ABCMdb

PMID: 23744072

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., [PubMed]

Sentences

No. Mutations Sentence Comment

68 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 69 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 79 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 89 ABCC8 p.Phe27Ser 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 ABCC7 p.Arg74Trp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Glu128Lys ABCC8 p.Asn24Lys ABCC8 p.Phe27Ser 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 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser Dose-response relationships were further determined for F27S, A116P, and V187D. Login to comment 127 ABCC7 p.Arg74Trp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Glu128Lys ABCC8 p.Asn24Lys ABCC8 p.Phe27Ser 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 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser Dose-response relationships were further determined for F27S, A116P, and V187D. Login to comment 129 ABCC8 p.Phe27Ser 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 131 ABCC8 p.Phe27Ser 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 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser The F27S mutant was further analyzed by surface staining and chemiluminescence assays. Login to comment 138 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 140 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 144 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 147 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 173 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser WT without any treatment and F27S mutant treated with DMSO (0.1%) for 16 h are shown for comparison. Login to comment 178 ABCC8 p.Phe27Ser WT without any treatment and F27S mutant treated with DMSO (0.1%) for 16 h are shown for comparison. Login to comment 181 ABCC8 p.Phe27Ser F27S treated with DMSO for 16 h is shown for comparison. Login to comment 183 ABCC8 p.Phe27Ser F27S treated with DMSO for 16 h is shown for comparison. Login to comment 186 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 188 ABCC8 p.Phe27Ser 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 189 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser This result indicates that carbamazepine does prevent rescued F27S channels from being activated by metabolic inhibition. Login to comment 191 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 193 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser *, p b0d; 0.001 comparing F27S vehicle with various treatment groups by one-way analysisofvariancewithBonferroniposthoctest.ErrorbarsrepresentS.E.Unt, untransfected cells. Login to comment 204 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser Carbamazepine increases F27S mutant channel surface expression by improving processing and maturation of the channel complex during biogenesis. Login to comment 205 ABCC8 p.Phe27Ser 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 206 ABCC8 p.Phe27Ser Carbamazepine increases F27S mutant channel surface expression by improving processing and maturation of the channel complex during biogenesis. Login to comment 207 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser *, 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 221 ABCC8 p.Phe27Ser 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 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 225 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 228 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 238 ABCC8 p.Phe27Ser 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 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 256 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser *, 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 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 262 ABCC8 p.Phe27Ser 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 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 267 ABCC8 p.Ala116Pro ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser This suggests that diazoxide may antagonize the inhibitory effect of carbamazepine to potentiate the function of rescued F27S channels. Login to comment 292 ABCC8 p.Phe27Ser This suggests that diazoxide may antagonize the inhibitory effect of carbamazepine to potentiate the function of rescued F27S channels. Login to comment 296 ABCC8 p.Phe27Ser 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 298 ABCC8 p.Phe27Ser 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 ABCC8 p.Phe27Ser 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 303 ABCC8 p.Phe27Ser 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