being harvested for immunoblotting. Login to comment
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
223 We used human islets and beta-cells as well as rat insulinoma INS-1 cells for these experiments. Human islets obtained through the Integrated Islet Distribution Program were co-infected overnight with adenoviruses carrying Kir6.2 and WT, F27S, or A116P f-SUR1 cDNAs. Login to comment
226 As was observed in COSm6 cells, overnight glibenclamide and carbamazepine treatments led to a marked increase in the upper SUR1 band in the F27S mutant and an obvious albeit weaker increase in the upper band in the A116P mutant in whole islet lysates (Fig. 7A, panel i). Login to comment
237 Carbamazepine as a Novel KATP Channel Corrector JULY 19, 2013ߦVOLUME 288ߦNUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20949 Together, these results demonstrate that carbamazepine effectively improved the processing and surface expression of the F27S and A116P SUR1 trafficking-impaired mutant KATP channels in pancreatic beta-cells. Login to comment
265 A, panel i, representative SUR1 blots from uninfected human islets (probed with anti-SUR1 antibody) and human islets infected with adenoviruses carrying WT Kir6.2 and WT or F27S or A116P mutant f-SUR1 cDNAs (probed with anti-FLAG antibody) and treated with DMSO, 5 òe;M glibenclamide (Glib), or 10 òe;M carbamazepine (CBZ) for 16 h. Panel ii, representative whole-cell patch clamp recordings measuring KATP current density in control and drug-treated human beta-cells infected with the F27S mutant viruses (recordings are from two cells with similar membrane capacitance of b03;10 picofarads (pF)). Login to comment
69 Groups of islets (100 islet equivalents) in each well of a 12-well plate were infected with Ad-tTA (m.o.i., 500), Ad-Kir6.2 (m.o.i., 2000), and either Ad-f-SUR1 (m.o.i., 1000) or mutant Ad-A116P f-SUR1 (m.o.i., 1000) or Ad-F27S f-SUR1 (m.o.i., 500) for 16 h in 0.5 ml of Opti-MEM (Invitrogen) at 37 &#b0;C. The islets were then incubated for an additional 24 h in RPMI 1640 medium with 10% FBS containing either DMSO, glibenclamide, or carbamazepine before being harvested for immunoblotting. 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
225 We used human islets and beta-cells as well as rat insulinoma INS-1 cells for these experiments. Human islets obtained through the Integrated Islet Distribution Program were co-infected overnight with adenoviruses carrying Kir6.2 and WT, F27S, or A116P f-SUR1 cDNAs. Login to comment
228 As was observed in COSm6 cells, overnight glibenclamide and carbamazepine treatments led to a marked increase in the upper SUR1 band in the F27S mutant and an obvious albeit weaker increase in the upper band in the A116P mutant in whole islet lysates (Fig. 7A, panel i). Login to comment
239 Carbamazepine as a Novel KATP Channel Corrector JULY 19, 2013ߦVOLUME 288ߦNUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20949 Together, these results demonstrate that carbamazepine effectively improved the processing and surface expression of the F27S and A116P SUR1 trafficking-impaired mutant KATP channels in pancreatic beta-cells. Login to comment
267 A, panel i, representative SUR1 blots from uninfected human islets (probed with anti-SUR1 antibody) and human islets infected with adenoviruses carrying WT Kir6.2 and WT or F27S or A116P mutant f-SUR1 cDNAs (probed with anti-FLAG antibody) and treated with DMSO, 5 òe;M glibenclamide (Glib), or 10 òe;M carbamazepine (CBZ) for 16 h. Panel ii, representative whole-cell patch clamp recordings measuring KATP current density in control and drug-treated human beta-cells infected with the F27S mutant viruses (recordings are from two cells with similar membrane capacitance of b03;10 picofarads (pF)). Login to comment

[hide] Ibuprofen rescues mutant cystic fibrosis transmemb... J Cyst Fibros. 2015 Jan;14(1):16-25. doi: 10.1016/j.jcf.2014.06.001. Epub 2014 Jun 25. Carlile GW, Robert R, Goepp J, Matthes E, Liao J, Kus B, Macknight SD, Rotin D, Hanrahan JW, Thomas DY
Ibuprofen rescues mutant cystic fibrosis transmembrane conductance regulator trafficking.
J Cyst Fibros. 2015 Jan;14(1):16-25. doi: 10.1016/j.jcf.2014.06.001. Epub 2014 Jun 25., [PMID:24974227]

Abstract [show]
BACKGROUND: Small molecules as shown by VX809 can rescue the mislocalization of F508del-CFTR. The aim of this study was to identify correctors with a clinical history and their targets of action. METHODS: CFTR correctors were screened using two F508del-CFTR expressing cell based HTS assays. Electrophysiological studies using CFBE41o(-) and HBE cells and in-vivo mouse assays confirmed CFTR rescue. The target of action was attained using pharmacological inhibitors and siRNA to specific genes. RESULTS: Ibuprofen was identified as a CFTR corrector. Ibuprofen treatment of polarized CFBE41o(-) monolayers increased the short-circuit current (Isc) response to stimulation. In vivo CF mice treatment with ibuprofen restored the CFTR trafficking. SiRNA knock down of cyclooxygenase expression caused partial F508del-CFTR correction. CONCLUSION: These studies show that ibuprofen is a CFTR corrector and that it causes correction by COX-1 inhibition. Hence ibuprofen may be suitable to be part of a future CF combination therapy.

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44 HEK cells stably expressing HA-tagged hERG G601S or wild-type hERG were given by Eckhard Ficker (Case Western Reserve University U.S.A.) Flag-tagged hamster SUR1 both the wild-type and A116P mutant form and rat Kir6.2 plasmids were given by Show-Ling Shyng (Oregon Health and Science University) and were reported previously [18]. Login to comment
158 Persistent hyperinsulinemic hypoglycemia of infancy mutation A116P in the sulfonylurea receptor 1 (SUR1) expressed in HeLa cells was treated (24 h) with ibuprofen. 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] Effects of single nucleotide polymorphisms in K(AT... Arch Med Res. 2012 May;43(4):317-23. doi: 10.1016/j.arcmed.2012.06.001. Epub 2012 Jun 13. Gonen MS, Arikoglu H, Erkoc Kaya D, Ozdemir H, Ipekci SH, Arslan A, Kayis SA, Gogebakan B
Effects of single nucleotide polymorphisms in K(ATP) channel genes on type 2 diabetes in a Turkish population.
Arch Med Res. 2012 May;43(4):317-23. doi: 10.1016/j.arcmed.2012.06.001. Epub 2012 Jun 13., [PMID:22704848]

Abstract [show]
BACKGROUND AND AIMS: ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells play a key role in glucose-stimulated insulin secretion mechanism. The Kir6.2 protein, forming the K(ATP) channel pore inwardly, and the SUR1 protein that surrounds it forming the outside part of the channel were encoded by ABCC8 and KCNJ11 genes, respectively. Recent studies reported that the single nucleotide polymorphisms (SNPs) established in these genes are associated with defects in insulin secretion and type 2 diabetes mellitus (T2DM). We aimed to investigate the allele profiles and the risk alleles of the ABCC8 and KCNJ11 genes and to highlight the associations with the disease in patients in Konya region of Turkey where T2DM is common. METHODS: In this study, 169 patients with T2DM and 119 healthy controls were included. A total of 29 SNPs in ABCC8 and KCNJ11 genes were screened by PCR-SSCP technique and sequenced. Biochemical parameters and genotype-phenotype relationships were analyzed using variance analysis. RESULTS: R1273R silent substitution in exon 31 and 16/-3t-->c substitution in noncoding region of exon 16 of ABCC8 gene showed a significant association (OR 4.8 [95% CI 2.41-9.77], p <0.001 and OR 3.5 [95% CI 1.64-7.40], p <0.001 under dominant and recessive models, respectively). We detected a significant association between E/K heterozygote genotype and reduced plasma insulin level in patients with T2DM (p <0.05). CONCLUSIONS: ABCC8 exons 16 and 31 variants increase susceptibility to T2DM and KCNJ11 E23K decreases insulin secretion in a Turkish population.

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102 genotype distributions of five SNPs (E23K in both study group, I337V, A116P, R1273R in case group) deviated from HWE ( p !0.05). Login to comment
116 c2 Test results for associations between SNPs in ABCC8 and KCNJ11 genes and risk of type 2 diabetes Rs id SNP Gene Genotype Case Control Additive Dominant Reccessive n (%) n (%) p p p C/C 100 (100) 109 (98.9) rs8192695 A110A ABCC8 C/T - 1 (1.1) O0.05a - - - - - C/C 68 (40.2) 50 (56.2) rs72559731 A116P ABCC8 C/T 101 (59.8) 39 (43.8) O0.05a - - - - - C/C 68 (40.5) 49 (47.1) 1799854 16 (3) ABCC8 C/T 88 (52.4) 33 (31.7) !0.05 O0.05 !0.05 T/T 12 (7.1) 22 (21.2) G/G 15 (11.1) 31 (37.8) rs1799859 R1273R ABCC8 G/A 110 (81.5) 39 (47.6) !0.05 !0.05 O0.05 A/A 10 (7.4) 12 (14.6) G/G 4 (3.4) 7 (6.8) rs757110 A1369S ABCC8 G/T 35 (29.9) 40 (38.8) O0.05 O0.05 O0.05 T/T 78 (66.7) 56 (54.4) A/A 139 (92.7) 106 (96.4) 437 - ABCC8 A/T 11 (7.3) 4 (3.6) O0.05a - - - - - G/G 52 (32.1) 31 (39.2) rs5219 E23K KCNJ11 G/A 110 (67.9) 48 (60.8) O0.05a - - - - - C/C 109 (71.7) 80 (70.8) rs5218 A190A KCNJ11 C/T 42 (27.6) 31 (27.4) O0.05 O0.05 O0.05 T/T 1 (0.7) 2 (1.8) C/C 144 (97.3) 96 (94.1) rs5216 L267L KCNJ11 C/G 4 (2.7) 6 (5.9) O0.05a - - - - - C/C 68 (45.6) 60 (51.7) rs1800467 L270V KCNJ11 C/G 54 (36.3) 44 (37.9) O0.05 O0.05 O0.05 G/G 27 (18.1) 12 (10.4) A/A 59 (44.4) 56 (50.0) rs5215 I337V KCNJ11 A/G 31 (23.3) 44 (39.3) 0.07 O0.05 O0.05 G/G 43 (32.3) 12 (10.7) a These SNPs had only two genotypes; therefore, 11 vs. 12 (or 12 vs. 22) presented as additive model. Login to comment

[hide] Engineered Kir6.2 mutations that correct the traff... Channels (Austin). 2013 Jul-Aug;7(4):313-7. Epub 2013 May 21. Zhou Q, Pratt EB, Shyng SL
Engineered Kir6.2 mutations that correct the trafficking defect of K(ATP) channels caused by specific SUR1 mutations.
Channels (Austin). 2013 Jul-Aug;7(4):313-7. Epub 2013 May 21., [PMID:23695995]

Abstract [show]
KATP channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. The molecular interactions between SUR1 and Kir6.2 that govern channel gating and biogenesis are incompletely understood. In a recent study, we showed that a SUR1 and Kir6.2 mutation pair, E203K-SUR1 and Q52E-Kir6.2, at the SUR1/Kir6.2 interface near the plasma membrane increases the ATP-sensitivity of the channel by nearly 100-fold. Here, we report the finding that the same mutation pair also suppresses channel folding/trafficking defects caused by select SUR1 mutations in the first transmembrane domain of SUR1. Analysis of the contributions from individual mutations, however, revealed that the correction effect is attributed largely to Q52E-Kir6.2 alone. Moreover, the correction is dependent on the negative charge of the substituting amino acid at the Q52 position in Kir6.2. Our study demonstrates for the first time that engineered mutations in Kir6.2 can correct the biogenesis defect caused by specific mutations in the SUR1 subunit.

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16 Of the three TMD0 mutations tested, F27S and A116P showed a clear upper band in addition to the lower immature band in the E203K//Q52E background; by contrast, the same trafficking mutations placed in the background without the E203K//Q52E mutations only exhibited the lower band (Fig. 2), indicating the proteins were retained in the ER as reported previously.25,26 Another TMD0 mutation, E128K, as well as three other previously identified, congenital hyperinsulinism-causing SUR1 trafficking mutations outside of TMD0 (R495Q, F686S and L1350Q),25 however, showed no improvement in their processing efficiency when combined with E203K//Q52E (data not shown). Login to comment
21 Positions of SUR1-E203 and Kir6.2-Q52 residues (open squares) as well as the two TMD0 trafficking mutations F27S and A116P (open circles) are indicated. Login to comment
25 E203K//Q52E mutation pair in correcting the processing defect of F27S and A116P. Login to comment
31 Note in the case of Q52K-Kir6.2, the pairing with E203 residue in SUR1 would represent a reverse-switch of charge at the two positions in relation to the E203K//Q52E mutation pair, and yet unlike E203K//Q52E, E203//Q52K failed to correct the trafficking defect caused by F27S and A116P. Login to comment
32 These results suggest that correction of the trafficking defects of F27S and A116P in the E203K//Q52E background is unlikely a consequence of electrostatic interactions between amino acids at the 203-SUR1 and 52-Kir6.2 positions, and that a negatively charged amino acid at position 52 of Kir6.2 is the major driving factor for expression rescue. Login to comment
39 Close physical proximity of the two residues is further supported by the observation that in inside-out patch-clamp recording of E203C-SUR1// Q52C-Kir6.2 channels, application of the oxidizing reagent H2 O2 to induce disulfide bond formation locked the channels in a closed state that was reversible by the reducing agent dithiothreotol.21 Given this, we considered the possibility that cross-linking of E203C//Q52C may rescue the folding/assembly defect caused by F27S- or A116P-SUR1 by stabilizing the mutant SUR1-Kir6.2 interface at this location. Login to comment
47 Similar observations were made for the A116P mutation (data Figure 2. Login to comment
48 The E203K//Q52E mutation pair suppresses the processing defect caused by the F27S or A116P SUR1 mutation. 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
215 Further, F1388-, A116P-SUR1 and W91R-Kir6.2 all showed accelerated degradation (Crane and Aguilar-Bryan, 2004; Yan et al., 2004, 2005). 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
262 Metabolic pulse-chase experiments demonstrated that glibenclamide slowed A116P-SUR1 degradation even in the absence of Kir6.2 and promoted maturation of the mutant SUR1 when Kir6.2 was co-expressed (Yan et al., 2004), providing evidence that sulfonylureas facilitate folding and/or prevent misfolding of mutant channels during assembly in the ER. Login to comment
266 Accordingly, mutation of S1238 to tyrosine abolished tolbutamide rescue of SUR1 mutants A116P and V187D. Login to comment
286 Interestingly, a recent study by Zhou et al. showed that a point mutation in Kir6.2, Q52E, located in the N-terminus of the protein just before the slide helix, partially compensated for the trafficking defects caused by SUR1-TMD0 mutations F27S and A116P, indicating that altered molecular interactions with Kir6.2 can overcome impaired channel folding/assembly caused by TMD0 mutations (Zhou et al., 2013). 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
168 A likely explanation is that CBZ protects the misfolded SUR1 proteins against ER-associated degradation, which would be consistent with our previous metabolic pulse-chase study showing that GBC also slows down the degradation rate of A116P-SUR1 expressed alone without Kir6.2 (20). Login to comment
312 It is worth noting that we have recently found that substitution of glutamine at the N-terminal amino acid position 52 of Kir6.2 by glutamate or aspartate suppresses the processing defect caused by F27S or A116P mutations in the TMD0 of SUR1 (67), consistent with a model of coupled conformational maturation between SUR1 and Kir6.2. Login to comment