ABCMdb

ABCC8 p.Tyr230Ala

Predicted by SNAP2:	A: D (75%), C: D (66%), D: D (91%), E: D (91%), F: N (72%), G: D (85%), H: D (71%), I: D (71%), K: D (91%), L: D (75%), M: D (66%), N: D (80%), P: D (91%), Q: D (91%), R: D (91%), S: D (85%), T: D (80%), V: D (71%), W: D (66%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, 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,

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[hide] Toward linking structure with function in ATP-sens... Diabetes. 2004 Dec;53 Suppl 3:S104-12. Bryan J, Vila-Carriles WH, Zhao G, Babenko AP, Aguilar-Bryan L
Toward linking structure with function in ATP-sensitive K+ channels.
Diabetes. 2004 Dec;53 Suppl 3:S104-12., [PMID:15561897]

Abstract [show]
Advances in understanding the overall structural features of inward rectifiers and ATP-binding cassette (ABC) transporters are providing novel insight into the architecture of ATP-sensitive K+ channels (KATP channels) (KIR6.0/SUR)4. The structure of the K(IR) pore has been modeled on bacterial K+ channels, while the lipid-A exporter, MsbA, provides a template for the MDR-like core of sulfonylurea receptor (SUR)-1. TMD0, an NH2-terminal bundle of five alpha-helices found in SURs, binds to and activates KIR6.0. The adjacent cytoplasmic L0 linker serves a dual function, acting as a tether to link the MDR-like core to the KIR6.2/TMD0 complex and exerting bidirectional control over channel gating via interactions with the NH2-terminus of the KIR. Homology modeling of the SUR1 core offers the possibility of defining the glibenclamide/sulfonylurea binding pocket. Consistent with 30-year-old studies on the pharmacology of hypoglycemic agents, the pocket is bipartite. Elements of the COOH-terminal half of the core recognize a hydrophobic group in glibenclamide, adjacent to the sulfonylurea moiety, to provide selectivity for SUR1, while the benzamido group appears to be in proximity to L0 and the KIR NH2-terminus.

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162 Results for the wild-type (WT) control, the Tyr230Ala (Y230A), and Trp231Ala (W232A) substitutions are shown. 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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184 First, a truncated SUR1 of TMD0 alone containing the A116P or V187D trafficking mutations failed to respond to sulfonylurea rescue. Login to comment
185 Second, two point mutations, Y230A and S1238Y, that are located downstream of TMD0 and are known to diminish or abolish glibenclamide and tolbutamide binding accordingly affected the ability of the drugs to rescue channel trafficking defects caused by TMD0 mutations (31,36,37). 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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5 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. Login to comment
38 Deletion of this cytoplasmic loop leads to loss of [3 H]glibenclamide binding in recombinant SUR1 expressed in insect cells (21) and mutation of tyrosine 230 in L0 to alanine (Y230A) abolishes photoaffinity labeling of SUR1 by [125 I]azidoglibenclamide (14). 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
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
127 Indeed, the Y230A mutation reduced the rescue effect of glibenclamide, as assessed by immunostaining (Fig. 3) as well as chemiluminescence assays (Fig. 5A). Login to comment
128 We then asked whether this reduction is attributable to loss of binding of FIGURE 3. Impact of the sulfonylurea binding mutations on the effectiveness of glibenclamide to rescue KATP channel trafficking defects in the presence of Kir6.2, using immunocytochemistry analysis. A, top panel, surface staining of COSm6 cells transiently transfected with Kir6.2 and WT-, Y230A-, S1238Y-, or Y230A/S1238Y-fSUR1, using the M2 anti-FLAG mouse monoclonal antibodies followed by Cy3-conjugated anti-mouse secondary antibody. 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
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
143 Finally, introducing the Y230A and S1238Y mutations simultaneously completely abolished the rescue effect by tolbutamide, repaglinide, or glibenclamide (Figs. 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
163 On the Y230A mutation background, however, the glibenclamide rescue effect was attenuated although still significant (p Ͻ 0.01), whereas the effects of repaglinide and tolbutamide were both abolished. 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
166 Comparison of the Effects of Sulfonylurea Binding Site Mutations on Trafficking Rescue Versus Channel Activity Block by Sulfonylureas-Given the somewhat unexpected result that Y230A not only attenuated and abolished channel trafficking rescue by glibenclamide and repaglinide, respectively, but also abrogated the rescue effect of tolbutamide, we sought to further characterize the role of Tyr230 in channel response to sulfonylureas by examining how Y230A affects the ability of glibenclamide and tolbutamide to inhibit KATP channel activity, the best characterized effect of these drugs. Login to comment
167 Inside-out patch clamp recording was used to monitor tolbutamide or glibenclamide block of channel activity in cells expressing Y230A, S1238Y, or Y230A/Y1238Y mutant channels. Login to comment
169 In the Y230A mutant, we observed reduced tolbutamide block at 10 and 300 ␮M, indicating a role of Tyr230 in mediating tolbutamide response both in the context of channel trafficking rescue and channel activity block. Login to comment
170 As expected, Y230A also reduced glibenclamide block at 10 nM and 1 ␮M. Login to comment
171 However, Y230A did not render glibenclamide inhibition reversible; this is in contrast to the S1238Y mutation that caused reduced block by 10 nM glibenclamide and rendered the block reversible upon washout (Fig. 7A) as reported previously by others (20). Login to comment
173 Finally, combining Y230A and S1238Y had a greater effect on preventing tolbutamide and glibenclamide block than either mutation alone (Fig. 7). 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
187 The Y230A/S1238Y double mutation completely abolished the rescue effects by glibenclamide (A), repaglinide (B), and tolbutamide (C). Login to comment
192 Effect of the Y230A and S1238Y mutations on channel block by sulfonylureas. Login to comment
201 The relative current amplitudes of Y230A or S1238Y in 10 or 300 ␮M tolbutamide are not significantly different from WT (p Ͼ 0.05), whereas that of Y230A/S1238Y in either 10 or 300 ␮M tolbutamide is significantly different from WT (p Ͻ 0.05). Login to comment
202 For glibenclamide inhibition, the response of Y230A, S1238Y, and Y230A/S1238Y are all significantly different from WT in either 10 nM or 1 ␮M (p Ͻ 0.05). Login to comment
245 Consistent with this picture, we found that mutation S1238Y in the proposed A site abolished the effect of tolbutamide and attenuated the effect of glibenclamide on the trafficking mutants, whereas mutation Y230A in the proposed B site rendered repaglinide ineffective and glibenclamide less effective in rescuing the trafficking mutants. Login to comment
246 To our surprise, mutation Y230A in the proposed B site also prevented tolbutamide from rescuing the trafficking mutants; whereas this may suggest a role of Tyr230 in tolbutamide binding, an alternative possibility is that the residue is involved in post-binding events that are important for the rescue effect of the drug. Login to comment
248 The Y230A mutation might abolish the tolbutamide rescue effect on the trafficking mutants by disrupting the cross-talk between SUR1 and Kir6.2, which we have shown to be necessary for the sulfonylurea rescue effects. Login to comment
249 Interestingly, we observed parallel effects of S1238Y and/or Y230A on the ability of sulfonylureas to rescue trafficking mutants and block channel activity, suggesting the two processes likely share similar transduction mechanisms. 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
124 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
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
157 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
162 On the Y230A mutation background, however, the glibenclamide rescue effect was attenuated although still significant (p b0d; 0.01), whereas the effects of repaglinide and tolbutamide were both abolished. Login to comment
168 In the Y230A mutant, we observed reduced tolbutamide block at 10 and 300 òe;M, indicating a role of Tyr230 in mediating tolbutamide response both in the context of channel trafficking rescue and channel activity block. Login to comment
172 Finally, combining Y230A and S1238Y had a greater effect on preventing tolbutamide and glibenclamide block than either mutation alone (Fig. 7). Login to comment
184 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
191 Effect of the Y230A and S1238Y mutations on channel block by sulfonylureas. Login to comment
200 The relative current amplitudes of Y230A or S1238Y in 10 or 300 òe;M tolbutamide are not significantly different from WT (p b0e; 0.05), whereas that of Y230A/S1238Y in either 10 or 300 òe;M tolbutamide is significantly different from WT (p b0d; 0.05). Login to comment
243 Consistent with this picture, we found that mutation S1238Y in the proposed A site abolished the effect of tolbutamide and attenuated the effect of glibenclamide on the trafficking mutants, whereas mutation Y230A in the proposed B site rendered repaglinide ineffective and glibenclamide less effective in rescuing the trafficking mutants. Login to comment
244 To our surprise, mutation Y230A in the proposed B site also prevented tolbutamide from rescuing the trafficking mutants; whereas this may suggest a role of Tyr230 in tolbutamide binding, an alternative possibility is that the residue is involved in post-binding events that are important for the rescue effect of the drug. Login to comment
247 Interestingly, we observed parallel effects of S1238Y and/or Y230A on the ability of sulfonylureas to rescue trafficking mutants and block channel activity, suggesting the two processes likely share similar transduction mechanisms. Login to comment

[hide] Defining a binding pocket for sulfonylureas in ATP... FASEB J. 2007 Jan;21(1):18-25. Epub 2006 Nov 16. Vila-Carriles WH, Zhao G, Bryan J
Defining a binding pocket for sulfonylureas in ATP-sensitive potassium channels.
FASEB J. 2007 Jan;21(1):18-25. Epub 2006 Nov 16., [PMID:17110465]

Abstract [show]
Sulfonylurea receptors SUR1 and SUR2 are the regulatory subunits of K(ATP) channels. Their differential affinity for hypoglycemic sulfonylureas provides a basis for the selectivity of these compounds for different K(ATP) channel isoforms. Sulfonylureas have a 100- to 1000-fold greater affinity for SUR1 vs. SUR2. Structure-activity studies suggested a bipartite binding pocket. Chimeric SUR1 approximately SUR2 receptors have shown TMD2, the third bundle of transmembrane helices, to be part of an "A" site that confers SUR1 selectivity for sulfonylureas. The purpose of this study is to determine the position of the "B" site. Previous photoaffinity labeling studies have placed the B site on the amino-terminal third of SUR and colabeled the associated K(IR). In our study, deletion of TMD0, the first bundle of transmembrane helices, did not compromise labeling. Further deletions into the cytoplasmic linker, L0, eliminated binding and labeling. Alanine substitutions in L0 identified a limited number of conserved residues, Y230 and W232, important for affinity labeling. A fragment of K(IR)6.2, missing M2 and the entire carboxyl terminal, assembles with SUR1 and is affinity labeled, while deletion of 10 or more amino-terminal residues compromises labeling. These studies indicate that the B site involves L0 and the K(IR) amino terminus, elements that are critical for control of channel gating.

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104 The graph shows a decrease in photoaffinity labeling for SUR1 Y230A and W232A (Fig. 3C). Login to comment
126 The data are plotted as the ratio of label-toprotein in arbitrary units Ϯ sd. the SUR1, Y230A, and W232A substitutions. Login to comment
127 Both SUR1 and KIR6.2 in KATP channels are affinity-labeled by [125 I]-azido-glibenclamide and coexpression of SUR1, Y230A, and W232A with KIR6.2 resulted in labeling of the KIR (data not shown) and also in an increase of the mutant SUR1 labeling (Fig. 6). Login to comment
129 When SUR1 Y230A, and W232A were coexpressed with ⌵⌬33KIR6.2, a KIR6.2 construct that does not photolabel, none of the SUR1 mutants labeled (Fig. 6), indicating that the KIR6.2 N terminus play a role in the sulfonylurea binding pocket. Login to comment
184 When SUR1 and the SUR1 alanine point mutations Y230A and W232A were coexpressed with KIR, this resulted in an increase in [125 I]-azido-glibenclamide labeling and KIR cophotolabeling, consistent with KIR forming part of the B site. Login to comment
189 Wild-type SUR1, SUR1, Y230A, and W232A were expressed or coexpressed with either KIR6.2 or N⌬33KIR6.2 in COSm6 cells. Login to comment
192 However, coexpression of SUR1, Y230A, and W232A with N⌬33KIR6.2 did not increase labeling of SUR1 or the mutants. Login to comment
103 The graph shows a decrease in photoaffinity labeling for SUR1 Y230A and W232A (Fig. 3C). Login to comment
125 The data are plotted as the ratio of label-to- protein in arbitrary units afe; sd. the SUR1, Y230A, and W232A substitutions. Login to comment
128 When SUR1 Y230A, and W232A were coexpressed with टèc;33KIR6.2, a KIR6.2 construct that does not photolabel, none of the SUR1 mutants labeled (Fig. 6), indicating that the KIR6.2 N terminus play a role in the sulfonylurea binding pocket. Login to comment
183 When SUR1 and the SUR1 alanine point mutations Y230A and W232A were coexpressed with KIR, this resulted in an increase in [125 I]-azido-glibenclamide labeling and KIR cophotolabeling, consistent with KIR forming part of the B site. Login to comment
188 Wild-type SUR1, SUR1, Y230A, and W232A were expressed or coexpressed with either KIR6.2 or Nèc;33KIR6.2 in COSm6 cells. Login to comment
191 However, coexpression of SUR1, Y230A, and W232A with Nèc;33KIR6.2 did not increase labeling of SUR1 or the mutants. 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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268 Mutation of either site A (S1238Y) or site B (Y230A) diminished glibenclamide`s rescue effect, while simultaneous mutation of both completely abolished it, suggesting that the sulfonylurea and benzamido moieties both contribute to the rescue effect of glibenclamide. Login to comment
269 Interestingly, the site B mutation Y230A, in addition to attenuating the effect of glibenclamide and abolishing the effect of rapaglinide, also rendered tolbutamide ineffective at rescuing mutant channels. Login to comment
273 The results of S1238Y and/or Y230A mutants on the ability of sulfonylureas to rescue KATP trafficking mutants were also in parallel to their ability to block channel activity. 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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113 Point mutations in the proposed site A (S1238Y) or site B (Y230A) (Fig. 2B) render channels less sensitive to GBC block, and combining both mutations completely abolishes channel inhibition by GBC (39-41). Login to comment
116 First, we asked whether CBZ rescue of TMD0 trafficking mutants is disrupted by GBC binding mutations, Y230A, S1238Y, or both (Y230A/S1238Y). Login to comment
135 KATP Channel Pharmacological Chaperones MARCH 20, 2015ߦVOLUME 290ߦNUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7983 expected, the rescue effect of GBC was attenuated by Y230A or S1238Y and abolished by Y230A/S1238Y. Login to comment
141 As shown in Fig. 2D, Y230A rendered channels less sensitive to CBZ inhibition. Login to comment
143 Combining Y230A and S1238Y completely prevented CBZ from inhibiting channel activity. Login to comment
147 B, topology model of SUR1 showing the location of GBC binding site A mutation S1238Y and site B mutation Y230A. Login to comment
149 C, trafficking-defectiveSUR1mutantcontainingTMD0mutationF27SincombinationwithGBCbindingmutationY230A(left),S1238Y(middle),ordoublebinding mutation Y230A/S1238Y (right) was co-expressed with Kir6.2 and subjected to drug treatment (0.1% DMSO vehicle (V), 5 òe;M GBC (G), or 10 òe;M CBZ (C)) overnight (16 h) as indicated. Login to comment
152 COSm6 cells were transfected with Kir6.2 and WT-SUR1, Y230A-SUR1, S1238Y-SUR1, or Y230A/S1238Y-SUR1. Login to comment
157 Note that the inhibition was not reversible for WT and the Y230A mutant but was reversible for the S1238Y mutant. Login to comment
176 The Y230A/S1238Y double mutation in SUR1, which abolished the ability of both drugs to rescue the trafficking defects of TMD0-SUR1 mutations also rendered the drugs unable to correct the processing defect of SUR1 coexpressed with I296L-Kir6.2 (Fig. 3D). Login to comment