Different Binding Properties and Affinities for ATP and ADP among Sulfonylurea Receptor Subtypes, SUR1, SUR2A, and SUR2B
2000; Elsevier BV; Volume: 275; Issue: 37 Linguagem: Inglês
10.1074/jbc.m004818200
ISSN1083-351X
AutoresMichinori Matsuo, Kouichi Tanabe, Noriyuki Kioka, Teruo Amachi, Kazumitsu Ueda,
Tópico(s)Anesthesia and Neurotoxicity Research
ResumoATP-sensitive potassium (KATP) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle KATP channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of KATP channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[α-32P]ATP or 8-azido-[γ-32P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg2+ dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of KATP channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic KATP channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs. ATP-sensitive potassium (KATP) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle KATP channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of KATP channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[α-32P]ATP or 8-azido-[γ-32P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg2+ dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of KATP channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic KATP channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs. ATP-sensitive potassium ATP-binding cassette cystic fibrosis transmembrane conductance regulator multidrug resistance multidrug resistance-associated protein nucleotide-binding fold sulfonylurea receptor 1,2-cyclohexylenedinitrilotetraacetic acid ATP-sensitive potassium (KATP)1 channels are inwardly rectifying potassium channels, which are inhibited by ATP and stimulated by MgADP (1Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 2Babenko A.P. Aguilar-Bryan L. Bryan J. Annu. Rev. Physiol. 1998; 60: 667-687Crossref PubMed Scopus (483) Google Scholar, 3Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (456) Google Scholar). They play important roles by linking cellular metabolic level to membrane potential by sensing intracellular ATP and ADP levels in various tissues such as pancreatic β-cells, heart, brain, skeletal muscle, and vascular smooth muscle. The KATP channel is a hetero-octamer composed of sulfonylurea receptor (SURx) and Kir6.x subunits in 4:4 stoichiometry (4Inagaki N. Gonoi T. Seino S. FEBS Lett. 1997; 409: 232-236Crossref PubMed Scopus (248) Google Scholar, 5Shyng S.-L. Nichols C.G. J. Gen. Physiol. 1997; 110: 655-664Crossref PubMed Scopus (430) Google Scholar, 6Clement J.P., IV Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguliar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 7Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). SURx is a member of the ATP-binding cassette (ABC) superfamily including P-glycoprotein (MDR1), multidrug resistance-associated protein (MRP1), and the cystic fibrosis transmembrane conductance regulator (CFTR) (8Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar,9Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. González G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1289) Google Scholar), all of which have two nucleotide-binding folds (NBFs) per molecule; Kir6.x is a member of the inwardly rectifying potassium channel family (10Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1169Crossref PubMed Scopus (1623) Google Scholar, 11Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino S. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 12Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Crossref PubMed Scopus (402) Google Scholar). Both SURx and Kir6.x have a number of subtypes as follows: SUR1, SUR2A, and SUR2B and Kir6.1 and Kir6.2. SUR1 has been cloned as a high affinity binding protein for sulfonylurea (9Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. González G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1289) Google Scholar), the most commonly used drug for treatment of patients with type 2 diabetes. SUR2A shares 68% amino acid identity with SUR1, and SUR2B is a splicing variant of SUR2A differing only in its C-terminal 42 amino acids (13Inagaki N. Gonoi T. Clement J.P., IV Wang C.-Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar, 14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). The C-terminal 42 amino acids of SUR2B are similar to those of SUR1. Kir6.1 and Kir6.2 share 71% amino acid identity with each other, both of which have two putative transmembrane domains and an ion pore-forming (H5) region. Pancreatic β-cell KATP channels, composed of SUR1 and Kir6.2, regulate insulin secretion by altering the β-cell membrane potential (1Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (456) Google Scholar, 15Nichols 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, 16Aguilar-Bryan L. Clement J.P., IV Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (517) Google Scholar). Coexpression of SUR2A/Kir6.2, SUR2B/Kir6.2, and SUR2B/Kir6.1 has been reported to reconstitute cardiac, smooth muscle, and vascular smooth muscle KATP channels, respectively (2Babenko A.P. Aguilar-Bryan L. Bryan J. Annu. Rev. Physiol. 1998; 60: 667-687Crossref PubMed Scopus (483) Google Scholar, 3Seino S. Annu. Rev. Physiol. 1999; 61: 337-362Crossref PubMed Scopus (456) Google Scholar). These channels have different sensitivities to ATP and show different responses to sulfonylurea drugs and potassium channel openers (10Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1169Crossref PubMed Scopus (1623) Google Scholar, 11Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino S. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar, 17Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. (Lond.). 1997; 499: 715-720Crossref Scopus (343) Google Scholar, 18Shindo T. Yamada M. Isomoto S. Horio Y. Kurachi Y. Br. J. Pharmacol. 1998; 124: 985-991Crossref PubMed Scopus (67) Google Scholar). The IC50 (ATP) of SUR1/Kir6.2 KATP channels is about 10 μm, whereas those of SUR2A/Kir6.2 and SUR2B/Kir6.2 are about 100 and 300 μm, respectively (10Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1169Crossref PubMed Scopus (1623) Google Scholar, 11Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino S. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). SUR2B/Kir6.1 KATP channels are not inhibited by ATP but are stimulated by ADP and ATP (17Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. (Lond.). 1997; 499: 715-720Crossref Scopus (343) Google Scholar). SUR1/Kir6.2 KATP channels are inhibited by glibenclamide at K i ∼10 nm, and SUR2A/Kir6.2, SUR2B/Kir6.2, and SUR2B/Kir6.1 KATP channels are inhibited with K i values in the low micromolar range (10Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1169Crossref PubMed Scopus (1623) Google Scholar, 11Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino S. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar, 17Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. (Lond.). 1997; 499: 715-720Crossref Scopus (343) Google Scholar). Both SUR1/Kir6.2 and SUR2B/Kir6.2 KATP channels are stimulated by diazoxide, but SUR2A/Kir6.2 KATP channels are not (10Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1169Crossref PubMed Scopus (1623) Google Scholar, 11Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino S. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). The differences between these channels may be caused, at least in part, by differences in SUR subtype. However, it is not clear how SUR subtypes cause the different properties of KATP channel subtypes. We have already shown that NBF1 of SUR1 is a Mg2+-independent high affinity ATP-binding site, that NBF2 is a Mg2+-dependent low affinity ATP-binding site, and that MgADP binding at NBF2 stabilizes the 8-azido-ATP binding at NBF1 of SUR1 (19Matsuo M. Tucker S.J. Ashcroft F.M. Amachi T. Ueda K. FEBS Lett. 1999; 458: 292-294Crossref PubMed Scopus (9) Google Scholar, 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, 21Ueda 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). In this study, to determine the reasons for differences among KATP channel subtypes, we investigated the nucleotide-binding properties and the stabilization effect of three subtypes of SUR, SUR1, SUR2A, and SUR2B. Our results indicate that three subtypes of SUR have different nucleotide-binding properties, which may explain the differential regulation of KATPchannel subtypes. 8-Azido-[α-32P]ATP and 8-azido-[γ-32P]ATP were purchased from ICN Biomedicals. Hamster SUR1 (9Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P., IV Boyd III, A.E. González G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1289) Google Scholar), rat SUR2A (13Inagaki N. Gonoi T. Clement J.P., IV Wang C.-Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar), and mouse SUR2B (14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar) cDNAs were gifts from Dr. Joseph Bryan (Baylor College of Medicine), Dr. Susumu Seino (Chiba University), and Dr. Yoshihisa Kurachi (Osaka University), respectively. Antibodies against the C-terminal 21 amino acids of rat SUR1 (22Béguin P. Nagashima K. Nishimura M. Gonoi T. Seino S. EMBO J. 1999; 18: 4722-4732Crossref PubMed Scopus (148) Google Scholar) and of rat SUR2A were gifts from Dr. Susumu Seino. Membranes (20 μg of proteins) from COS-7 cells expressing SUR1, SUR2A, or SUR2B, prepared as described previously (23Ueda K. Inagaki N. Seino S. J. Biol. Chem. 1997; 272: 22983-22986Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), 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), TEE buffer (40 mm Tris-Cl (pH 7.5), 0.1 mm EGTA, 1 mm EDTA), or TEC buffer (40 mm Tris-Cl (pH 7.5), 0.1 mm EGTA, 1 mm CDTA) containing 2 mm ouabain. Proteins were UV-irradiated for 5 min (at 254 nm, 5.5 milliwatts/cm2) on ice before or after removing free 8-azido-[32P]ATP. To remove free 8-azido-[32P]ATP, ice-cold TEM buffer was added to the sample, and the supernatant was removed after centrifugation (15,000 × g, 5 min, 2 °C). Pellets were resuspended in TEE buffer containing 5 μg/ml trypsin and 250 mm sucrose to 10 μg of membrane proteins/μl and incubated for 15 min at 37 °C. Then 100 μl of RIPA 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 with the antibody raised against NBF1 or NBF2 of hamster SUR1 prepared as described (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 a 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 50 μm8-azido-[α-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-[α-32P]ATP was removed after centrifugation (15,000 × g, 5 min, 2 °C). Pellets were resuspended in 10 μl of TEM or TEE buffer containing 2 mm ouabain and 1 mm ATP or ADP. The mixture was incubated for 15 min at 37 °C and UV-irradiated on ice. Samples were electrophoresed on a 7% SDS-polyacrylamide gel and autoradiographed. Bound 8-azido-[α-32P]ATP in SURx was measured using the radioimaging analyzer as described above. We demonstrated previously that mild digestion of SUR1 with trypsin produces 35- and 65-kDa fragments containing NBF1 and NBF2, respectively, and that we can analyze the ATP-binding properties of NBF1 and NBF2 separately after immunoprecipitation (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 examined whether NBFs of SUR2A and SUR2B can be also separated by mild tryptic digestion followed by immunoprecipitation. When SUR1, photoaffinity-labeled with 50 μm 8-azido-[α-32P]ATP, was mildly digested with trypsin, 100- and 35-kDa labeled fragments were immunoprecipitated with the anti-NBF1 antibody, and 100- and 65-kDa labeled fragments were immunoprecipitated with the anti-NBF2 antibody as previously reported (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) (Fig.1 A). The 100- and 65-kDa fragments were detected with the anti-C terminus antibody on Western blotting (Fig. 2 A). These results suggest that SUR1 is first digested with trypsin at site 1 to produce the tryptic 100-kDa fragment, containing both NBF1 and NBF2, and that the 100-kDa fragment is further digested to the 35-kDa fragment, which contains NBF1, and the 65-kDa fragment, which contains NBF2, as shown in Fig. 2 D.Figure 2Limited digestion of SURx with trypsin.Membrane proteins (25 μg) from host COS-7 cells or COS-7 cells expressing SUR1 (A), SUR2A (B), or SUR2B (C) were digested with 5 μg/ml trypsin at 37 °C for the indicated periods and separated by 12% SDS-polyacrylamide gel electrophoresis. Limited tryptic fragments were detected with anti-C terminus of SUR1 (A and C) or SUR2A (B) antibody. Undigested SURx, 100-kDa, and 65-kDa tryptic fragments are indicated. Experiments were performed in duplicate.D, predicted diagram of limited trypsin digestion of SURx.View Large Image Figure ViewerDownload (PPT) When photoaffinity-labeled SUR2A and SUR2B were digested with trypsin under the same conditions, they produced 65- and 35-kDa fragments but very little 100-kDa fragment (Fig. 1, B and C). On Western blotting, 65-kDa fragments of SUR2A and SUR2B, but little 100-kDa fragment, were detected by antibodies against the C termini of SUR2A and SUR1, respectively (Fig. 2, B and C). The antibody raised against the C terminus of SUR1 recognized SUR2B as shown in Fig. 2 C, because the C-terminal 42 amino acids of SUR2B are similar to those of SUR1 (14Isomoto S. Kondo C. Yamada M. Matsumoto S. Higashiguchi O. Horio Y. Matsuzawa Y. Kurachi Y. J. Biol. Chem. 1996; 271: 24321-24324Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). These results suggest that SUR2A and SUR2B are digested with trypsin at site 1 and site 2 simultaneously to produce the tryptic 35-kDa fragment, which contains NBF1, and the 65-kDa fragment, which contains NBF2. It also indicated that antibodies raised against NBFs of SUR1 can be used to precipitate NBFs of SUR2A and SUR2B. To investigate the ATP-binding properties of NBFs of SURx, SURx was photoaffinity-labeled with 50 μm8-azido-[α-32P]ATP under various conditions and mildly digested with trypsin (Fig. 3). Tryptic fragments were immunoprecipitated with anti-NBF1 or anti-NBF2 antibody. NBF1 of SUR1 was labeled with 8-azido-[α-32P]ATP even in the presence of metal-chelating agent EDTA (Fig. 3 A, lane 1) or CDTA (lane 2), whereas NBF2 was labeled only in the presence of Mg2+ (lane 7). NBF1 was labeled with 8-azido-[α-32P]ATP by UV irradiation even after removing free ligand with excess cold buffer (lane 4), whereas NBF2 was not (lane 8), indicating that the bound 8-azido-[α-32P]ATP did not dissociate from NBF1 during washing of membranes with excess cold buffer. These results suggest that 8-azido-ATP binding to NBF1 is Mg2+-independent and very stable, whereas that to NBF2 is Mg2+-dependent and unstable as reported 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). In the case of SUR2A (Fig. 3 B) and SUR2B (Fig.3 C), both NBF1 and NBF2 were labeled with 8-azido-[α-32P]ATP either in the presence of EDTA (lanes 1 and 5) or Mg2+ (lanes 3 and 7). However, when Mg2+ was completely depleted by chelating with CDTA, which has stronger Mg2+-chelating ability than EDTA, NBF2 was not labeled with 8-azido-[α-32P]ATP (lane 6), whereas NBF1 was (lane 2). NBF1 was labeled with 8-azido-[α-32P]ATP by UV irradiation after removing free ligand with excess cold buffer (lane 4), whereas NBF2 was not (lane 8). These results suggest that 8-azido-ATP binding to NBF1 of SUR2A and SUR2B is Mg2+-independent and very stable, whereas that to NBF2 is Mg2+-dependent and unstable similar to SUR1. However, Mg2+ dependence of 8-azido-ATP binding to NBF2 of SUR2A and SUR2B is much lower than that of SUR1. In our previous study, it was suggested that NBF2 of SUR1 may have ATPase activity, because NBF2 of SUR1 was photoaffinity-labeled with 8-azido-[α-32P]ATP but not with 8-azido-[γ-32P]ATP as shown in Fig.4 A (lanes 2 and4). We examined whether NBFs of SUR2A and SUR2B could be labeled with 8-azido-[α-32P]ATP and 8-azido-[γ-32P]ATP (Fig. 4, B andC). NBF1s of SUR2A and SUR2B were photoaffinity-labeled with both 8-azido-[α-32P]ATP (lane 1) and 8-azido-[γ-32P]ATP (lane 3) in the presence of Mg2+. However, NBF2s of SUR2A and SUR2B were labeled with 8-azido-[α-32P]ATP (lane 2) but not with 8-azido-[γ-32P]ATP (lane 4) in the presence of Mg2+. NBF2s of SUR2A and SUR2B were labeled with both 8-azido-[α-32P]ATP and 8-azido-[γ-32P]ATP in the presence of EDTA but were not labeled in the presence of CDTA (data not shown). These results suggest that bound 8-azido-[32P]ATP is hydrolyzed and γ-phosphate dissociates from NBF2 of SURx in the presence of Mg2+, although NBF1 of SURx have no or little ATPase activity. We have suggested that NBF1 of SUR1 is a high affinity 8-azido-ATP-binding site and NBF2 is a low affinity 8-azido-ATP-binding site (19Matsuo M. Tucker S.J. Ashcroft F.M. Amachi T. Ueda K. FEBS Lett. 1999; 458: 292-294Crossref PubMed Scopus (9) Google Scholar, 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). To know the affinity for ATP and ADP of NBFs of SURx, we examined inhibition of 8-azido-ATP binding by ATP and ADP. When SURx was photoaffinity-labeled with 50 μm 8-azido-[α-32P]ATP in the presence of cold ATP or ADP, photoaffinity labeling of both NBFs of SURx was inhibited by ATP and ADP in a concentration-dependent manner (Fig.5), indicating that ADP as well as ATP binds to NBFs. The K i values for ATP and ADP of NBFs were calculated, and the values and nucleotide-binding properties are summarized in Table I. TheK i values of NBF1 for ATP were 4.4 ± 3.7, 110 ± 41, and 51 ± 13 μm in SUR1, SUR2A, and SUR2B, respectively. Those of NBF1 for ADP were 26 ± 8.6, 86 ± 23, and 66 ± 7.5 μm in SUR1, SUR2A, and SUR2B, respectively. Thus, the affinities of NBF1 for both ATP and ADP are significantly higher in SUR1 than in SUR2A and SUR2B. This is consistent with the concentration dependence of photoaffinity labeling of NBF1 of SURx with 8-azido-[32P]ATP; the degree of photoaffinity labeling was saturated at about 5 μm in SUR1, but saturation was seen at about 50 μm in SUR2A and SUR2B (data not shown). The affinity of NBF1 of SUR1 for ATP is significantly higher than that for ADP, although the affinities of NBF1 of SUR2A and SUR2B are not significantly different between ATP and ADP. The K i values of NBF2 for ATP were 60 ± 26, 120 ± 39, and 38 ± 26 μm in SUR1, SUR2A, and SUR2B, respectively. Those of NBF2 for ADP were 100 ± 26, 170 ± 70, and 67 ± 40 μm in SUR1, SUR2A, and SUR2B, respectively. Thus, the affinities of NBF2 of SUR2A for ATP and ADP are significantly lower than those of SUR2B.Table ICharacteristics of NBFs of SURxSUR subtypeNBFK i ATPK i ADPMg2+dependency1-aMg2+ dependency of nucleotide binding of NBF2 of SUR1 is high (++), and those of SUR2A and SUR2B are low (+). Nucleotide binding of NBF1 is Mg2+-independent (−).Slow dissociation1-b8-Azido-ATP binds to NBF1 of SURx very stably and does not dissociate at 0 °C.ATPase1-cNBF2 may have ATPase activity, whereas NBF1 has none or little ATPase activity.μm114.4 ± 3.726 ± 8.6−+−260 ± 26100 ± 26++−+2A1110 ± 4186 ± 23−+−2120 ± 39170 ± 70+−+2B151 ± 1366 ± 7.5−+−238 ± 2667 ± 40+−+1-a Mg2+ dependency of nucleotide binding of NBF2 of SUR1 is high (++), and those of SUR2A and SUR2B are low (+). Nucleotide binding of NBF1 is Mg2+-independent (−).1-b 8-Azido-ATP binds to NBF1 of SURx very stably and does not dissociate at 0 °C.1-c NBF2 may have ATPase activity, whereas NBF1 has none or little ATPase activity. Open table in a new tab We reported previously that ADP stabilizes the binding of prebound 8-azido-[32P]ATP at NBF1 on SUR1, either by direct binding to NBF2 or hydrolysis of bound ATP at NBF2 (21Ueda 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 this effect was impaired in mutant SUR1 (R1420C) found in Japanese persistent hyperinsulinemic hypoglycemia of infancy patients (24Tanizawa 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 examined whether two NBFs of SUR2A and SUR2B bind nucleotides cooperatively. When membranes from COS-7 cells expressing SUR2A or SUR2B are incubated with 50 μm8-azido-[α-32P]ATP, both NBF1 and NBF2 bind 8-azido-[α-32P]ATP. Because 8-azido-[α-32P]ATP bound to NBF2 dissociates during washing of membranes with excess buffer at 0 °C but that bound to NBF1 does not, we can estimate the stabilization effect of nucleotides bound to NBF2 on 8-azido-[α-32P]ATP binding to NBF1 during postincubation at 37 °C as shown in Fig.6. When membranes were postincubated in the absence of Mg2+, 8-azido-[α-32P]ATP quickly dissociated from NBF1 of SUR2A and SUR2B (Fig.7). When membranes were postincubated in the presence of Mg2+ without nucleotide, 8-azido-[α-32P]ATP gradually dissociated from NBF1 in a time-dependent manner as in the case of SUR1 (21Ueda 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). However, 8-azido-[α-32P]ATP did not dissociate from NBF1 in the presence of both Mg2+ and nucleotide (Fig. 7), indicating that MgATP and MgADP stabilize 8-azido-[α-32P]ATP binding at NBF1 as in the case of SUR1 (21Ueda 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).Figure 7Cooperative binding of nucleotides and 8-azido-[α-32P]ATP. Membrane proteins (20 μg) from COS-7 cells expressing SUR2A or SUR2B were incubated with 50 μm 8-azido-[α-32P]ATP, and free 8-azido-[α-32P]ATP was removed by washing membranes with excess cold buffer. Proteins were photoaffinity-labeled immediately or after postincubation with or without nucleotides in the presence or absence of Mg2+ for 15 min at 37 °C. The amount of photoaffinity-labeled 8-azido-[α-32P]ATP is shown as percentage of that of SUR2A (white bars) or SUR2B (black bars) UV-irradiated immediately after resuspension. Experiments were performed in triplicate, and the average values are represented with S.E.View Large Image Figure ViewerDownload (PPT) In the present study, we have compared the nucleotide-binding properties of each NBF of SURx to elucidate the molecular mechanisms responsible for the differential regulation of KATP channel subtypes in various organs. Some nucleotide-binding properties were found to be similar among all the SUR subtypes as follows: 1) NBF1 is a Mg2+-independent ATP- and ADP-binding site; 2) NBF2 is a Mg2+-dependent ATP- and ADP-binding site; 3) 8-azido-ATP binds to NBF1 very stably and does not dissociate at 0 °C; 4) MgATP or MgADP binding to NBF2 stabilizes 8-azido-ATP binding at NBF1; 5) NBF2 may have ATPase activity, whereas NBF1 has showed no or little ATPase activity. However, some properties are different among SUR subtypes as follows. 1) Nucleotide binding to NBF2 of SUR1 is highly Mg2+-dependent, whereas Mg2+ dependence of nucleotide binding to NBF2 of SUR2A and SUR2B is low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, are significantly higher than those of SUR2A and SUR2B. 3) The aff
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