ABCMdb

ABCC8 p.Phe27Ser

Predicted by SNAP2:	A: D (80%), C: D (71%), D: D (95%), E: D (91%), G: D (91%), H: D (91%), I: D (59%), K: D (91%), L: N (66%), M: D (75%), N: D (85%), P: D (95%), Q: D (85%), R: D (91%), S: D (85%), T: D (85%), V: D (80%), W: D (85%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N,

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[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
112 Of the five mutants, G7R, N24K, and F27S had WT-like or slightly increased ATP sensitivity, and either normal or reduced MgADP response that is commonly associated with CHI mutations (Fig. 1) (23). Login to comment
141 Scale bars: WT: 200 pA, 10 s; G7R: 200 pA, 10 s; N24K: 20 pA, 10 s; F27S: 50 pA, 10 s. B, quantification of channel response to ATP and MgADP. Login to comment
143 The ATP sensitivity of N24K is significantly higher than WT while the MgADP sensitivity of both N24K and F27S are significantly lower than WT channels (*, p Ͻ 0.05; Student`s t test). 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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47 TABLE 1 Genetic and clinical information on patients carrying the CHI mutations Mutation Disease Haplotype Diazoxide response References G7R Focal G7R No 44 N24K Diffuse N24K/R1215W No Not reported F27S Focal F27S No 39 R74W Focal R74W/R1215Q No 39,45,46 E128K Diffuse E128K No Not reported R495Q Diffuse R495Q/R1215Q No 39 E501K Focal E501K No 39 L503P Focal L503P No 44 F686S Focal F686S No 39 G716V* Diffuse G716V/G716V No 47,48 K1337N Not done g3992-9a/K1337N Yes 39 L1350Q Focal L1350Q No 44 S1387F Diffuse S1387F/NA No 9,24 L1390P NA L1390P/NA No Not reported D1472H Diffuse ⌬F1388/D1472H No 39 *Patient was from consanguineous mating and therefore was homozygous for the G716V mutation (48). Login to comment
94 The first group, including G7R, N24K, F27S, R74W, and E128K, is located in the first transmembrane domain TMD0; the second group, including R495Q, E501K, L503P, F686S, and G716V, is located in the second transmembrane domain TMD1 extending through the first nucleotide binding domain; the third group, including K1337N, L1350Q, S1387F, L1390P, and D1472H, is clustered in the second nucleotide binding domain and the COOH terminus of the protein. Login to comment
118 Results from this assay showed that F27S, R74W, E128K, R495Q, E501K, L503P, F686S, G716V, L1350Q, and D1472H mutant channels had greatly reduced surface expression (Ͻ20% of wild-type level)-whereas G7R and N24K mutant channels displayed modestly decreased surface expression level (Ͼ30% but Ͻ50% of wild-type level) and K1337N, S1378F, and L1390P exhibited normal or mildly reduced expression (Ͼ60% of wild-type level; Fig. 3A). Login to comment
130 In Western blots, several mutants, including G7R, N24K, F27S, R74W, and E128K, all located in TMD0, exhibited increased complex-glycosylated SUR1 in cells coexpressing Kir6.2 on overnight treatment with 1 ␮mol/l glibenclamide (Fig. 5A). Login to comment
48 TABLE 1 Genetic and clinical information on patients carrying the CHI mutations Mutation Disease Haplotype Diazoxide response References G7R Focal G7R No 44 N24K Diffuse N24K/R1215W No Not reported F27S Focal F27S No 39 R74W Focal R74W/R1215Q No 39,45,46 E128K Diffuse E128K No Not reported R495Q Diffuse R495Q/R1215Q No 39 E501K Focal E501K No 39 L503P Focal L503P No 44 F686S Focal F686S No 39 G716V* Diffuse G716V/G716V No 47,48 K1337N Not done g3992-9a/K1337N Yes 39 L1350Q Focal L1350Q No 44 S1387F Diffuse S1387F/NA No 9,24 L1390P NA L1390P/NA No Not reported D1472H Diffuse èc;F1388/D1472H No 39 *Patient was from consanguineous mating and therefore was homozygous for the G716V mutation (48). Login to comment
95 The first group, including G7R, N24K, F27S, R74W, and E128K, is located in the first transmembrane domain TMD0; the second group, including R495Q, E501K, L503P, F686S, and G716V, is located in the second transmembrane domain TMD1 extending through the first nucleotide binding domain; the third group, including K1337N, L1350Q, S1387F, L1390P, and D1472H, is clustered in the second nucleotide binding domain and the COOH terminus of the protein. Login to comment
119 Results from this assay showed that F27S, R74W, E128K, R495Q, E501K, L503P, F686S, G716V, L1350Q, and D1472H mutant channels had greatly reduced surface expression (b0d;20% of wild-type level)-whereas G7R and N24K mutant channels displayed modestly decreased surface expression level (b0e;30% but b0d;50% of wild-type level) and K1337N, S1378F, and L1390P exhibited normal or mildly reduced expression (b0e;60% of wild-type level; Fig. 3A). Login to comment

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

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

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54 Gene Haplotype Calcium (␮U/ml) Leucine (␮U/ml) Glucose (␮U/ml) Tolbutamide (␮U/ml) Diazoxide responsive Diffuse HI 1 SUR1 delF1388/D1472H 6 2 13 -2 No 2 Kir6.2 G134A/P266L 20 3 36 -2 No 3 SUR1 g3992-9a/g1630ϩ1a 11 16 -2 No 4 SUR1 N188S/D1472N 7 1 7 7 No 5 SUR1 R598X/R999X 32 1 72 27 No 6 SUR1 R495Q/R1215Q -2 15 44 30 No 7 SUR1 R74W/R1215Q 52 28 20 98 No 8 SUR1 g3992-9a/K1337N 2 18 39 33 Yes Focal HI 9 SUR1 F27S 17 -1 16 29 No 10 SUR1 F686S 12 2 27 12 No 11 SUR1 E501K 6 3 9 10 No 12 SUR1 3576delg 9 6 9 12 No 13 SUR1 g3992-9a 5 8 25 9 No 14 SUR1 g3992-9a 3 8 40 21 No 15 SUR1 c2924-10a 4 8 67 29 No 16 Kir6.2 A101D 1 8 177 88 No 17 SUR1 R1215W 7 9 15 6 No 18 Kir6.2 R136L 8 10 115 21 No 19 SUR1 g3992-9a 40 15 35 -0.3 No 20 SUR1 6aa insertion in exon 5 6 16 22 15 No 21 SUR1 R1215W 38 47 58 15 No 22 Kir6.2 R301H 16 55 75 14 No Controls (␮U/ml, mean Ϯ SD) KATP HI (n ϭ 7) 28 Ϯ 16 5 Ϯ 8 12 Ϯ 9 4 Ϯ 6 No GDH-HI (n ϭ 7) 2.3 Ϯ 5.4 42 Ϯ 27 120 Ϯ 52 94 Ϯ 56 Yes Normal (n ϭ 6) 3 Ϯ 4 1.4 Ϯ 2.8 56 Ϯ 26 48 Ϯ 32 Yes a To convert insulin (␮U/ml to pmol/liter), multiply by 6.0. identified in other patients. Login to comment
107 Degree of residual channel function in KATP mutations Null Indeterminate Partial SUR1 g3992-9a g1630ϩ1a R598X/R999X delF1388 N188S/D1472N R495Q/R1215Q F27S 3576delg R74W/R1215Q F686S K1337N E501K 6 aa insertion in exon 5 c2924-10a R1215W Kir6.2 G134A/P266L R301H A101D R136L FIG. 1. 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
78 Immunofluorescence Staining-COSm6 cells transfected with WT Kir6.2 and WT or F27S f-SUR1were plated on coverslips 1 day before the experiment and treated overnight with DMSO, tolbutamide, or carbamazepine. Login to comment
88 Metabolic Labeling and Immunoprecipitation-COSm6 cells were plated on 35-mm dishes and transfected with WT Kir6.2 and F27S f-SUR1. 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
129 Time Course and Duration of the Carbamazepine Rescue Effect-To characterize the carbamazepine effect further, we determined the time course and duration of the rescue effect using the F27S mutation as an example. 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
137 The F27S mutant was further analyzed by surface staining and chemiluminescence assays. 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
139 In addition, we quantified the amount of SUR1 surface expression using a chemiluminescence assay as described under "Materials and Methods" and observed a pronounced increase in surface expression of F27S mutant channels in response to 10 or 50 òe;M carbamazepine treatment (Fig. 3C). Login to comment
142 To test this, we conducted metabolic pulse-chase experiments to monitor the kinetics of F27S mutant SUR1 maturation from the core-glycosylated to the complex-glycosylated band in cells treated with carbamazepine or the vehicle DMSO. Login to comment
145 Note that the low maturation efficiency of SUR1 from the core-glycosylated to the complex-glycosylated form observed in the carbamazepine-treated F27S mutant (b03;20% of pulse-labeled SUR1) is similar to the value obtained previously for WT SUR1 in experiments where pulse-labeled SUR1 was chased for up to 24 h (29, 30, 36). Login to comment
171 A, COSm6 cells were transiently transfected with WT Kir6.2 and WT or F27S mutant SUR1 cDNAs and treated with 10 or 50 òe;M carbamazepine (C) or 5 òe;M glibenclamide (G) for 0, 1, 2, 4, 6, 10, 12, or 16 h. Panel i, blots showing cells treated with glibenclamide or carbamazepine for 1 h (left) when an effect on the upper band signal was first detected and for 6 h (right) when the effect began to plateau. Login to comment
176 WT without any treatment and F27S mutant treated with DMSO (0.1%) for 16 h are shown for comparison. Login to comment
181 F27S treated with DMSO for 16 h is shown for comparison. Login to comment
186 We chose the F27S mutant for this analysis because it exhibited the greatest response to rescue by carbamazepine and has been shown previously to have WT-like gating properties (31). Login to comment
187 In F27S-transfected cells treated overnight with 10 òe;M carbamazepine, metabolic inhibition induced only a low level of efflux similar to that seen in vehicle-treated cells. Login to comment
189 This result indicates that carbamazepine does prevent rescued F27S channels from being activated by metabolic inhibition. Login to comment
191 A progressive increase in efflux through rescued F27S channels over a 40-min period was observed with longer washout; the efflux level was nearly comparable with that seen in WT channels with 60-min washout (Fig. 6B). Login to comment
202 *, p b0d; 0.001 comparing F27S vehicle with various treatment groups by one-way analysisofvariancewithBonferroniposthoctest.ErrorbarsrepresentS.E.Unt, untransfected cells. Login to comment
204 Carbamazepine increases F27S mutant channel surface expression by improving processing and maturation of the channel complex during biogenesis. Login to comment
205 A, COSm6 cells transfected with F27S SUR1 and WT Kir6.2 were pulse-labeled with Tran35 S-Label for an hour and chased for 0-4 h in regular medium. Login to comment
211 *, p b0d; 0.001 by Student`s t test. Error bars represent S.E. Carbamazepine as a Novel KATP Channel Corrector 20948 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 29ߦJULY 19, 2013 at SEMMELWEIS UNIV OF MEDICINE on December , upon extensive washout (b03;1 h), physiological function of rescued surface F27S channels was recovered nearly completely. Login to comment
213 We tested this idea by examining the effects of carbamazepine and the KATP channel opener diazoxide on F27S channels using the 86 Rbaf9; assay. Login to comment
215 However, combination of metabolic inhibitors and 200 òe;M diazoxide resulted in a significantly higher efflux through the F27S channels in cells treated overnight with 10 òe;M carbamazepine compared with cells treated with vehicle alone without washout (Fig. 6C). Login to comment
217 To ensure that diazoxide and carbamazepine can be co-administered without compromising the rescue effect of carbamazepine, we compared the processing efficiency of F27S SUR1 in cells treated overnight with diazoxide, carbamazepine, or both together. Login to comment
219 Our results show that although diazoxide abolished the chaperoning effect of either glibenclamide or tolbutamide it did not interfere with the ability of carbamazepine to correct the processing defect of F27S SUR1 (Fig. 6D). 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
234 A, Western blot of SUR1 from COSm6 cells transfected with F27S SUR1 and WT Kir6.2 and treated with carbamazepine (CBZ), chloroquine (Chloroq), or both for 16 h (upper panel) or carbamazepine, Ly294002, or both for 16 h (lower panel) as indicated. Login to comment
236 B, Western blot of SUR1 from COSm6 cells transfected with F27S SUR1 and WT Kir6.2 and treated with carbamazepine or rapamycin (Rapa) or Liaf9; , both of which are autophagy inducers, for 16 h as indicated. 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
254 F27S cells were treated overnight (O.N.) with 10 òe;M carbamazepine, and carbamazepine was removed 15, 30, or 60 min (washout) prior to incubation with metabolic inhibitors. Login to comment
258 *, p b0d; 0.001 comparing F27S vehicle (Veh) with various treatment groups by one-way analysis of variance with Bonferroni post hoc test. Error bars represent S.E. D, Western blots show the effects of different drug combinations on the processing efficiency of F27S SUR1 co-expressed with WT Kir6.2 in COSm6 cells. Login to comment
260 Note that although diazoxide nearly abolished the rescue effect of glibenclamide or tolbutamide it had no effect on the ability of carbamazepine to correct the processing defect of F27S SUR1. 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
290 This suggests that diazoxide may antagonize the inhibitory effect of carbamazepine to potentiate the function of rescued F27S channels. Login to comment
296 Feasibility of the carbamazepine and diazoxide combination therapy strategy is further supported by our finding that the two drugs can be co-applied to cells without compromising the ability of carbamazepine to rescue the trafficking defects of the F27S mutant (Fig. 6D). Login to comment
301 Although our study focused mostly on one mutation (F27S) that responded well to carbamazepine, there are many additional congenital hyperinsulinism-associated SUR1 TMD0 mutations waiting to be tested (4). 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
79 Immunofluorescence Staining-COSm6 cells transfected with WT Kir6.2 and WT or F27S f-SUR1were plated on coverslips 1 day before the experiment and treated overnight with DMSO, tolbutamide, or carbamazepine. Login to comment
89 Metabolic Labeling and Immunoprecipitation-COSm6 cells were plated on 35-mm dishes and transfected with WT Kir6.2 and F27S f-SUR1. 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
131 Time Course and Duration of the Carbamazepine Rescue Effect-To characterize the carbamazepine effect further, we determined the time course and duration of the rescue effect using the F27S mutation as an example. Login to comment
140 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
141 In addition, we quantified the amount of SUR1 surface expression using a chemiluminescence assay as described under "Materials and Methods" and observed a pronounced increase in surface expression of F27S mutant channels in response to 10 or 50 òe;M carbamazepine treatment (Fig. 3C). Login to comment
144 To test this, we conducted metabolic pulse-chase experiments to monitor the kinetics of F27S mutant SUR1 maturation from the core-glycosylated to the complex-glycosylated band in cells treated with carbamazepine or the vehicle DMSO. Login to comment
147 Note that the low maturation efficiency of SUR1 from the core-glycosylated to the complex-glycosylated form observed in the carbamazepine-treated F27S mutant (b03;20% of pulse-labeled SUR1) is similar to the value obtained previously for WT SUR1 in experiments where pulse-labeled SUR1 was chased for up to 24 h (29, 30, 36). Login to comment
173 A, COSm6 cells were transiently transfected with WT Kir6.2 and WT or F27S mutant SUR1 cDNAs and treated with 10 or 50 òe;M carbamazepine (C) or 5 òe;M glibenclamide (G) for 0, 1, 2, 4, 6, 10, 12, or 16 h. Panel i, blots showing cells treated with glibenclamide or carbamazepine for 1 h (left) when an effect on the upper band signal was first detected and for 6 h (right) when the effect began to plateau. Login to comment
178 WT without any treatment and F27S mutant treated with DMSO (0.1%) for 16 h are shown for comparison. Login to comment
183 F27S treated with DMSO for 16 h is shown for comparison. Login to comment
188 We chose the F27S mutant for this analysis because it exhibited the greatest response to rescue by carbamazepine and has been shown previously to have WT-like gating properties (31). Login to comment
193 A progressive increase in efflux through rescued F27S channels over a 40-min period was observed with longer washout; the efflux level was nearly comparable with that seen in WT channels with 60-min washout (Fig. 6B). Login to comment
206 Carbamazepine increases F27S mutant channel surface expression by improving processing and maturation of the channel complex during biogenesis. Login to comment
207 A, COSm6 cells transfected with F27S SUR1 and WT Kir6.2 were pulse-labeled with Tran35 S-Label for an hour and chased for 0-4 h in regular medium. Login to comment
221 Our results show that although diazoxide abolished the chaperoning effect of either glibenclamide or tolbutamide it did not interfere with the ability of carbamazepine to correct the processing defect of F27S SUR1 (Fig. 6D). 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
238 B, Western blot of SUR1 from COSm6 cells transfected with F27S SUR1 and WT Kir6.2 and treated with carbamazepine or rapamycin (Rapa) or Liaf9; , both of which are autophagy inducers, for 16 h as indicated. 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
256 F27S cells were treated overnight (O.N.) with 10 òe;M carbamazepine, and carbamazepine was removed 15, 30, or 60 min (washout) prior to incubation with metabolic inhibitors. Login to comment
262 Note that although diazoxide nearly abolished the rescue effect of glibenclamide or tolbutamide it had no effect on the ability of carbamazepine to correct the processing defect of F27S SUR1. 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
292 This suggests that diazoxide may antagonize the inhibitory effect of carbamazepine to potentiate the function of rescued F27S channels. Login to comment
298 Feasibility of the carbamazepine and diazoxide combination therapy strategy is further supported by our finding that the two drugs can be co-applied to cells without compromising the ability of carbamazepine to rescue the trafficking defects of the F27S mutant (Fig. 6D). Login to comment
303 Although our study focused mostly on one mutation (F27S) that responded well to carbamazepine, there are many additional congenital hyperinsulinism-associated SUR1 TMD0 mutations waiting to be tested (4). Login to comment

[hide] Molecular and immunohistochemical analyses of the ... Mod Pathol. 2006 Jan;19(1):122-9. Suchi M, MacMullen CM, Thornton PS, Adzick NS, Ganguly A, Ruchelli ED, Stanley CA
Molecular and immunohistochemical analyses of the focal form of congenital hyperinsulinism.
Mod Pathol. 2006 Jan;19(1):122-9., [PMID:16357843]

Abstract [show]
Congenital hyperinsulinism is a rare pancreatic endocrine cell disorder that has been categorized histologically into diffuse and focal forms. In focal hyperinsulinism, the pancreas contains a focus of endocrine cell adenomatous hyperplasia, and the patients have been reported to possess paternally inherited mutations of the ABCC8 and KCNJ11 genes, which encode subunits of an ATP-sensitive potassium channel (K(ATP)). In addition, the hyperplastic endocrine cells show loss of maternal 11p15, where imprinted genes such as p57(kip2) reside. In order to evaluate whether all cases of focal hyperinsulinism are caused by this mechanism, 56 pancreatectomy specimens with focal hyperinsulinism were tested for the loss of maternal allele by two methods: immunohistochemistry for p57(kip2) (n=56) and microsatellite marker analysis (n=27). Additionally, 49 patients were analyzed for K(ATP) mutations. Out of 56 focal lesions, 48 demonstrated clear loss of p57(kip2) expression by immunohistochemistry. The other eight lesions similarly showed no nuclear labeling, but the available tissue was not ideal for definitive interpretation. Five of these eight patients had paternal K(ATP) mutations, of which four demonstrated loss of maternal 11p15 within the lesion by microsatellite marker analysis. All of the other three without a paternal K(ATP) mutation showed loss of maternal 11p15. K(ATP) mutation analysis identified 32/49 cases with paternal mutations. There were seven patients with nonmaternal mutations whose paternal DNA material was not available, and one patient with a mutation that was not present in either parent's DNA. These eight patients showed either loss of p57(kip2) expression or loss of maternal 11p15 region by microsatellite marker analysis, as did the remaining nine patients with no identifiable K(ATP) coding region mutations. The combined results from the immunohistochemical and molecular methods indicate that maternal 11p15 loss together with paternal K(ATP) mutation is the predominant causative mechanism of focal hyperinsulinism.

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93 KATP mutationsa Nuclear labeling of p57kip2 Microsatellite marker analysis at 11p15 Remarks on histology Lesion Islets in normal area 1 g3992-9a/ + ND 2 R1494Q/ + ND 3 V21D/ + ND 4 g3992-9a/ + ND 5 3576 del g/ Small lesion + ND 6 R74W/ Small normal area and weak Loss of maternal allele 7 C717X/ + Loss of maternal allele 8 1874 del c/ + ND 9 Q954X/ + ND 10 g3992-9g/ + Loss of maternal allele 11 E501K/ + Loss of maternal allele 12 R136Lb / Weak Loss of maternal allele 13 c2924-9a/ + Loss of maternal allele Focal lesion occupies large area of pancreas 14 g3992-9a/ + ND 15 3084 del g/ + ND 16 R302Hb / + Loss of maternal allele 17 g3992-9a/ + ND 18 536-539 del atgg/ + ND 19 R1215W/ + Loss of maternal allele 20 R999X/ + ND 21 L1350Q/ + ND 22 G1401R/ Weak Loss of maternal allele 23 g2041-21a/ + Loss of maternal allele 24 G7R/ Weak Loss of maternal allele 25 g3992-9a/ + Loss of maternal allele Rare nonadjacent large islet cell nuclei 26 g3992-9a/ + ND 27 Q954X/ + ND 28 delF1388/ + ND 29 Q472X/ + ND 30 G40Db / + Loss of maternal allele 31 S116Pb / + ND 32 g3992-9a/ + ND 33 g2116+1t, nonmaternal + ND 34 A101Db , nonmaternal Small normal area Loss of maternal allele Focal lesion occupies large area of pancreas 35 F27S, nonmaternal Weak Loss of maternal allele 36 G1379R, nonmaternal + ND 37 1631 del t, nonmaternal + ND 38 R1215W, nonmaternal + Loss of maternal allele 39 L503P, nonmaternal + Loss of maternal allele 40 F686S, de novo + Loss of maternal allele 41 1332+4 del c, maternalc + Loss of maternal allele 42 / + Loss of maternal allele 43 / + ND 44 / Small lesion + Loss of maternal allele 45 / + Loss of maternal allele 46 / + Loss of maternal allele 47 / + ND 48 / + Loss of maternal allele 49 / + ND 50 ND + ND 51 ND + ND 52 ND + Loss of maternal allele Rare nonadjacent large islet cell nuclei 53 ND + Loss of maternal allele Focal lesion occupies large area of pancreas All 10 pancreatic specimens studied from patients with diffuse hyperinsulinism did not show loss of p57kip2 labeling of the islet cell nuclei (data not shown). 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
26 This scenario differs somewhat from that observed for gating regulation whereby E203K-SUR1 does not affect channel ATP-sensitivity and Q52E-Kir6.2 increases ATP-sensitivity by ~5-fold but E203K//Q52E increases ATP-sensitivity by ~100-fold.21 Moreover, while crosslinking of E203C//Q52C induces channel closure21 it does not appear to rescue the trafficking defect caused by F27S, at least under the experimental conditions we have tested. Login to comment
30 Although the precise mechanism underlying our findings remains to be determined, the charge-dependence of the effect of Q52-Kir6.2 mutation on F27S-SUR1 processing leads us to speculate that the negative charge at this position may not shown). 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
36 Analysis of the contribution from individual mutations revealed that the processing defect caused by F27S-SUR1 is little affected by E203K-SUR1 but is significantly alleviated by the Q52E and D mutations in Kir6.2 alone. 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
40 We attempted to test this hypothesis by treating cells expressing F27S/E203C//Q52C with H2 O2 . Login to comment
41 While we were able to observe a crosslinked SUR1-Kir6.2 species on immunoblots within 10 min of H2 O2 exposure, no significant correction of the F27S processing defect was detected even after 30 min or overnight H2 O2 exposure (data not shown). Login to comment
44 Next, we tested the role of individual E203K-SUR1 or Q52E-Kir6.2 mutations in F27S-SUR1 mutant processing. Login to comment
45 Surprisingly, while the E203K-SUR1 mutation had little effect on F27S-SUR1 processing, co-expression of F27S-SUR1 with Q52E-Kir6.2 was sufficient to increase the upper F27S-SUR1 band, and surface F27S-SUR1 detected by surface biotinylation was nearly as abundant as F27S/E203K//Q52E (Fig. 3A). Login to comment
46 Moreover, we found that while Q52D-Kir6.2 similarly improved the processing and surface expression of F27S-SUR1, Q52K-Kir6.2 did not (Fig. 3B). 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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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
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

[hide] Carbamazepine inhibits ATP-sensitive potassium cha... Channels (Austin). 2014;8(4):376-82. Zhou Q, Chen PC, Devaraneni PK, Martin GM, Olson EM, Shyng SL
Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP.
Channels (Austin). 2014;8(4):376-82., [PMID:24849284]

Abstract [show]
In pancreatic beta-cells, K(ATP) channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K(ATP) channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1. In addition, sulfonylureas have been shown to function as pharmacological chaperones to correct channel biogenesis and trafficking defects. Recently, we reported that carbamazepine, an anticonvulsant known to inhibit voltage-gated sodium channels, has profound effects on K(ATP) channels. Like sulfonylureas, carbamazepine corrects trafficking defects in channels bearing mutations in the first transmembrane domain of SUR1. Moreover, carbamazepine inhibits the activity of K(ATP) channels such that rescued mutant channels are unable to open when the intracellular ATP/ADP ratio is lowered by metabolic inhibition. Here, we investigated the mechanism by which carbamazepine inhibits K(ATP) channel activity. We show that carbamazepine specifically blocks channel response to MgADP. This gating effect resembles that of sulfonylureas. Our results reveal striking similarities between carbamazepine and sulfonylureas in their effects on K(ATP) channel biogenesis and gating and suggest that the 2 classes of drugs may act via a converging mechanism.

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14 Mutations that render channels less sensitive to ATP inhibition, or more sensitive to MgADP stimulation, cause neonatal diabetes and in some cases also DEND syndrome.3,12-14 By contrast, mutations which render channels unresponsive to the stimulatory effect of MgADP result in loss of channel function and are frequently found in patients with congenital hyperinsulinism, which is characterized by persistent insulin secretion despite life-threatening hypoglycemia.15,16 Aside from gating defects, channel biogenesis and trafficking defects which prevent expression of functional channels in the b2;-cell plasma membrane are also major causes of congenital hyperinsulinism.17 Sulfonylureas such as tolbutamide and glibenclamide inhibit KATP channel activity to stimulate insulin secretion and are thus effective in treating type II diabetes as well as some cases of neonatal diabetes/ DEND syndrome caused by hyperactive KATP channels.3 The inhibitory effect of sulfonylureas on KATP channel activity is largely attributed to an inhibition of channel response to MgADP.18 In addition to inhibiting channel activity, sulfonylureas have been shown to act as KATP channel pharmacological chaperones and correct trafficking defects caused by a subset of mutations in SUR1, specifically those in the first transmembrane domain TMD0.19,20 We have previously shown that mutant channels rescued to the cell surface by the high-affinity sulfonylurea glibenclamide are unable to open in response to metabolic inhibition in intact cells as assessed by 86 Rb+ efflux assays.19 Closer examination by inside-out patch-clamp recording revealed that the rescued channels failed to respond to MgADP, likely because the drug remained bound to the channel even after extensive washout.19 However, a lower affinity sulfonylurea tolbutamide could be washed out from rescued surface channels to recover channel response to MgADP and to allow channels to open upon metabolic inhibition in 86 Rb+ efflux assays.19,20 In a recent study, we identified carbamazepine as a novel KATP channel 'corrector` able to rescue trafficking defects caused by the same set of TMD0 mutations rescued by sulfonylureas.21 Interestingly, functional analysis of a SUR1-TMD0 trafficking mutant, F27S, showed that channels rescued to the cell surface by carbamazepine also failed to open upon metabolic inhibition.21 However, mutant channel activity gradually recovered as carbamazepine was washed out, with activity near the level observed in wild-type (WT) channels after 90 min washout.21 In this study, we investigated how carbamazepine affects KATP channel gating to prevent rescued channels from opening in metabolically stressed cells. Login to comment
17 We therefore determined the response of carbamazepine-rescued F27S mutant channels to ATP and ADP by inside-out patch-clamp recording. Login to comment
19 Note although the F27S mutation severely hinders channel trafficking to the cell surface, sufficient currents (~10% of averaged WT channel current amplitude) can be detected in a small number of cells to allow analysis of ATP and MgADP sensitivity. Login to comment
20 Compared with WT channels, mutant F27S channels exhibited comparable sensitivity to ATP inhibition and MgADP stimulation (Fig. 1), indicating that the F27S mutation per se does not have a significant effect on channel response to intracellular nucleotides. Login to comment
21 Next, we recorded F27S mutant channels from cells treated overnight with Figure 1. Login to comment
22 The F27S mutation in SUR1 does not alter KATP channel gating property. Login to comment
23 (A) Representative recordings testing MgADP responses of WT and F27S channels expressed in COSm6 cells without overnight carbamazepine treatment. Login to comment
25 There is no statistical significance in MgADP response between F27S and WT channels. Login to comment
29 As the MgADP stimulatory effect is crucial for KATP channels to open upon glucose deprivation,15,16 this explains why carbamazepine-rescued F27S mutant channels were unable to open upon metabolic inhibition, as observed in our previous study.21 Interestingly, washout of carbamazepine by incubating cells in fresh media without the drug for 2 h prior to recording recovered the channel`s response to MgADP, suggesting carbamazepine impairs MgADP response. Login to comment
31 As observed in the F27S mutant, WT channels recorded from cells treated overnight with carbamazepine also had little MgADP response; upon washout for more than 2 h, the MgADP response recovered to nearly the extent seen in channels not previously exposed to carbamazepine (Fig. 2). Login to comment
34 We found that both WT and F27S mutant channels from cells treated overnight with carbamazepine also exhibited greatly reduced response to diazoxide; and again, after washout, diazoxide response recovered to the level comparable to that reported previously for WT channels (Fig. 3). Login to comment
43 (A) Representative inside-out patch-clamp recordings testing MgADP responses of WT and F27S channels expressed in COSm6 cells treated overnight with 10 &#b5;M carbamazepine with (right) or without (left) 2 h of washout prior to recording. Login to comment
61 (A) Representative inside-out patch-clamp recordings testing diazoxide responses of WT and F27S channels expressed in cells treated overnight with 10 &#b5;M carbamazepine with (right) or without (left) 2 h of washout prior to recording. 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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117 The F27S SUR1-TMD0 mutation previously shown to respond to GBC and CBZ rescue (22) was used as an example. Login to comment
120 By contrast, only the immature band was observed in cells co-expressing F27S-SUR1 and Kir6.2, a defect that was efficiently corrected by GBC and CBZ. Login to comment
136 Remarkably, both single binding mutations alone and the double binding mutation also rendered CBZ ineffective in rescuing F27S (Fig. 2C). Login to comment
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
150 For comparison, WT control (WT-SUR1 af9; WT-Kir6.2) and F27S in WT-SUR1 background (F27S-SUR1 af9; WT-Kir6.2) with or without drugtreatmentwasincludedineachblot.Tubulinblotsservedasloadingcontrols.Thefilledandopencirclesinthisandsubsequentblotsindicatethecomplex- and core-glycosylated SUR1 proteins, respectively. Login to comment
189 Using F27S-SUR1, we found that whereas processing of the mutant SUR1 in cells co-expressing WT-Kir6.2 was fully normalized by both drugs, as evidenced by the abundant upper band, this was not the case when F27S-SUR1 was co-expressed with èc;N30-Kir6.2, where the upper band in the drug-treated cells was barely detectable (Fig. 4A). Login to comment
216 A, effects of Kir6.2 Nand C-terminal deletions on pharmacological rescue of trafficking-defective SUR1-TMD0 mutant F27S. Login to comment
217 Shown are SUR1 blots of cells expressing F27S-SUR1 along with WT-, èc;N30-, or èc;C25-Kir6.2 and treated overnight with DMSO (0.1%) (V), 5 òe;M GBC (G), or 10 òe;M CBZ (C). Login to comment
219 C, Kir6.2 blots from cells co-expressing WTor F27S-SUR1 and WTor èc;N30-Kir6.2 and treated with DMSO, 5 òe;M GBC, or 10 òe;M CBZ. Login to comment
243 D, Kir6.2Y12AzF cross-links to F27S-SUR1 only in cells treated overnight with 5 òe;M GBC to rescue the trafficking defect caused by F27S. Login to comment
249 We then tested whether pharmacological chaperones will induce cross-linking of Kir6.2 to the TMD0-SUR1 trafficking mutant F27S. Login to comment
307 Our observation that in the absence of pharmacological chaperones, F27S-SUR1 fails to confer cross-linking suggests that F27S disrupts SUR1-Kir6.2 interactions, perhaps due to misfolding of TMD0. 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