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• W2048406951 abstract "The pancreatic ATP-sensitive potassium (KATP) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to β-cell membrane potential. Sulfonylureas inhibit KATP channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue KATP channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional KATP channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain KATP channel trafficking mutations. The pancreatic ATP-sensitive potassium (KATP) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to β-cell membrane potential. Sulfonylureas inhibit KATP channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue KATP channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional KATP channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain KATP channel trafficking mutations. ATP-sensitive potassium (KATP) 1The abbreviations used are: KATP, ATP-sensitive potassium; PHHI, persistent hyperinsulinemia hypoglycemia of infancy; SUR1, sulfonylurea receptor 1; ER, endoplasmic reticulum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; WT, wild type; TM, transmembrane domain; RLU, relative luminescence unit. 1The abbreviations used are: KATP, ATP-sensitive potassium; PHHI, persistent hyperinsulinemia hypoglycemia of infancy; SUR1, sulfonylurea receptor 1; ER, endoplasmic reticulum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; WT, wild type; TM, transmembrane domain; RLU, relative luminescence unit. channels present in the plasma membrane of pancreatic β-cells play a central role in mediating glucose-induced insulin secretion (1Miki T. Nagashima K. Tashiro F. Kotake K. Yoshitomi H. Tamamoto A. Gonoi T. Iwanaga T. Miyazaki J. Seino S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10402-10406Crossref PubMed Scopus (435) Google Scholar, 2Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (619) Google Scholar, 3Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 4Huopio H. Shyng S.-L. Otonkoski T. Nichols C.G. Am. J. Physiol. 2002; 283: E207-E216Crossref PubMed Scopus (97) Google Scholar). The activity of KATP channels, which regulates β-cell membrane potential, is determined by the relative concentrations of intracellular ATP and ADP. When the blood glucose level rises, the increased intracellular [ATP/ADP] ratio favors KATP channel closure, resulting in membrane depolarization, Ca2+ influx, and insulin secretion. When the blood glucose level falls, the above molecular events reverse, and insulin release is stopped. In the event where KATP channels fail to open during glucose starvation, β-cell membrane potential remains depolarized, and insulin secretion persists, leading to severe hypoglycemia. These symptoms are found in patients suffering from congenital hyperinsulinism (5Stanley C.A. J. Clin. Endocrinol. Metab. 2002; 87: 4857-4859Crossref PubMed Scopus (69) Google Scholar), also known as persistent hyperinsulinemia hypoglycemia of infancy (PHHI) (6Sharma N. Crane A. Gonzalez G. Bryan J. Aguilar-Bryan L. Kidney Int. 2000; 57: 803-808Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Indeed, mutations in the KATP channel genes, sulfonylurea receptor 1 (SUR1) and the inward rectifier potassium channel Kir6.2, that lead to a loss of channel function have been shown to be major causes of PHHI (4Huopio H. Shyng S.-L. Otonkoski T. Nichols C.G. Am. J. Physiol. 2002; 283: E207-E216Crossref PubMed Scopus (97) Google Scholar, 6Sharma N. Crane A. Gonzalez G. Bryan J. Aguilar-Bryan L. Kidney Int. 2000; 57: 803-808Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).The pancreatic KATP channel complex consists of four poreforming Kir6.2 subunits and four regulatory SUR1 subunits (7Clement J.P.t. 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 (622) Google Scholar, 8Inagaki N. Gonoi T. Clement J.P.t. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1607) Google Scholar, 9Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (244) Google Scholar, 10Shyng S. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (418) Google Scholar). Gating of KATP channels occurs as a result of 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 (11Gribble F.M. Tucker S.J. Ashcroft F.M. EMBO J. 1997; 16: 1145-1152Crossref PubMed Scopus (310) Google Scholar, 12Gribble F.M. Tucker S.J. Haug T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7185-7190Crossref PubMed Scopus (147) Google Scholar, 13Nichols C.G. Shyng S.-L. Nestorowicz A. Glaser B. Clement J.P.T. Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (467) Google Scholar, 14Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar). SUR1 is a member of the ATP-binding cassette transporter family; it has three transmembrane domains: TM0, TM1, and TM2, and two large cytoplasmic nucleotide binding domains: NBD1 and NBD2 (15Conti 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, 16Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P.t. Boyd 3rd, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1278) Google Scholar). Structure-function studies suggest that the chemical energy derived from nucleotide binding and hydrolysis at the NBDs is relayed through TM0 to the pore subunit Kir6.2 to induce channel opening (17Matsuo M. Tanabe K. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 28757-28763Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 18Schwappach B. Zerangue N. Jan Y.N. Jan L.Y. Neuron. 2000; 26: 155-167Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 19Ueda K. Komine J. Matsuo M. Seino S. Amachi T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1268-1272Crossref PubMed Scopus (132) Google Scholar, 20Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 21Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). When intracellular [ATP/ADP] ratio is high, ATP inhibition predominates, and channel activity decreases. Conversely, when intracellular [ATP/ADP] ratio is low, MgADP stimulation predominates, and channel activity increases. Importantly, many PHHI-associated SUR1 mutations specifically reduce or abolish the ability of the channel to be stimulated by MgADP (13Nichols C.G. Shyng S.-L. Nestorowicz A. Glaser B. Clement J.P.T. Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (467) Google Scholar, 22Matsuo M. Trapp S. Tanizawa Y. Kioka N. Amachi T. Oka Y. Ashcroft F.M. Ueda K. J. Biol. Chem. 2000; 275: 41184-41191Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 23Shyng S.-L. Ferrigni T. Shepard J.B. Nestorowicz A. Glaser B. Permutt M.A. Nichols C.G. Diabetes. 1998; 47: 1145-1151Crossref PubMed Scopus (139) Google Scholar), indicating that the primary mechanism for channel activation during glucose starvation is a rise in ADP. In addition to conferring MgADP stimulation, SUR1 also mediates channel regulation by sulfonylureas and the potassium channel opener diazoxide (2Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (619) Google Scholar, 3Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 24Ashcroft S.J. J. Membr. Biol. 2000; 176: 187-206Crossref PubMed Scopus (69) Google Scholar). Sulfonylureas, such as glibenclamide and tolbutamide, inhibit channel activity by binding to SUR1; they are clinically effective in the treatment of type II diabetes (25Ashcroft F.M. Horm. Metab. Res. 1996; 28: 456-463Crossref PubMed Scopus (133) Google Scholar, 26Doyle M.E. Egan J.M. Pharmacol. Rev. 2003; 55: 105-131Crossref PubMed Scopus (196) Google Scholar). Diazoxide, by contrast, stimulates channel activity when bound to SUR1 and has been successfully used to treat some cases of PHHI (4Huopio H. Shyng S.-L. Otonkoski T. Nichols C.G. Am. J. Physiol. 2002; 283: E207-E216Crossref PubMed Scopus (97) Google Scholar, 27Glaser B. Semin. Perinatol. 2000; 24: 150-163Crossref PubMed Scopus (38) Google Scholar).Aside from functional regulation, SUR1 and Kir6.2 subunits are also both required for cell surface expression of the channel. Assembly of the KATP channel complex occurs in the endoplasmic reticulum (ER) (7Clement J.P.t. 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 (622) Google Scholar). To ensure that only correctly assembled functional channels are exported to the plasma membrane, the cell has developed a quality control mechanism involving a RKR tripeptide motif present in both SUR1 and Kir6.2 (28Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar). It has been proposed that in preassembled and partially assembled channel proteins, the RKR motif is exposed, providing a signal for ER retention. Upon complete assembly of the channel complex, the RKR motifs become shielded to allow the channel to pass the ER quality control checkpoint and traffic forward to the plasma membrane. Inactivation of the motif by deletion or mutation to AAA leads to unregulated surface expression of individual subunits and partially assembled channel complexes (14Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (675) Google Scholar, 28Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar). Although trafficking signals other than the RKR sequence motif have been reported to regulate the abundance of KATP channels in the plasma membrane, their mechanisms are not as well understood (21Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 29Chan K.W. Zhang H. Logothetis D.E. EMBO J. 2003; 22: 3833-3843Crossref PubMed Scopus (139) Google Scholar, 30Sharma N. Crane A. Clement J.P.t. Gonzalez G. Babenko A.P. Bryan J. Aguilar-Bryan L. J. Biol. Chem. 1999; 274: 20628-20632Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar).Defective protein trafficking caused by genetic mutations underlies many human diseases. Examples include the cystic fibrosis transmembrane conductance regulator, the HERG (human ether-a-go-go-related gene) potassium channel and the gap junction protein connexin (31Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1407) Google Scholar, 32Deschenes S.M. Walcott J.L. Wexler T.L. Scherer S.S. Fischbeck K.H. J. Neurosci. 1997; 17: 9077-9084Crossref PubMed Google Scholar, 33Zhou Z. Gong Q. Epstein M.L. January C.T. J. Biol. Chem. 1998; 273: 21061-21066Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Recent studies show that such a mechanism also explains how some SUR1 mutations lead to loss of channel function and consequently the disease PHHI (30Sharma N. Crane A. Clement J.P.t. Gonzalez G. Babenko A.P. Bryan J. Aguilar-Bryan L. J. Biol. Chem. 1999; 274: 20628-20632Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 34Cartier 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, 35Partridge C.J. Beech D.J. Sivaprasadarao A. J. Biol. Chem. 2001; 276: 35947-35952Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 36Taschenberger 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). So far, the SUR1 mutations that have been reported to cause channel trafficking defects are located within or downstream of the second nucleotide-binding domain of the protein. Here, we report that two PHHI-associated SUR1 mutations, A116P and V187D (2Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (619) Google Scholar, 21Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 29Chan K.W. Zhang H. Logothetis D.E. EMBO J. 2003; 22: 3833-3843Crossref PubMed Scopus (139) Google Scholar, 37Otonkoski T. Ammala C. Huopio H. Cote G.J. Chapman J. Cosgrove K. Ashfield R. Huang E. Komulainen J. Ashcroft F.M. Dunne M.J. Kere J. Thomas P.M. Diabetes. 1999; 48: 408-415Crossref PubMed Scopus (137) Google Scholar), located in the first transmembrane domain (TM0), prevent trafficking of KATP channels from the ER to the plasma membrane. Unlike the previously reported trafficking mutants, trafficking defects caused by these two mutations can be corrected by sulfonylureas, and the rescued channels are fully functional.EXPERIMENTAL PROCEDURESMolecular Biology—FLAG epitope (DYKDDDDK) was inserted at the N terminus of the hamster SUR1 cDNA by sequential overlap extension PCR. Point mutations of SUR1 were introduced into hamster SUR1 cDNA in the pECE plasmid using a QuikChange site-directed mutagenesis kit (Stratagene). The FLAG epitope tag and mutations were confirmed by DNA sequencing. All SUR1-Kir6.2 fusion constructs were also in pECE vector (22Matsuo M. Trapp S. Tanizawa Y. Kioka N. Amachi T. Oka Y. Ashcroft F.M. Ueda K. J. Biol. Chem. 2000; 275: 41184-41191Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Rat Kir6.2 cDNA is in pCDNA3 vector. Mutant clones from multiple PCRs were analyzed in all experiments to avoid false results caused by undesired mutations introduced by PCR.86Rb+ Efflux Assay—The Cells were incubated for 24 h in culture medium containing 86RbCl (1 μCi/ml) 2–3 days after transfection with SUR1 and Kir6.2. Before measurement of Rb efflux, the cells were incubated for 30 min at 25 °C in Krebs-Ringer solution with metabolic inhibitors (2.5 μg/ml oligomycin plus 1 mm 2-deoxy-d-glucose). At selected time points, the solution was aspirated from the cells and replaced with fresh solution. At the end of a 40-min period, the cells were lysed in 2% SDS-Ringer solutions. The 86Rb+ in the aspirated solution and the cell lysate were counted. The percentage of efflux at each time point was calculated as the cumulative counts in the aspirated solution divided by the total counts from the solutions and the cell lysates.Immunofluorescence Staining—COSm6 cells were plated in 6-well tissue culture plates, transfected with 0.6 μg of SUR1 and 0.4 μg of Kir6.2/well using FuGENE 6 (Roche Applied Science) according to the manufacturer's directions. The cells were analyzed 48–72 h post-transfection. For surface staining, the 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, and then incubated with Cy-3-conjugated donkey anti-mouse secondary antibodies (Jackson) for 30 min at 4 °C. After three 5-min washes in ice-cold PBS, the cells were fixed with 4% paraformaldehyde and viewed using an Olympus Fluoview confocal microscope. For total cellular staining of FLAG-tagged SUR1, the cells were fixed with cold (–20 °C) methanol for 5 min. The fixed cells were incubated with the anti-FLAG M2 monoclonal antibody (10 μg/ml PBS containing 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—Immunoblotting analyses were performed using COS-1 cells instead of COSm6 cells to reduce background (22Matsuo M. Trapp S. Tanizawa Y. Kioka N. Amachi T. Oka Y. Ashcroft F.M. Ueda K. J. Biol. Chem. 2000; 275: 41184-41191Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Transfection was carried out as described above. The cells were lysed 48–72 h post-transfection in a buffer (referred to as the lysis buffer) containing 20 mm HEPES, pH 7.0, 5 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, and CompleteTR protease inhibitors (Roche Applied Science). The proteins in cell lysates were separated by SDS/PAGE (8%), transferred to nitrocellulose membrane, analyzed by M2 anti-FLAG antibody followed by horseradish peroxidase-conjugated anti-mouse secondary antibodies (Amersham Biosciences), and visualized by chemiluminescence (Super Signal West Femto; Pierce).Chemiluminescence Assay—COSm6 cells were plated in 35-mm dishes and transfected with KATP channel subunits using FuGENE 6. Drug treatment was initiated 32–40 h post-transfection and lasted for 4–24 h. The cells were then processed for chemiluminescence assays as described previously (36Taschenberger 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, 38Cartier E.A. Shen S. Shyng S.-L. J. Biol. Chem. 2003; 278: 7081-7090Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Briefly, the cells were fixed with 2% paraformaldehyde for 30 min at 4 °C, preblocked in PBS with 0.1% BSA for 30 min, incubated in M2 anti-FLAG antibody (10 μg/ml) for an hour, washed four times for 30 min in PBS with 0.1% BSA, incubated in horseradish peroxidase-conjugated anti-mouse (Jackson, 1:1000 dilution) for 20 min, and washed again four times for 30 min in PBS with 0.1% BSA. Chemiluminescence of each dish was quantified in a TD-20/20 luminometer (Turner Designs) following 5 s of incubation in Power Signal ELISA 32 Femto luminol solution (Pierce). All steps after fixation were carried out at room temperature.Metabolic Labeling and Immunoprecipitation—COS1 cells grown in 35-mm dishes were transfected with fSUR1 and Kir6.2 for 24 h. The cells were starved in methionine/cysteine-free Dulbecco's modified Eagle's medium supplemented with 5% dialyzed fetal bovine serum for 30 min prior to labeling with l-[35S]methionine (ICN Tran35S-Label, 150–250 μCi/ml) for 30 min. Labeled cultures were chased for various times in regular medium supplemented with 10 mm methionine at 37 °C. At the end of the chase, the cells were lysed in 500 μl of the lysis buffer (see above). For immunoprecipitation, 500 μl of cell lysate was incubated with 10 μg of anti-FLAG M2 antibodies for 1 h at 4 °C and then with protein A-Sepharose 4B (Bio-Rad) for 2 h at 4 °C. The precipitate was washed three times in the lysis buffer, and the proteins were eluted using the Laemmli sample buffer. The eluted proteins were separated by 8% SDS-PAGE, and the gel was subjected to fluorography. The dried gels were analyzed using a Bio-Rad PhosphorImager.Patch Clamp Recordings—COSm6 cells were transfected using FuGENE 6 and plated onto coverslips. The cDNA for the green fluorescent protein was co-transfected with SUR1 and Kir6.2 to facilitate identification of positively transfected cells. Patch clamp recordings were made 36–72 h post-transfection. All of the experiments were performed at room temperature as previously described. Micropipettes were pulled from nonheparinized Kimble glass (Fisher) on a horizontal puller (Sutter Instrument, Co., Novato, CA). Electrode resistance was typically 1–2 MΩ when filled with K-INT solution (below). Inside-out patches were voltage-clamped with an Axopatch 1-D amplifier (Axon Inc., Foster City, CA). The standard bath (intracellular) and pipette (extracellular) solution (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. All currents were measured at a membrane potential of –50 mV (pipette voltage = +50 mV). The data were analyzed using pCLAMP8 software (Axon Instrument). Off-line analysis was performed using Microsoft Excel programs. The data were presented as the means ± S.E.RESULTSBoth the A116P and V187D Mutations in SUR1 Prevent Normal Cell Surface Expression of KATP Channels—Several recent studies have shown that defective KATP channel trafficking is an underlying mechanism of congenital hyperinsulinism. The trafficking mutations reported to date are all located in the NBD2 and the distal C-terminal region of the molecule, including ΔF1388, R1394H, and L1544P (34Cartier 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, 35Partridge C.J. Beech D.J. Sivaprasadarao A. J. Biol. Chem. 2001; 276: 35947-35952Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 36Taschenberger 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). To examine whether mutations located in other parts of the molecule also affect channel trafficking, we focused our attention to two mutations, A116P and V187D, that are located in the first transmembrane domain, or TM0, of SUR1 (Fig. 1), and that have previously been reported to not form functional channels when co-expressed with Kir6.2 (6Sharma N. Crane A. Gonzalez G. Bryan J. Aguilar-Bryan L. Kidney Int. 2000; 57: 803-808Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 37Otonkoski T. Ammala C. Huopio H. Cote G.J. Chapman J. Cosgrove K. Ashfield R. Huang E. Komulainen J. Ashcroft F.M. Dunne M.J. Kere J. Thomas P.M. Diabetes. 1999; 48: 408-415Crossref PubMed Scopus (137) Google Scholar). To investigate how the A116P and V187D mutations lead to loss of functional KATP channels, we first performed Western blot analysis. Both mutations were engineered into a SUR1 construct that has been tagged with a FLAG epitope at its extracellular N terminus (referred to as fSUR1 from here on) to facilitate detection (34Cartier 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). The N-terminal FLAG tag has been tested in many SUR1 constructs in previous studies and has been shown not to affect channel phenotype (34Cartier 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, 36Taschenberger 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, 38Cartier E.A. Shen S. Shyng S.-L. J. Biol. Chem. 2003; 278: 7081-7090Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 39Conti L.R. Radeke C.M. Vandenberg C.A. J. Biol. Chem. 2002; 277: 25416-25422Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The SUR1 protein has two N-linked glycosylation sites. As the core-glycosylated immature form (the lower band) traffics through the Golgi, its N-linked oligosaccharides become further modified to yield the complex glycosylated mature form (the upper band). Fig. 2A shows that, although both the immature and mature forms were seen with cells co-expressing WT-fSUR1 and Kir6.2, only the immature form was evident in cells co-expressing Kir6.2 and the A116P- or the V187D-fSUR1 mutants. These results demonstrate that the mutant protein is synthesized and suggest that the mutant channel may fail to traffic to the cell surface. The lack of surface expression is confirmed by immunofluorescent staining experiments. In contrast to the abundant surface staining observed in cells transfected with Kir6.2 and WT-fSUR1, surface staining in cells transfected with Kir6.2 and A116P-fSUR1 or V187D-fSUR1 was barely detectable (Fig. 2B, top panels). However, upon fixation and membrane permeabilization, both mutant proteins were found inside the cell, with fluorescent signals concentrated in the perinuclear region suggesting ER retention. These results led us to conclude that the A116P and V187D mutations cause loss of functional KATP channels by preventing channels from trafficking to the cell surface.Fig. 2Analysis of the A116- and V187D-SUR1 mutants by immunoblotting and immunofluorescent staining experiments. A, Western blot analysis of fSUR1. In cells expressing Kir6.2 and WT-fSUR1, two bands are observed: the lower core glycosylated band, or the immature band (solid arrow), and the upper complex glycosylated band, or the mature band (open arrow). In contrast, only the immature band is observed in cells expressing Kir6.2 and A116P- or V187D-fSUR1. The total steady-state protein level of A116P- and V187D-fSUR1 also appears less than that of WT-fSUR1. Molecular mass markers (kDa) are indicated on the right. B, top panels, surface staining of COSm6 cells transiently transfected with Kir6.2 and either WT-, A116P-, or V187D-fSUR1 using the M2 anti-FLAG mouse monoclonal antibodies followed by Cy-3-conjugated anti-mouse secondary antibody. Staining was performed in living cells at 4 °C, and the cells were then fixed with 4% paraformaldehyde and viewed by confocal microscopy (Olympus Fluoview 300). Whereas cells expressing WT-fSUR1 channels have abundant surface staining, those expressing A116P- or V187D-fSUR1 channels have barely detectable staining. Bottom panels, total cellular expression of WT or mutant fSUR1. Cells co-transfected with Kir6.2 and the various fSUR1 constructs were fixed and permeabilized with methanol and stained for the FLAG epitope using the M2 anti-FLAG mouse monoclonal antibodies followed by Cy-3-conjugated anti-mouse secondary antibody. Both A116P- and V187D-fSUR1 were detected inside the cell, with a perinuclear staining pattern. fWT, WT-fSUR1; fA116P, A116P-fSUR1; fV187D, V187D-fSUR1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Trafficking Defects of the A116P and V187D Mutants Are Intrinsic to SUR1—One potential explanation for the trafficking defects seen with the A116P- and V187D-fSUR1 mutations is that the SUR1 mutants are unable to associate with Kir6.2. Because a prerequisite for either SUR1 or Kir6.2 to exit the ER is proper assembly of the two subunits into the octameric KATP channel complex that shields the RKR ER retention/retrieval signals, failure of SUR1 to associate with Kir6.2 would result in the absence of SUR1 in the plasma membrane, as we have observed. To address this possibility, we used a heterotandem dimer construct in which the C terminus of the mutant fSUR1 has been fused to the N terminus of Kir6.2 (referred to as A116P- or V187D-fSUR1/Kir6.2 fusion) to achieve obligatory physical association between the two subunits; similar SUR1/Kir6.2 fusion constructs have been used previously by a number of groups for structure-function and trafficking studies (7Clement J.P.t. 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 (622) Google Scholar, 9Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (244) Google Scholar, 10Shyng S. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (418) Google Scholar, 28Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar, 34Cartier E" @default.
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• W2048406951 title "Sulfonylureas Correct Trafficking Defects of ATP-sensitive Potassium Channels Caused by Mutations in the Sulfonylurea Receptor" @default.
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