Title: Sulfonylureas Correct Trafficking Defects of Disease-causing ATP-sensitive Potassium Channels by Binding to the Channel Complex
Abstract: ATP-sensitive potassium (KATP) channels mediate glucose-induced insulin secretion by coupling metabolic signals to β-cell membrane potential and the secretory machinery. Reduced KATP 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 KATP channel surface expression defects caused by channel mutations. ATP-sensitive potassium (KATP) channels mediate glucose-induced insulin secretion by coupling metabolic signals to β-cell membrane potential and the secretory machinery. Reduced KATP 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 KATP channel surface expression defects caused by channel mutations. Regulation of insulin secretion by blood glucose relies on expression of functional ATP-sensitive potassium (KATP) channels at the β-cell membrane. The β-cell KATP channel is an octameric protein complex comprising four pore-forming Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) 2The abbreviations used are: SUR1, sulfonylurea receptor 1; CHI, congenital hyperinsulinism; PNDM, permanent neonatal diabetes mellitus; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; WT, wild type. 2The abbreviations used are: SUR1, sulfonylurea receptor 1; CHI, congenital hyperinsulinism; PNDM, permanent neonatal diabetes mellitus; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; WT, wild type. subunits (1Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 2Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (242) Google Scholar, 3Shyng S. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (416) Google Scholar). Channel activity is determined by the interplay between both channel subunits and intracellular ATP and ADP: binding of ATP to the Kir6.2 subunit inhibits channel activity, whereas binding of Mg2+-complexed ATP or ADP to the SUR1 subunit stimulates channel activity (4Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (617) Google Scholar, 5Ashcroft F.M. Biochem. Soc. Trans. 2006; 34: 243-246Crossref PubMed Scopus (78) Google Scholar, 6Nichols C.G. Nature. 2006; 440: 470-476Crossref PubMed Scopus (647) Google Scholar). In this way, channel activity serves as a reporter of intracellular ATP and ADP concentrations during glucose metabolism to control β-cell excitability, hence insulin secretion. Dysfunction of β-cell KATP channels because of mutations in the channel subunits Kir6.2 or SUR1 underlies congenital insulin secretion disorders (4Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (617) Google Scholar, 5Ashcroft F.M. Biochem. Soc. Trans. 2006; 34: 243-246Crossref PubMed Scopus (78) Google Scholar). Whereas mutations causing loss of channel function lead to excessive insulin secretion and hypoglycemia as seen in patients with congenital hyperinsulinism (CHI), those causing gain of channel function lead to insufficient insulin secretion and permanent neonatal diabetes mellitus (PNDM). In congenital hyperinsulinism, two prominent mechanisms accounting for loss of channel function are loss of channel expression at the cell surface and loss of channel sensitivity to stimulation by MgADP (7Huopio H. Shyng S.L. Otonkoski T. Nichols C.G. Am. J. Physiol. 2002; 283: E207-E216Crossref PubMed Scopus (97) Google Scholar). In contrast, gain of channel function associated with heterozygous mutations in Kir6.2 or SUR1 in neonatal diabetes is thought to result from reduced channel inhibition at physiological concentrations of MgATP (8Hattersley A.T. Ashcroft F.M. Diabetes. 2005; 54: 2503-2513Crossref PubMed Scopus (365) Google Scholar, 9Proks P. Arnold A.L. Bruining J. Girard C. Flanagan S.E. Larkin B. Colclough K. Hattersley A.T. Ashcroft F.M. Ellard S. Hum. Mol. Genet. 2006; 15: 1793-1800Crossref PubMed Scopus (180) Google Scholar). Interestingly, we recently showed that some PNDM-causing Kir6.2 mutations also reduce the efficiency of channel expression at the cell surface, although the loss of expression effect is masked by the ATP gating defect, resulting in a net gain of channel function, thereby the diabetes phenotype (10Lin C.W. Lin Y.W. Yan F.F. Casey J. Kochhar M. Pratt E.B. Shyng S.L. Diabetes. 2006; 55: 1738-1746Crossref PubMed Scopus (35) Google Scholar). Sulfonylureas such as tolbutamide and glibenclamide are oral hypoglycemic drugs commonly used for treating type II diabetes; they do so by binding to the channel, primarily to the SUR1 subunit, and inhibiting channel activity (4Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (617) Google Scholar, 11Gribble F.M. Reimann F. Diabetologia. 2003; 46: 875-891Crossref PubMed Scopus (248) Google Scholar, 12Melander A. Diabetes. 2004; 53: S151-S155Crossref PubMed Scopus (46) Google Scholar). Glibenclamide differs from tolbutamide in that it has an additional benzamido moiety attached to the sulfonylurea moiety. Glibenclamide binds to SUR1 with ∼1,000-fold higher affinity than tolbutamide, and blocks channel activity at a much lower concentration with the block being irreversible (11Gribble F.M. Reimann F. Diabetologia. 2003; 46: 875-891Crossref PubMed Scopus (248) Google Scholar). Another class of compounds known as glinides, for example, meglitinide and repaglinide, which are structurally more related to the nonsulfonylurea half of glibenclamide, also bind SUR1 to inhibit channel activity (11Gribble F.M. Reimann F. Diabetologia. 2003; 46: 875-891Crossref PubMed Scopus (248) Google Scholar, 13Quast U. Stephan D. Bieger S. Russ U. Diabetes. 2004; 53: 156-S164Crossref PubMed Scopus (90) Google Scholar, 14Bryan J. Vila-Carriles W.H. Zhao G. Babenko A.P. Aguilar-Bryan L. Diabetes. 2004; 53: S104-S112Crossref PubMed Scopus (91) Google Scholar). The binding pocket of glibenclamide is therefore proposed to comprise two sites: site A, which binds ligands with the short chain sulfonylurea moiety like tolbutamide and site B, which binds the non-sulfonylurea half of glibenclamide, as well as glinides such as repaglinide, as shown in Fig. 1 (13Quast U. Stephan D. Bieger S. Russ U. Diabetes. 2004; 53: 156-S164Crossref PubMed Scopus (90) Google Scholar, 15Brown G.R. Foubister A.J. J. Med. Chem. 1984; 27: 79-81Crossref PubMed Scopus (41) Google Scholar). Sites A and B are thought to overlap to accommodate the negative charge and the central phenyl ring present in both sulfonylureas and glinides (Fig. 1) (15Brown G.R. Foubister A.J. J. Med. Chem. 1984; 27: 79-81Crossref PubMed Scopus (41) Google Scholar). SUR1 belongs to the ATP-binding cassette (ABC) transporter protein family (16Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd 3rd, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar); it has three transmembrane domains, TMD0, TMD1, and TMD2 each containing 5, 6, and 6 transmembrane segments, and two intracellular nucleotide-binding domains (Fig. 1) (17Conti L.R. Radeke C.M. Shyng S.L. Vandenberg C.A. J. Biol. Chem. 2001; 276: 41270-41278Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Tusnady G.E. Bakos E. Varadi A. Sarkadi B. FEBS Lett. 1997; 402: 1-3Crossref PubMed Scopus (215) Google Scholar). Structure-function and binding studies using recombinant channel proteins reveal two SUR1 regions that are critical for binding (14Bryan J. Vila-Carriles W.H. Zhao G. Babenko A.P. Aguilar-Bryan L. Diabetes. 2004; 53: S104-S112Crossref PubMed Scopus (91) Google Scholar, 16Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd 3rd, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar, 19Babenko A.P. Gonzalez G. Bryan J. FEBS Lett. 1999; 459: 367-376Crossref PubMed Scopus (54) Google Scholar, 20Ashfield R. Gribble F.M. Ashcroft S.J. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar, 21Mikhailov M.V. Mikhailova E.A. Ashcroft S.J. FEBS Lett. 2001; 499: 154-160Crossref PubMed Scopus (84) Google Scholar, 22Mikhailov M.V. Ashcroft S.J. J. Biol. Chem. 2000; 275: 3360-3364Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The first region, likely representing the A site, involves loop linking transmembrane segments 15 and 16 of TMD2. Mutation of a single serine residue (Ser1237 of rat SUR1) in this region abolishes [3H]glibenclamide binding to SUR1 and in electrophysiological experiments, the same mutation abolishes channel inhibition by tolbutamide and renders channel inhibition by glibenclamide readily reversible (20Ashfield R. Gribble F.M. Ashcroft S.J. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar). The second region involves the cytoplasmic loop between TMD0 and TMD1 (referred to as L0 by Bryan et al. (14Bryan J. Vila-Carriles W.H. Zhao G. Babenko A.P. Aguilar-Bryan L. Diabetes. 2004; 53: S104-S112Crossref PubMed Scopus (91) Google Scholar)) where the B site may be located. Deletion of this cytoplasmic loop leads to loss of [3H]glibenclamide binding in recombinant SUR1 expressed in insect cells (21Mikhailov M.V. Mikhailova E.A. Ashcroft S.J. FEBS Lett. 2001; 499: 154-160Crossref PubMed Scopus (84) Google Scholar) and mutation of tyrosine 230 in L0 to alanine (Y230A) abolishes photoaffinity labeling of SUR1 by [125I]azidoglibenclamide (14Bryan J. Vila-Carriles W.H. Zhao G. Babenko A.P. Aguilar-Bryan L. Diabetes. 2004; 53: S104-S112Crossref PubMed Scopus (91) Google Scholar). Although the effect of sulfonylureas on KATP channel activity has been known for a long time, their effect on KATP channel expression/trafficking was only recently appreciated. We have previously reported that sulfonylureas rescue surface expression defects of KATP channels caused by two CHI-associated SUR1 mutations, A116P and V187D (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). More recently, we found that several Kir6.2 mutations identified in PNDM also reduce channel surface expression and that the reduced surface expression is improved upon sulfonylurea treatment (10Lin C.W. Lin Y.W. Yan F.F. Casey J. Kochhar M. Pratt E.B. Shyng S.L. Diabetes. 2006; 55: 1738-1746Crossref PubMed Scopus (35) Google Scholar). In this work, we investigated the mechanism by which sulfonylureas correct the channel surface expression defects caused by the A116P or V187D mutations and by the PNDM-associated Kir6.2 mutations. Our results indicate that both the sulfonylurea and the glinide moieties contribute to the rescue effect and show that SUR1 mutations known to interfere with drug binding also interfere with the ability of these drugs to rescue the mutant channel surface expression defect. Interestingly and somewhat unexpectedly, we found that Kir6.2 is required for sulfonylureas to rescue the A116P and V187D mutant SUR1 at the cell surface. Conversely, sulfonylurea binding at SUR1 is necessary for the drug to improve surface expression of PNDM mutant channels harboring Kir6.2 mutations. These results suggest that sulfonylureas exert chaperoning effects on the SUR1·Kir6.2 complex rather than mutant SUR1 or Kir6.2 alone to correct KATP channel surface expression defects. Molecular Biology—FLAG epitope (DYKDDDDK) was inserted at the N terminus of the hamster SUR1 cDNA by sequential overlap extension PCR (referred to as fSUR1), as described previously (24Cartier E.A. Conti L.R. Vandenberg C.A. Shyng S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2882-2887Crossref PubMed Scopus (144) Google Scholar). Point mutations of SUR1 were introduced into hamster SUR1 cDNA in the pECE plasmid using the QuikChange site-directed mutagenesis kit (Stratagene). The FLAG epitope tag and mutations were confirmed by DNA sequencing. Rat Kir6.2 cDNA is in the pcDNAI vector. Mutant clones from multiple PCR were analyzed in all experiments to avoid false results caused by undesired mutations introduced by PCR. Immunofluorescence Staining—COSm6 cells were plated in 6-well tissue culture plates, transfected with 0.6 μg of fSUR1 and 0.4 μg of Kir6.2 per well using FuGENE 6 (Roche) according to the manufacturer's directions. Transfected cells in each well were equally split and replated onto three coverslips 24 h post-transfection. Two coverslips were used for surface staining and one was used for total staining of fSUR1. For surface staining, cells were pretreated with or without 5 μm glibenclamide (diluted in culture medium from 10 mm stock dissolved in dimethyl sulfoxide (Me2SO)) 24 h prior to the staining. Staining was carried out as described previously (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, cells were incubated with anti-FLAG M2 mouse monoclonal antibody (Sigma, diluted to 10 μg/ml in Opti-MEM containing 0.1% BSA) for 1 h at 4°C, washed with ice-cold PBS, then incubated with Cy3-conjugated donkey anti-mouse secondary antibodies (Jackson) for 30 min at 4 °C. After three 5-min washes in ice-cold PBS, cells were fixed with 4% paraformaldehyde, and viewed using an Olympus Fluoview confocal microscope. For total cellular staining of fSUR1, cells were fixed with cold (-20 °C) methanol for 5 min. Fixed cells were incubated with the anti-FLAG M2 monoclonal antibody (10 μg/ml PBS containing 0.1% BSA) at room temperature for 1 h, washed in PBS, incubated with Cy3-conjugated donkey anti-mouse secondary antibodies for 30 min at room temperature, and washed again in PBS before imaging. Immunoblotting—COSm6 cells were plated in 35-mm dishes and transfected with KATP channel subunits using FuGENE 6 as previously described (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Cells were lysed 48-72 h post-transfection in a buffer containing 20 mm Hepes (pH 7.0), 5 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, and Complete™ protease inhibitors (Roche). Proteins in cell lysates were separated by SDS-PAGE (8%), transferred to nitrocellulose membrane, incubated with the M2 anti-FLAG antibody (Sigma) followed by horseradish peroxidase-conjugated anti-mouse secondary antibodies (Amersham Biosciences), and visualized by chemiluminescence (Super Signal West Femto; Pierce). Chemiluminescence Assay—Transfection of COSm6 cells was carried out as described above. Drug treatment was initiated 32-40 h post-transfection and lasted for 24 h. Cells were then processed for chemiluminescence assays as described previously (25Taschenberger G. Mougey A. Shen S. Lester L.B. LaFranchi S. Shyng S.L. J. Biol. Chem. 2002; 277: 17139-17146Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Briefly, cells were fixed with 2% paraformaldehyde for 30 min at 4 °C, preblocked in PBS, 0.1% BSA for 30 min, incubated in M2 anti-FLAG antibody (10 μg/ml) for 1 h, washed four times for 30 min in PBS, 0.1% BSA, incubated in horseradish peroxidase-conjugated anti-mouse antibody (Jackson, 1:1000 dilution) for 20 min, and washed again four times for 30 min in PBS, 0.1% BSA. Chemiluminescence of each dish was quantified in a TD-20/20 luminometer (Turner Designs) following a 5-s incubation in Power Signal Elisa Femto luminol solution (Pierce). All steps after fixation were carried out at room temperature. Electrophysiology—COSm6 cells were transfected using FuGENE 6 and plated onto coverslips. The cDNA for the green fluorescent protein was cotransfected with SUR1 and Kir6.2 to facilitate identification of positively transfected cells. Patch clamp recordings were made 36-72 h post-transfection. All experiments were performed at room temperature as previously described (24Cartier E.A. Conti L.R. Vandenberg C.A. Shyng S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2882-2887Crossref PubMed Scopus (144) Google Scholar). Micropipettes were pulled from non-heparinized Kimble glass (Fisher Scientific) on a horizontal puller (Sutter Instrument, Co., Novato, CA). Electrode resistance was typically 1.4-1.6 MΩ when filled with K-INT solution (below). Inside-out patches were voltage-clamped with an Axopatch 1D amplifier (Axon Inc., Foster City, CA). The standard bath (intracellular) and pipette (extracellular) solutions (K-INT) had the following composition: 140 mm KCl, 10 mm K-HEPES, 1 mm K-EGTA (pH 7.3). ATP was added as the potassium salt. Tolbutamide and glibenclamide were dissolved in Me2SO at 300 or 10 mm, respectively, and diluted further in K-INT. Control experiments (see supplemental Fig. S1) confirm that Me2SO at the final concentrations used does not affect KATP channel activity (26Cartier E.A. Shen S. Shyng S.L. J. Biol. Chem. 2003; 278: 7081-7090Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). All currents were measured at a membrane potential of -50 mV (pipette voltage = +50 mV), and inward currents shown as upward deflections. Data were analyzed using pCLAMP8 software (Axon Instrument). Off-line analysis was performed using Origin 6.1 and Microsoft Excel programs. Data Analysis—Data were presented as mean ± S.E. Statistical analysis was performed using independent two-population two-tailed Student's t test, with p < 0.05 considered statistically significant. TMD0 of SUR1 Does Not Confer the Rescue Effect of Sulfonylureas on Mutant Expression—Many CHI-causing SUR1 mutations have been reported to reduce surface expression of KATP channels by causing ER retention of the mutant channel (they are referred to as trafficking mutations hereafter for their inability to traffic normally to the plasma membrane) (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 24Cartier E.A. Conti L.R. Vandenberg C.A. Shyng S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2882-2887Crossref PubMed Scopus (144) Google Scholar, 25Taschenberger G. Mougey A. Shen S. Lester L.B. LaFranchi S. Shyng S.L. J. Biol. Chem. 2002; 277: 17139-17146Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 27Chan K.W. Zhang H. Logothetis D.E. EMBO J. 2003; 22: 3833-3843Crossref PubMed Scopus (137) Google Scholar). Of these, two mutations, A116P and V187D, were rescued by the pharmacological agent sulfonylurea (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). We hypothesize that sulfonylurea may rescue these mutants by binding to the mutant protein and acting as chemical chaperones to facilitate channel biogenesis and trafficking. Because both A116P and V187D are located in TMD0, we first tested if sulfonylureas bind directly to TMD0 to facilitate mutant protein biogenesis and trafficking, even though TMD0 has not been implicated in sulfonylurea binding in prior studies (21Mikhailov M.V. Mikhailova E.A. Ashcroft S.J. FEBS Lett. 2001; 499: 154-160Crossref PubMed Scopus (84) Google Scholar). We compared surface expression of recombinant SUR1-TMD0 (amino acids 1-197) containing either the A116P or the V187D mutation in the presence or absence of 5 μm glibenclamide. Studies by others have shown that co-expression of SUR1-TMD0 with Kir6.2 C-terminal deletion mutant Kir6.2ΔC25, which lacks the -RKR-ER retention motif, reconstitutes "mini" KATP channels at the cell surface (27Chan K.W. Zhang H. Logothetis D.E. EMBO J. 2003; 22: 3833-3843Crossref PubMed Scopus (137) Google Scholar, 28Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Consistently, we observed cell surface expression of SUR1-TMD0 (with a FLAG epitope tag placed at the N terminus; referred to as fSUR1-TMD0) in COSm6 cells transiently transfected with the construct and Kir6.2ΔC25 using chemiluminescence assays (Fig. 2). By contrast, when the A116P or V187D mutations were introduced into fSUR1-TMD0, surface expression was greatly reduced (by >70%), even though the total mutant recombinant proteins were abundantly expressed as assessed by Western blots and immunofluorescent staining of permeabilized cells (not shown). Treating cells with 5 μm glibenclamide did not improve surface expression of the mutant fSUR1-TMD0. These results demonstrate that the TMD0 domain of SUR1 alone is insufficient to confer the sulfonylurea rescue effect. Restoration of A116P- and V187D-mutant Channel Expression by Sulfonylureas Is Dependent on Intact Sulfonylurea Binding Sites in SUR1—Several studies indicate that the high affinity tolbutamide binding site in SUR1 resides in transmembrane segments 13-16 (19Babenko A.P. Gonzalez G. Bryan J. FEBS Lett. 1999; 459: 367-376Crossref PubMed Scopus (54) Google Scholar, 20Ashfield R. Gribble F.M. Ashcroft S.J. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar). Specifically, mutation of a serine residue in this region (Ser1237 of rat SUR1) to tyrosine abolishes the high affinity block of channel activity by tolbutamide (20Ashfield R. Gribble F.M. Ashcroft S.J. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar). The same mutation also reduces [3H]glibenclamide binding to SUR1 and renders channel inhibition by glibenclamide readily reversible (20Ashfield R. Gribble F.M. Ashcroft S.J. Ashcroft F.M. Diabetes. 1999; 48: 1341-1347Crossref PubMed Scopus (164) Google Scholar). If sulfonylureas rescue the A116P and V187D trafficking mutants by binding to the channel protein, then introducing the S1237Y mutation should also reduce or abolish the ability of sulfonylureas to correct the trafficking defect. We made the equivalent sulfonylurea binding site mutation (S1238Y) in hamster SUR1 (16Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd 3rd, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1273) Google Scholar) and examined how it affects the response of the A116P- or V187D-mutant channels to sulfonylureas. Initial assessment by immunofluorescent staining indicates that the S1238Y mutation by itself does not affect fSUR1 surface expression when coexpressed with Kir6.2; however, when combined with the A116P or V187D mutation, it indeed reduced or prevented the ability of glibenclamide to rescue the surface expression defect caused by the A116P and V187D mutations (Fig. 3). Staining of permeabilized cells transfected with the various mutants revealed similar total mutant protein expression levels. Consistent results were obtained using Western blot analysis. A representative blot of A116P fSUR1 in the WT or S1238Y background from cells treated with or without 5 μm glibenclamide for 24 h is shown in Fig. 4A. We next sought to quantify mutant channel expression at the cell surface in the presence or absence of sulfonylurea treatment. Although KATP current density provides a measure for surface expression of the SUR1-Kir6.2 octameric channel complex, it is not suitable for assessing the effect of glibenclamide on surface channel expression because glibenclamide, even after extensive washes, remains bound to channels rescued to the cell surface and inhibits channel activity (see supplemental Figs. S2 and S3) (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The inhibition by prebound glibenclamide in electrophysiological experiments causes underestimation of channel expression. We therefore resorted to chemiluminescence assays that have been used extensively in prior studies to quantify surface channel expression (23Yan F. Lin C.W. Weisiger E. Cartier E.A. Taschenberger G. Shyng S.L. J. Biol. Chem. 2004; 279: 11096-11105Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25Taschenberger G. Mougey A. Shen S. Lester L.B. LaFranchi S. Shyng S.L. J. Biol. Chem. 2002; 277: 17139-17146Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 26Cartier E.A. Shen S. Shyng S.L. J. Biol. Chem. 2003; 278: 7081-7090Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 29Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar). As shown in Fig. 4B, channels containing the S1238Y-SUR1 mutation alone had surface expression levels comparable with that of the WT channels (see also Fig. 3 and supplemental Fig. S3A). However, when combined with the A116P- or V187D-SUR1 trafficking mutations, S1238Y completely abolished the rescue effect of tolbutamide at 300 (Fig. 4B) and 600 μm (not shown). The S1238Y mutation also significantly reduced the potency of glibenclamide rescue (Fig. 4C). In the WT background (no sulfonylurea binding mutation), 24-h treatment with 1 μm glibenclamide increased surface expression of the A116P mutant from 3.1 ± 0.7 to 34.5 ± 7.2% and the V187D mutant from 12.8 ± 1.6 to 49.2 ± 6.8% of WT channels. But in the S1238Y background, the same treatment only slightly increased surface expression of the A116P and V187D mutants, from 0.7 ± 1.0 to 6.6 ± 2.1% and 9.8 ± 1.4 to 15.6 ± 2.3% of WT, respectively. Increasing the concentration of glibenclamide to 5 μm led to a much greater effect on surface expression of the mutants (to 21.8 and 23.9% of WT for A116P and V187D, respectively; Fig. 4C). Thus, whereas tolbutamide response was abolished by the S1238Y mutation, the glibenclamide response appears to be only partially affected. Note that the Me2SO present in the sulfonylurea stock solutions had no effect on surface expression of the channel (data not shown).FIGURE 4Effect of the S1238Y mutation on the ability of sulfonylureas to rescue KATP channel trafficking defects in the presence of Kir6.2, analysis by immunoblotting and chemiluminescence assays. A, Western blot analysis of fSUR1. In cells expressing Kir6.2 and WT-fSUR1, two bands were observed: the lower core glycosylated band, or the immature band (open arrow) and the upper complex glycosylated band, or the mature band (solid arrow). For the A116P-fSUR1, however, only the immature band was detected in untreated cells (A116P); upon treatment with 1 μm glibenclamide for 24 h (A116P+Glib), the upper A116P-fSUR1 band became apparent, indicating rescue of the mutant protein out of the ER. When the S1238Y mutation was introduced into A116P-fSUR1 (A116P/S1238Y), the same glibenclamide treatment was less effective in promoting expression of the upper band (A116P/S1238Y+Glib). B, quantification of channel expression at the cell surface by chemiluminescence assays. COSm6 cells were co-transfected with Kir6.2 and one of the fSUR1 constructs indicated below the x-axis, and treated with or without 300 μm tolbutamide (Tolb) for 24 h prior to the assay. Surface expression of tolbutamide-treated cells was significantly higher than untr