Functional Analysis of a Mutant Sulfonylurea Receptor, SUR1-R1420C, That Is Responsible for Persistent Hyperinsulinemic Hypoglycemia of Infancy

The ATP-sensitive potassium (KATP+) channel is crucial for the regulation of insulin secretion from the pancreatic β-cell, and mutations in either the sulfonylurea receptor type 1 (SUR1) or Kir6.2 subunit of this channel can cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1. Mild tryptic digestion of SUR1 after photoaffinity labeling allowed analysis of the nucleotide-binding properties of NBF1 and NBF2. Labeling of NBF1 with 8-azido-[α-32P]ATP was inhibited by MgATP and MgADP with similar Ki for wild-type SUR1 and SUR1-R1420C. However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1. MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide. However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC50 for MgADP activation from 74 to 197 μm. We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller KATP+ current in R1420C-PHHI β-cells and thereby to the enhanced insulin secretion. We also propose a new model for nucleotide activation of KATP+ channels. The ATP-sensitive potassium (KATP+) channel is crucial for the regulation of insulin secretion from the pancreatic β-cell, and mutations in either the sulfonylurea receptor type 1 (SUR1) or Kir6.2 subunit of this channel can cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1. Mild tryptic digestion of SUR1 after photoaffinity labeling allowed analysis of the nucleotide-binding properties of NBF1 and NBF2. Labeling of NBF1 with 8-azido-[α-32P]ATP was inhibited by MgATP and MgADP with similar Ki for wild-type SUR1 and SUR1-R1420C. However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1. MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide. However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC50 for MgADP activation from 74 to 197 μm. We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller KATP+ current in R1420C-PHHI β-cells and thereby to the enhanced insulin secretion. We also propose a new model for nucleotide activation of KATP+ channels. nucleotide-binding fold hyperinsulinemic hypoglycemia of infancy ATP-sensitive potassium (KATP+) channels link the metabolic state of the cell to its membrane potential in many tissues including pancreatic β-cells, heart, brain, and skeletal muscle (1Nichols C.G. Shyng S.-L. Nestorowicz A. Glaser B. Clement J.P., IV Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (471) Google Scholar, 2Ashcroft F.M. Gribble F.M. 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Elevation of blood glucose concentration increases glucose uptake and metabolism by the β-cell producing changes in cytosolic nucleotide concentrations that result in closure of the KATP+ channels. This leads to depolarization of the β-cell membrane potential and thus to activation of voltage-gated calcium channels and Ca2+influx. The resulting rise in the intracellular Ca2+concentration triggers insulin release. The β-cell KATP+ channel is a hetero-octamer composed of pore-forming Kir6.2 subunits and regulatory sulfonylurea receptor (SUR1) subunits that coassemble with 4:4 stoichiometry (5Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (248) Google Scholar, 6Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (430) Google Scholar, 7Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. 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Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is an autosomal recessive disorder characterized by inappropriate insulin secretion despite severe hypoglycemia. In the absence of clinical treatment, PHHI may be lethal or result in irreversible neurologic sequelae. To date, three mutations responsible for PHHI have been identified in the Kir6.2 gene (21Thomas P. Ye Y. Lightner E. Hum. Mol. Genet. 1996; 5: 1809-1812Crossref PubMed Scopus (389) Google Scholar, 22Nestorowicz A. Inagaki N. Gonoi T. Schoor K.P. Wilson B.A. Glaser B. Landau H. Stanley C.A. Thornton P.S. Seino S. Permutt M.A. Diabetes. 1997; 46: 1743-1748Crossref PubMed Scopus (0) Google Scholar, 23Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (625) Google Scholar) and in numerous mutations in the SUR1 gene (1Nichols C.G. Shyng S.-L. Nestorowicz A. Glaser B. Clement J.P., IV Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. 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Med. 1997; 336: 703-706Crossref PubMed Scopus (229) Google Scholar, 29Shyng S.-L. Ferrigni T. Shepard J.B. Nestorowicz A. Glaser B. Permutt M.A. Nichols C.G. Diabetes. 1998; 47: 1145-1151Crossref PubMed Scopus (142) Google Scholar, 30Otonkoski T. Ämmälä 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 (139) Google Scholar). Some of theSUR1 mutations are nonsense or frameshift mutations that produce a truncated form of SUR1. Others are missense mutations, many of which are found within the NBFs of SUR1 and impair KATP+ channel activity by affecting Mg2+-nucleotide interactions with SUR1 (1Nichols C.G. Shyng S.-L. Nestorowicz A. Glaser B. Clement J.P., IV Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (471) Google Scholar, 29Shyng S.-L. Ferrigni T. Shepard J.B. Nestorowicz A. Glaser B. Permutt M.A. Nichols C.G. Diabetes. 1998; 47: 1145-1151Crossref PubMed Scopus (142) Google Scholar). As a consequence, the KATP+ channel remains closed even in the absence of glucose, which results in persistent and unregulated insulin secretion. Recently, we identified a missense SUR1 mutation (R1420C) in Japanese PHHI patients (31Tanizawa Y. Matsuda K. Matsuo M. Ohta Y. Ochi N. Adachi M. Koga M. Mizuno S. Kajita M. Tanaka Y. Tachibana K. Inoue H. Furukawa S. Amachi T. Ueda K. Oka Y. Diabetes. 2000; 49: 114-120Crossref PubMed Scopus (46) Google Scholar). They were siblings from a consanguineous family and homozygous for the mutation, and their clinical characteristics consisted of a mild form of PHHI. Verkarreet al. (32Verkarre V. Fournet J.C. de Lonlay P. Gross-Morand M.S. Devillers M. Rahier J. Brunelle F. Robert J.J. Nihoul-Fekete C. Saudubray J.M. Junien C. J. Clin. Invest. 1998; 102: 1286-1291Crossref PubMed Scopus (267) Google Scholar) also reported this mutation in a patient with focal adenomatous hyperplasia of pancreatic islets (32Verkarre V. Fournet J.C. de Lonlay P. Gross-Morand M.S. Devillers M. Rahier J. Brunelle F. Robert J.J. Nihoul-Fekete C. Saudubray J.M. Junien C. J. Clin. Invest. 1998; 102: 1286-1291Crossref PubMed Scopus (267) Google Scholar). Arginine 1420 is located between the Walker A motif and the SGGQ signature sequence of NBF2. An 86Rb+ efflux study revealed that KATP+ channels composed of SUR1-R1420C and Kir6.2 are not activated by metabolic inhibition as much as wild-type channels. This may be related to the fact that the expression level of SUR1-R1420C was only about half that of the wild-type channel when it was transiently expressed in COS-7 cells (31Tanizawa Y. Matsuda K. Matsuo M. Ohta Y. Ochi N. Adachi M. Koga M. Mizuno S. Kajita M. Tanaka Y. Tachibana K. Inoue H. Furukawa S. Amachi T. Ueda K. Oka Y. Diabetes. 2000; 49: 114-120Crossref PubMed Scopus (46) Google Scholar). We also reported that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes the binding of 8-azido-ATP at NBF1 of wild-type SUR1 (19Ueda K. Komine J. Matsuo M. Seino S. Amachi T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1268-1272Crossref PubMed Scopus (135) Google Scholar), and that the R1420C mutation impairs this effect without altering high affinity 8-azido-ATP binding to NBF1 (31Tanizawa Y. Matsuda K. Matsuo M. Ohta Y. Ochi N. Adachi M. Koga M. Mizuno S. Kajita M. Tanaka Y. Tachibana K. Inoue H. Furukawa S. Amachi T. Ueda K. Oka Y. Diabetes. 2000; 49: 114-120Crossref PubMed Scopus (46) Google Scholar). However, it is not clear whether the mutation lowers the affinity of NBF2 for nucleotides or if it affects cooperative interactions between the two NBFs. In this study, we examined the nucleotide-binding properties of the two NBFs of SUR1-R1420C by photoaffinity labeling experiments and studied the properties of Kir6.2/SUR1-R1420C channels by electrophysiological analysis. Our results suggest that the R1420C mutation increases the EC50 for MgADP-dependent channel activation by reducing the nucleotide-binding affinity of NBF2. This property, together with the lower expression of the mutant channel, may be responsible for PHHI. 8-Azido-[α-32P]ATP and 8-azido-[γ-32P]ATP were purchased from ICN Biomedicals. Membranes from COS-7 cells expressing mouse SUR1 were prepared as described (17Ueda K. Inagaki N. Seino S. J. Biol. Chem. 1997; 272: 22983-22986Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Crude membranes, containing similar amounts of wild-type SUR1 and SUR1-R1420C as determined by Western blotting, were incubated with 50 μm8-azido-[32P]ATP in the presence or absence of 0.1–1000 μm ATP or ADP in 3 μl of TEM buffer (40 mmTris-Cl (pH 7.5), 0.1 mm EGTA, 1 mmMgSO4) containing 2 mm ouabain. Proteins were UV-irradiated for 5 min (at 254 nm, 5.5 milliwatts/cm2) on ice. Ice-cold TEM buffer was then added to the sample, and the supernatant was removed after centrifugation (15,000 ×g, 5 min, 2 °C). Pellets were resuspended in 40 mm Tris-Cl (pH 7.5) buffer containing 0.1 mmEGTA, 250 mm sucrose, and 10 μg/ml trypsin to 10 μg of membrane proteins/μl and incubated for 5 min at 37 °C. 100 μl of radioimmune precipitation buffer (20 mm Tris-Cl (pH 7.5), 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) containing 100 μg/ml (p-amidinophenyl)methanesulfonyl fluoride was added to the samples to terminate proteolysis, and membrane proteins were solubilized for 30 min at 4 °C. After centrifugation for 15 min at 15,000 × g, tryptic fragments were immunoprecipitated from the supernatant using an antibody raised against NBF1 or NBF2 of hamster SUR1 as described previously (20Matsuo M. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 37479-37482Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Samples were electrophoresed on 12% SDS-polyacrylamide gel and autoradiographed. Bound 8-azido-[32P]ATP in NBF1 or NBF2 was measured by scanning with a radioimaging analyzer (BAS2000, Fuji Photo Film Co.). Membranes (20 μg of proteins) were incubated with 10 μm8-azido-[32P]ATP in 3 μl of TEM buffer containing 2 mm ouabain for 3 min at 37 °C. The reactions were stopped by adding ice-cold TEM buffer, and free 8-azido-[32P]ATP was removed after centrifugation (15,000 × g, 5 min, 2 °C). Pellets were resuspended in 10 μl of TEM buffer containing 2 mm ouabain and 0–1000 μm ATP or ADP. The mixture was UV-irradiated immediately after the resuspension or after postincubation for 15 min at 37 °C. Samples were electrophoresed on 7% SDS-polyacrylamide gel and autoradiographed. Bound 8-azido-[32P]ATP in SUR1 was measured by radioimaging analyzer as described above. Wild-type or mutant Kir6.2 was coexpressed with wild-type or mutant SUR1 inXenopus oocytes as described previously (33Gribble F.M. Ashfield R. Ämmälä C. Ashcroft F. J. Physiol. ( Lond. ). 1997; 498: 87-98Crossref PubMed Scopus (192) Google Scholar). Briefly, immature stage V-VI Xenopus oocytes were incubated for 60 min with 1.0 mg/ml collagenase (Sigma, type V) and manually defolliculated. They were then coinjected with ∼0.1 ng of mouse Kir6.2 and ∼2 ng of mouse SUR1, giving a 1:20 ratio. Isolated oocytes were maintained in Barth's solution and studied 1–4 days after injection (33Gribble F.M. Ashfield R. Ämmälä C. Ashcroft F. J. Physiol. ( Lond. ). 1997; 498: 87-98Crossref PubMed Scopus (192) Google Scholar). Whole-cell currents were recorded using a two-electrode voltage clamp (33Gribble F.M. Ashfield R. Ämmälä C. Ashcroft F. J. Physiol. ( Lond. ). 1997; 498: 87-98Crossref PubMed Scopus (192) Google Scholar) in a high potassium extracellular solution containing (mm): 90 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH 7.4 with KOH) at 20–24 °C. The holding potential was −10 mV and current amplitudes were measured in response to a −100-mV, 300-ms voltage pulse. Currents were filtered at 1 kHz and digitized at 4 kHz. Macroscopic currents were recorded from giant excised inside-out patches at a holding potential of 0 mV and at 20–24 °C in response to 3-s voltage ramps from −110 mV to +100 mV (33Gribble F.M. Ashfield R. Ämmälä C. Ashcroft F. J. Physiol. ( Lond. ). 1997; 498: 87-98Crossref PubMed Scopus (192) Google Scholar). The pipette (external) solution contained (mm): 140 KCl, 1.2 MgCl2, 2.6 CaCl2, and 10 HEPES (pH 7.4 with KOH). The standard intracellular (bath) solution contained (mm): 107 KCl, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES (pH 7.2 with KOH; final [K+] ∼140 mm), and nucleotides as indicated. Tolbutamide was made up as a 0.05 m stock solution in 0.1 m KOH and diazoxide as a 0.02 m stock solution in 0.1 mKOH. Rapid exchange of solutions was achieved by positioning the patch in the mouth of one of a series of adjacent inflow pipes placed in the bath. Single-channel currents were recorded from small inside-out patches, filtered at 1 kHz, and sampled at 3 kHz. Macroscopic currents were filtered at 0.5 kHz, digitized at 1 kHz using a Digidata 1200 Interface, and analyzed using pClamp software (Axon Instruments, Foster City, CA). The slope conductance was measured by fitting a straight line to the current-voltage relation between −20 mV and −100 mV; the average of five consecutive ramps was calculated in each solution. Nucleotide concentration-response relationships were measured by alternating the control solution with a test concentration of nucleotide. The conductance (G) was expressed as a fraction of the mean of the value obtained in the control solution before and after application of the nucleotide (Gc). Concentration-response curves for ATP inhibition were fit by the Hill equation (Equation 1),G/Gc=1/(1+([ATP]/IC50) h)Equation 1 where [ATP] is the ATP concentration, IC50 is the concentration at which inhibition is half-maximal, and h is the slope factor (Hill coefficient). Concentration-response curves for ADP activation were fitted to a modified version of the Hill equation (Equation 2),G/Gc=a/(1+(EC50/[ADP])h)+1Equation 2 where [ADP] is the ADP concentration, EC50 is the concentration at which activation is half-maximal, a is the maximal conductance (relative to control), and h is the slope factor (Hill coefficient). Data are given as mean ± S.E., and thesymbols in the figures indicate the mean and thevertical bars indicate S.E. To examine the biochemical properties of SUR1-R1420C, we investigated 8-azido-[32P]ATP photoaffinity labeling of NBF1 and NBF2 (20Matsuo M. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 37479-37482Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Crude membranes from COS-7 cells expressing SUR1 or SUR1-R1420C were incubated with 50 μm8-azido-[α-32P]ATP followed by mild trypsin digestion. Labeled tryptic fragments containing NBF1 or NBF2 of both wild-type and mutant SUR1 were immunoprecipitated with anti-NBF1 (Fig. 1, lanes 1 and 3) or NBF2 antibody (Fig. 1, lanes 2 and 4), respectively. This indicates that both NBFs of SUR1-R1420C can bind 8-azido-ATP, as is found for wild-type SUR1.Figure 1Photoaffinity labeling of the NBFs of SUR1 and SUR1-R1420C with 8-azido-[α-32P]ATP and 8-azido-[γ-32P]ATP. Membrane proteins (10–20 μg) from COS-7 cells expressing wild-type SUR1 (lanes 1, 2, 5, and 6) or SUR1-R1420C (lanes 3, 4, 7, and 8) were incubated with 50 μm 8-azido-[α-32P]ATP (lanes 1–4) or 8-azido-[γ-32P]ATP (lanes 5–8) for 10 min at 37 °C and then UV-irradiated. Photoaffinity-labeled proteins were subject to mild trypsin digestion. The tryptic fragments were immunoprecipitated with anti-NBF1 (lanes 1, 3, 5, and 7) or anti-NBF2 (lanes 2, 4, 6, and 8) antibody and separated by 12% polyacrylamide gel electrophoresis. A 65-kDa fragment containing NBF2 and a 35-kDa fragment containing NBF1 are indicated. Experiments were performed in duplicate.View Large Image Figure ViewerDownload (PPT) We have previously suggested that NBF2 of SUR1 might hydrolyze ATP, because NBF2 of SUR1 is photoaffinity-labeled with 8-azido-[α-32P]ATP but not with 8-azido-[γ-32P]ATP (20Matsuo M. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 37479-37482Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). We therefore examined whether SUR1-R1420C is photoaffinity-labeled with 8-azido-[γ-32P]ATP. Both NBF1 of SUR1 and of SUR1-R1420C were photoaffinity-labeled with 8-azido-[γ-32P]ATP (Fig. 1, lanes 5 and7), indicating that NBF1 either does not have ATPase activity or has little ATPase activity under our experimental conditions. In contrast, NBF2s of SUR1 and SUR1-R1420C were not photoaffinity-labeled with 8-azido-[γ-32P]ATP (Fig. 1,lanes 6 and 8), although they were photoaffinity-labeled by 8-azido-[α-32P]ATP (Fig. 1,lanes 2 and 4). These results suggest that the γ-phosphate dissociates from 8-azido-[32P]ATP bound at NBF2 and thus that NBF2 of SUR1-R1420C might have ATPase activity as is found for wild-type SUR1. To characterize the biochemical properties of SUR1-R1420C further, we investigated the affinities of both NBF1 and NBF2 for ATP and ADP. When crude membranes from COS-7 cells expressing SUR1 or SUR1-R1420C were incubated with 50 μm 8-azido-[α-32P]ATP in the presence of ATP or ADP followed by mild trypsin digestion, photoaffinity labeling of tryptic fragments containing either NBF1 or NBF2 was inhibited in a concentration-dependent manner (Fig. 2). This indicates that SUR1 and SUR1-R1420C can bind ATP and ADP. The data were fit by the Hill equation, and the Ki values obtained are shown in Table I. The Ki values of NBF1 of SUR1 for ATP and ADP were 1.6 ± 0.64 (n = 3) and 17 ± 7.9 μm(n = 3) respectively, and those of SUR1-R1420C were 1.5 ± 0.26 (n = 3) and 13 ± 6.8 μm (n = 3), respectively. This indicates that the affinity of NBF1 for nucleotides is not significantly different between wild-type SUR1 and SUR1-R1420C and that the affinity for ATP is significantly higher than that for ADP. We have shown previously that the Ki values for ATP and ADP binding to NBF1 of hamster SUR1 are 4.4 ± 3.7 and 26 ± 8.6 μm, respectively, and those of NBF2 are 60 ± 26 μm and 100 ± 26 μm, respectively (34Matsuo M. Tanabe K. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 28757-28763Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These values are in good agreement with the affinities obtained in this study for mouse SUR1, indicating that there are no significant differences between hamster and mouse SUR1. However, theKi values of NBF2 of SUR1-R1420C for ATP and ADP (350 ± 36 (n = 3) and 290 ± 66 μm (n = 3), respectively) were significantly higher than those of wild-type SUR1 (64 ± 4.7 (n = 3) and 65 ± 16 μm(n = 3), respectively). These results demonstrate that the R1420C mutation decreases the affinity of NBF2 for both ATP and ADP.Table IKi values for interaction of ATP and ADP with the NBFs of SUR1 and SUR1-R1420CKiWild-typeR1420CμmμmNBF1-ATP1.6 ± 0.641.5 ± 0.26NBF1-ADP17 ± 7.913 ± 6.8NBF2-ATP64 ± 4.7350 ± 36NBF2-ADP65 ± 16290 ± 66The data of Fig. 2 were fit by the Hill equation, andKi values were obtained. Open table in a new tab The data of Fig. 2 were fit by the Hill equation, andKi values were obtained. To explore the possibility that the impaired cooperative nucleotide binding observed for SUR1-R1420C is due to the low nucleotide-binding affinity of NBF2, we examined the dependence of cooperative binding on nucleotide concentration. As we have shown previously (19Ueda K. Komine J. Matsuo M. Seino S. Amachi T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1268-1272Crossref PubMed Scopus (135) Google Scholar), prebound 8-azido-[α-32P]ATP at NBF1 of wild-type SUR1 gradually dissociated during postincubation at 37 °C in the presence of Mg2+ without nucleotide, and MgATP or MgADP stabilized 8-azido-ATP binding in a concentration dependent manner (Fig. 3). This cooperative nucleotide binding was also observed when SUR1 was photoaffinity-labeled with 8-azido-[γ-32P]ATP (data not shown). EC50values for the stabilization effect of MgATP and MgADP were 59 ± 2.9 and 50 ± 18 μm, respectively, for SUR1 and are in good agreement with the binding affinities of MgATP and MgADP at NBF2 (64 ± 4.7 and 65 ± 16 μm, respectively) (Table I). This supports the idea that MgATP and MgADP bind at NBF2 to stabilize 8-azido-ATP binding at NBF1. In contrast, neither ATP nor ADP (1 mm) stabilized 8-azido-[32P]ATP binding at NBF1 of SUR1-R1420C despite the fact that 1 mm MgATP and MgADP inhibited photoaffinity labeling of NBF2 (68 ± 3.2 (n = 3) and 76 ± 2.9% (n = 3), respectively) as efficiently as that of wild-type SUR1 (74 ± 10 (n = 3) and 73 ± 1.5% (n = 3), respectively) (Fig. 2). These results suggest that neither MgATP nor MgADP bound at NBF2 can stabilize 8-azido-ATP binding at NBF1 of SUR1-R1420C. To examine the functional properties of the mutant KATP+ channels, we coexpressed SUR1-R1420C with Kir6.2 in Xenopus oocytes. As observed for the wild-type channel (Kir6.2/SUR1), no significant current was detected for Kir6.2/SUR1-R1420C channels in the cell-attached configuration, but large currents developed following patch excision into nucleotide-free solution. The mean current amplitude at −100 mV was 3.0 ± 1.0 nA (n = 10) for Kir6.2/SUR1 and 1.5 ± 0.6 nA (n = 15) for Kir6.2/SUR1-R1420C channels. The smaller currents observed with the mutant channel are probably due to reduced expression of the mutant SUR (31Tanizawa Y. Matsuda K. Matsuo M. Ohta Y. Ochi N. Adachi M. Koga M. Mizuno S. Kajita M. Tanaka Y. Tachibana K. Inoue H. Furukawa S. Amachi T. Ueda K. Oka Y. Diabetes. 2000; 49: 114-120Crossref PubMed Scopus (46) Google Scholar). A quantitatively similar reduction in SUR1-R1420C protein expression was observed in COS-7 cells, indicating that the lower expression of mutant SUR1 is confined to the oocyte expression system. We first examined the effect of the R1420C mutation on the activation of the channel by the potassium channel opener diazoxide (Fig. 4) and its inhibition by the sulfonylurea tolbutamide. Diazoxide was tested in the presence of MgATP because its stimulatory action is dependent upon the presence of intracellular hydrolyzable nucleotides (35Kozlowski R.Z. Hales C.N. Ashford M.L.J. Br. J. Pharmacol. 1989; 97: 1039-1050Crossref PubMed Scopus (85) Google Scholar). In the presence of 100 μm ATP, both 200 μmdiazoxide and 100 μm ADP led to a pronounced increase in both wild-type and mutant KATP+currents. Diazoxide increased Kir6.2/SUR1 currents by 694 ± 40% (n = 4) and Kir6.2/SUR1-R1420C currents by 499 ± 126% (n = 7). The sulfonylurea tolbutamide blocked Kir6.2/SUR1 currents by 53 ± 2% (n = 3) and Kir6.2/SUR1-R1420C currents by 68 ± 7% (n = 3). Thus the R1420C mutation does not affect the pharmacological properties of the channel. Inhibition of the channel by ATP was not affected by the R1420C mutation. Kir6.2/SUR1 currents were inhibited with an IC50 of 13 ± 2 μm and a Hill coefficient of 0.99 ± 0.18 (n = 5), whereas Kir6.2/SUR1-R1420C currents were blocked with an IC50 of 12 ± 2 μm and a Hill coefficient of 0.90 ± 0.14 (n = 6; Fig. 5). ADP produced a mean current increase of 402 ± 82% (n = 3) and 347 ± 52% (n = 6) for Kir6.2/SUR1 and Kir6.2/SUR1-R1420C, respectively, when tested in the presence of 100 μm MgATP. ADP and GDP also activated both mutant and wild-type KATP+ channels when applied in the absence of ATP (Fig. 4); surprisingly, the extent of this activation was somewhat greater for mutant than for wild-type channels. It is well known that nucleotides such as ADP exert two opposing effects on the KATP+ channel: they stimulate channel activity by interacting (in a magnesium-dependent fashion) with the NBFs o