Localization of the ATP/Phosphatidylinositol 4,5 Diphosphate-binding Site to a 39-Amino Acid Region of the Carboxyl Terminus of the ATP-regulated K+ Channel Kir1.1
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m208679200
ISSN1083-351X
AutoresKe Dong, Lieqi Tang, Gordon G. MacGregor, Steven Hébert,
Tópico(s)Mechanical Circulatory Support Devices
ResumoIntracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP2(PI(4,5)P2), regulate the activity of ATP-sensitive K+ (KATP) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP2 competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP2 antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP2. A truncated COOH-terminal fusion protein construct, MBP_1.1CΔC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH2-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP2 competed TNP-ATP binding to MBP_1.1CΔC170 with an EC50 of 10.9 μm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and KATP channels and are involved in ATP and/or PIP2 effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1ΔC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP2 competition of TNP-ATP (EC50 = 5.7 μm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP2 binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K+ channels. Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP2(PI(4,5)P2), regulate the activity of ATP-sensitive K+ (KATP) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP2 competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP2 antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP2. A truncated COOH-terminal fusion protein construct, MBP_1.1CΔC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH2-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP2 competed TNP-ATP binding to MBP_1.1CΔC170 with an EC50 of 10.9 μm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and KATP channels and are involved in ATP and/or PIP2 effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1ΔC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP2 competition of TNP-ATP (EC50 = 5.7 μm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP2 binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K+ channels. ATP-sensitive K+(KATP) channels couple cell metabolism to K+ channel activity (3Ashcroft S.J.H. Ashcroft F.M. Cell. Signal. 1990; 2: 197-214Crossref PubMed Scopus (674) Google Scholar, 4Ashcroft S.J. J. Membr. Biol. 2000; 176: 187-206Crossref PubMed Scopus (69) Google Scholar, 5Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar, 6Baukrowitz T. Fakler B. Eur. J. Biochem. 2000; 267: 5842-5848Crossref PubMed Scopus (66) Google Scholar, 7Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. (Cell Physiol.). 1998; 274: C25-C37Crossref PubMed Google Scholar). KATP channels are formed by four pore-forming subunits (Kir6.x) in association with four subunits of sulfonylurea receptors (8Aguilar-Bryan L. Clement J.P. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (510) Google Scholar), SUR1 or SUR2a/b. Kir1.1 has many properties similar to these KATP channels and has been suggested to interact with the cystic fibrosis transmembrane conductance regulator or SUR2b to form a glibenclamide-sensitive and ATP-inhibited channel (9Ruknudin A. Schulze D.H. Sullivan S.K. Lederer W.J. Welling P.A. J. Biol. Chem. 1998; 273: 14165-14171Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Tanemoto M. Vanoye C.G. Dong K. Welch R. Abe T. Hebert S.C. Xu J.Z. Am. J. Physiol. Renal Physiol. 2000; 278: F659-F666Crossref PubMed Google Scholar). Nucleotides can both inhibit and activate KATP channels (3Ashcroft S.J.H. Ashcroft F.M. Cell. Signal. 1990; 2: 197-214Crossref PubMed Scopus (674) Google Scholar,4Ashcroft S.J. J. Membr. Biol. 2000; 176: 187-206Crossref PubMed Scopus (69) Google Scholar, 11Wang W. Hebert S.C. Giebisch G. Ann. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (175) Google Scholar). Inhibition of KATP channel activity is mediated by binding of ATP to the Kir subunits, whereas channel activation by ADP occurs upon interaction with the SUR subunits. Phosphatidylinositol phosphates (e.g. phosphatidylinositol 4,5 diphosphate, PIP2) 1The abbreviations used are: PIP2, phosphatidylinositol 4,5 diphosphate; MBP, maltose binding protein; a.u., arbitrary unit; WT, wild type; TNP, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene); ROMK, renal outer medulla potassium channel1The abbreviations used are: PIP2, phosphatidylinositol 4,5 diphosphate; MBP, maltose binding protein; a.u., arbitrary unit; WT, wild type; TNP, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene); ROMK, renal outer medulla potassium channel compete with ATP for gating of KATP channels (12Huang C.-L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (760) Google Scholar, 13Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (483) Google Scholar, 14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar, 15Fan Z. Makielski J.C. J. Biol. Chem. 1997; 272: 5388-5395Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 16Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (438) Google Scholar). The effect of PIP2 to antagonize the inhibitory action of ATP provides a mechanism for opening KATP channels in intact cells in the presence of cytosolic ATP concentrations that would otherwise be inhibitory (13Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (483) Google Scholar, 16Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (438) Google Scholar, 17Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (557) Google Scholar, 18Fan Z. Makielski J.C. J. Gen. Physiol. 1999; 114: 251-269Crossref PubMed Scopus (76) Google Scholar).We recently localized the ATP-binding region to the COOH terminus in each of the three ATP-regulated channels: Kir1.1, Kir6.1, and Kir6.2 (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). We found that maltose-binding (MBP) fusion proteins containing Kir1.1 or Kir6.x COOH termini are efficiently produced in bacteria, are soluble without detergent, and directly bind TNP-ATP with a stoichiometry of at least one ATP per COOH-terminal monomer. In addition, we showed that phosphatidylinositol phospholipids competed for ATP binding to the COOH termini of Kir6.x and Kir1.1 channels (2MacGregor G.G. Dong K. Vanoye C.G. Tang L. Giebisch G. Hebert S.C. Proc. Natl. Acad. Sci., U. S. A. 2002; 99: 2726-2731Crossref PubMed Scopus (115) Google Scholar), providing a mechanism for PIP2 antagonism of ATP effects.Kir1.1 or ROMK, originally cloned from rat kidney outer medulla (20Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (831) Google Scholar), forms the small conductance ATP-regulated K+ channel involved in apical K+ recycling in thick ascending limb and in K+ secretion in principal cells of the mammalian kidney (11Wang W. Hebert S.C. Giebisch G. Ann. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (175) Google Scholar). Mutations in the human ROMK gene (Kcnj1) that cause loss of channel function give rise to Bartter's syndrome, which is characterized by volume depletion resulting from renal salt wasting (21Karolyil L. Konrad M. Kockerling A. Ziegler A. Zimmermann D.K. Roth B. Wieg C. Grzeschik K.-H. Koch M.C. Seyberth H.W. Vargus R. Forestier L. Jean G. Deschaux M. Rizzoni G.F. Niaudet P. Antignac C. Feldman D. Lorridon F. Cougoureux E. Laroze F. Alessandri J.-L. David L. Saunier P. Deschenes G. Hildebrandt F. Vollmer M. Proesmans W. Brandis M. van den Heuvell L.P.W.J. Lemmink H.H. Nillesen W. Monnens L.A.H. Knoers N.V.A.M. Guay-Woodford L.M. Wright C.J. Madrigal G. Hebert S.C. Hum. Mol. Genet. 1997; 6: 17-26Crossref PubMed Scopus (186) Google Scholar, 22Simon D.B. Karet F.E. Rodriguez-Soriano J. Hamdan J.H. DiPietro A. Trachtman H. Sanjad S.A. Lifton R.P. Nat. Genet. 1996; 14: 152-156Crossref PubMed Scopus (725) Google Scholar). Mice with deletion of the ROMK (Kir1.1) gene exhibit a phenotype consistent with Bartter's syndrome with polyuria, increased urinary Na+ loss, and extracellular fluid volume depletion (23Lu M. Wang T. Yan Q. Yang X. Dong K. Knepper M.A. Wang W. Giebisch G. Shull G.E. Hebert S.C. J. Biol. Chem. 2002; 277: 37881-37887Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The ROMK-deficient mice have no small conductance channel activity on apical membranes of thick ascending limb or principal cells, consistent with ROMK encoding the small conductance K+ recycling/secretory channel in kidney.In the present study, we further localized the ATP binding within the COOH terminus of Kir1.1. We assessed TNP-ATP binding and its antagonism by PIP2 to purified maltose-binding fusion proteins with deletions of the cytosolic COOH terminus of Kir1.1. The ATP-PIP2 binding domain was localized to the first 39 amino acids of the COOH terminus of Kir1.1. We also defined specific arginine residues within this 39-amino acid domain that are critical for TNP-ATP binding. Because these arginine residues are conserved in the Kir6.x channels, similar domains in these channels are likely to also form ATP-binding pockets.DISCUSSIONOur previous study provided direct evidence that TNP-ATP can bind to the COOH termini of Kir1.1 and the ATP-sensitive K+channels, Kir6.1 and Kir6.2 (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). Because TNP-ATP did not bind to the NH2 termini of Kir1.1 or Kir6.1, the COOH terminus of ATP-regulated K+ channels was both necessary and sufficient to bind nucleotides. The present observations support the suggestion that ATP inhibition of KATP channel activity is mediated by interaction of ATP with the COOH terminus of the Kir subunit. Here we have further localized the nucleotide binding segment of Kir1.1 to the initial 39 amino acid residues of the COOH terminus. In addition, we show that specific residues within this segment are critical to nucleotide binding. Finally, we demonstrate that this 39-residue segment also retains the critical sequences necessary for PIP2 competition of TNP-ATP binding.Several lines of evidence indicate that the initial 39 amino acid residues of the COOH terminus of Kir1.1 is the minimal segment necessary for the high affinity binding of TNP-ATP and for PIP2 competition and that this segment is the only significant adenine nucleotide-binding region along the COOH terminus of Kir1.1. First, MBP_1.1CΔC170 bound TNP-ATP with higher affinity than the entire COOH terminus (Figs. 1 and 2, MBP_1.1C-WT) or any of the other truncation constructs (Figs. Figure 1, Figure 2, Figure 3, Figure 4). In addition, TNP-ATP binding to the construct with a 10-residue greater truncation (MBP_1.1CΔC180) was dramatically reduced compared with MBP_1.1CΔC170 (Fig. 1). Moreover, MBP_1.1ΔC170 exhibited a significantly higher γ (Fig. 2 C) than for MBP_1.1C-WT, consistent with an improved protein environment for TNP-ATP binding. Second, the 170-amino acid segment of the COOH terminus beyond the initial 39 amino acid residues (MBP_1.1CΔN39) did not exhibit significant TNP-ATP binding (Fig. 3 D), demonstrating that nucleotide binding is limited to the first 39 amino acids of the COOH terminus. Third, truncations of as few as 5 amino acids from the NH2-terminal end of the COOH terminus (Fig. 3 C) virtually abolished TNP-ATP binding. Fourth, mutation of any one of three individual residues (Fig. 6, Arg188,Arg203, or Arg217) within the 39-amino acid MBP_1.1CΔC170 construct abolished TNP-ATP binding, indicating that they are directly involved in the formation of the TNP-ATP binding structure of Kir1.1. The observation that R212A did not alter TNP-ATP binding (Fig. 6 C) indicates that the effects of R188A (Fig. 6 A), R203A (Fig. 6 B), and R217A (Fig. 6 D) on TNP-ATP binding are unlikely to be nonspecific.All three arginine residues that affect TNP-ATP binding in Kir1.1 (Arg188, Arg203, andArg217) are conserved in Kir6.x channels (Fig.5) as well as most Kir channels, the only exception being that Arg203 is a histidine residue in Kir4.1. Mutation of any of these three residues in Kir6.2 (Arg177,Arg192, or Arg206; Fig.5) has been shown to alter channel gating by ATP and/or PIP2 (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar). Because ATP and PIP2 are competitive regulators of KATP channel gating and we have shown that TNP-ATP and PIP2 interactions occur within the same 39-amino acid segment (Fig. 7), this mutational study in Kir6.2 is fully consistent with our observations of the critical roles of Arg188 and Arg217 in nucleotide binding (Fig.6). Interestingly, the R201A mutation in Kir6.2 (Fig. 5 A), corresponding to R212A in Kir1.1, reduces Kir6.2 channel gating by ATP and PIP2, whereas this mutation in Kir1.1 has no effect on TNP-ATP binding (Fig. 6 C) and enhances competition of TNP-ATP binding by PIP2 (Fig. 7 B). This suggests that Arg201 in Kir6.2 functions differently than Arg212 in Kir1.1 for ATP binding or that Arg201in Kir6.2 affects channel gating by a mechanism other than modification of direct binding of ATP. Finally, a number of other residues in the first 39 amino acids of Kir6.2 have also been shown to affect ATP and/or PIP2 gating of channel activity (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar, 27Trapp S. Proks P. Tucker S.J. Ashcroft F.M. J. Gen. Physiol. 1998; 112: 333-349Crossref PubMed Scopus (149) Google Scholar, 28Tucker S.J. Gribble F.M. Proks P. Trapp S. Ryder T.J. Haug T. Reimann F. Ashcroft F.M. EMBO J. 1998; 17: 3290-3296Crossref PubMed Scopus (198) Google Scholar, 29Drain P. Li L. Wang J. Proc. Natl. Acad. Sci., U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (171) Google Scholar) or the binding of 8-azido-[γ32P]ATP (30Tanabe T. Tucker S.J. Matsuo M. Proks P. Ashcroft F.M. Seino S. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 3931-3933Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), consistent with the role of the initial region of the Kir6.2 COOH terminus, and by analogy the Kir1.1 COOH terminus, in ATP/PIP2 effects.We previously suggested that TNP-ATP binds to the COOH termini of Kir1.1 and the Kir6.x ATP-sensitive channels with a stoichiometry of at least one molecule of ATP binding per COOH-terminal protein (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). The TNP-ATP/protein stoichiometry of 0.78 for the entire Kir1.1 COOH terminus (MBP_1.1C-WT) confirms our previous observations of a single adenine nucleotide binding site for each of the four Kir subunits forming the kidney ATP-regulated K+ channel. The present results provide additional support for this model by demonstrating that TNP-ATP binding is confined to a single short segment in the COOH terminus of Kir1.1. Although the No for MBP_1.1CΔC170 (Fig. 2 B) was reduced compared with WT, the stoichiometry of 0.5 is still consistent with one binding site per protein molecule. The reduced stoichiometry was also observed in MBP_1.1CΔC84 and MBP_1.1CΔC160, suggesting that amino acids in the distal COOH terminus may be involved in the stabilization of the proximal 39-residue adenine nucleotide binding pocket.One of the major regulatory mechanisms of Kir1.1 and KATPchannels, as well as other Kir K+ channels, is channel gating (opening) by phosphatidylinositol phospholipids like PIP2. PIP2 competition of the inhibitory action of ATP provides a mechanism for opening KATP channels in intact cells in the presence of millimolar concentrations of cytosolic ATP. We have recently reported that PIP2 competed with TNP-ATP binding to the COOH terminus of Kir1.1 and Kir6.x channels. Our present results show that the 39-amino acid nucleotide binding domain in the COOH termini of Kir1.1 retains the ability of PIP2to compete off TNP-ATP. Thus, the initial 39-amino acid residue segment in the COOH terminus of Kir1.1 represents both the ATP binding domain and a PIP2 binding segment. The enhancement, rather than diminution, of PIP2 competition of TNP-ATP binding in the R212A mutant suggests that the effect of mutation of the similar residue in Kir6.2 (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar) on PIP2-ATP interactions may involve a mechanism other than ATP-PIP2 competition for binding. It should be noted, however, that although the initial COOH-terminal sequences of Kir1.1 and Kir6.2 are similar, they are not identical. Thus, it is possible that certain distinct amino acid residues in Kir6.2 may change the role of this arginine residue in PIP2binding compared with Kir1.1.In summary, we have defined a 39-amino acid segment in the Kir1.1 (ROMK) COOH terminus that forms an ATP-PIP2 binding domain and have identified three arginine residues that significantly alter TNP-ATP binding. Because this region is similar and the three critical arginine residues are conserved in Kir6.x channels (Fig.5 B), it seems likely that the initial COOH-terminal segment in KATP channels also forms an ATP-PIP2 binding domain. ATP-sensitive K+(KATP) channels couple cell metabolism to K+ channel activity (3Ashcroft S.J.H. Ashcroft F.M. Cell. Signal. 1990; 2: 197-214Crossref PubMed Scopus (674) Google Scholar, 4Ashcroft S.J. J. Membr. Biol. 2000; 176: 187-206Crossref PubMed Scopus (69) Google Scholar, 5Edwards G. Weston A.H. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 597-637Crossref PubMed Scopus (528) Google Scholar, 6Baukrowitz T. Fakler B. Eur. J. Biochem. 2000; 267: 5842-5848Crossref PubMed Scopus (66) Google Scholar, 7Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. (Cell Physiol.). 1998; 274: C25-C37Crossref PubMed Google Scholar). KATP channels are formed by four pore-forming subunits (Kir6.x) in association with four subunits of sulfonylurea receptors (8Aguilar-Bryan L. Clement J.P. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (510) Google Scholar), SUR1 or SUR2a/b. Kir1.1 has many properties similar to these KATP channels and has been suggested to interact with the cystic fibrosis transmembrane conductance regulator or SUR2b to form a glibenclamide-sensitive and ATP-inhibited channel (9Ruknudin A. Schulze D.H. Sullivan S.K. Lederer W.J. Welling P.A. J. Biol. Chem. 1998; 273: 14165-14171Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 10Tanemoto M. Vanoye C.G. Dong K. Welch R. Abe T. Hebert S.C. Xu J.Z. Am. J. Physiol. Renal Physiol. 2000; 278: F659-F666Crossref PubMed Google Scholar). Nucleotides can both inhibit and activate KATP channels (3Ashcroft S.J.H. Ashcroft F.M. Cell. Signal. 1990; 2: 197-214Crossref PubMed Scopus (674) Google Scholar,4Ashcroft S.J. J. Membr. Biol. 2000; 176: 187-206Crossref PubMed Scopus (69) Google Scholar, 11Wang W. Hebert S.C. Giebisch G. Ann. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (175) Google Scholar). Inhibition of KATP channel activity is mediated by binding of ATP to the Kir subunits, whereas channel activation by ADP occurs upon interaction with the SUR subunits. Phosphatidylinositol phosphates (e.g. phosphatidylinositol 4,5 diphosphate, PIP2) 1The abbreviations used are: PIP2, phosphatidylinositol 4,5 diphosphate; MBP, maltose binding protein; a.u., arbitrary unit; WT, wild type; TNP, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene); ROMK, renal outer medulla potassium channel1The abbreviations used are: PIP2, phosphatidylinositol 4,5 diphosphate; MBP, maltose binding protein; a.u., arbitrary unit; WT, wild type; TNP, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene); ROMK, renal outer medulla potassium channel compete with ATP for gating of KATP channels (12Huang C.-L. Feng S. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (760) Google Scholar, 13Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (483) Google Scholar, 14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar, 15Fan Z. Makielski J.C. J. Biol. Chem. 1997; 272: 5388-5395Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 16Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (438) Google Scholar). The effect of PIP2 to antagonize the inhibitory action of ATP provides a mechanism for opening KATP channels in intact cells in the presence of cytosolic ATP concentrations that would otherwise be inhibitory (13Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (483) Google Scholar, 16Baukrowitz T. Schulte U. Oliver D. Herlitze S. Krauter T. Tucker S.J. Ruppersberg J.P. Fakler B. Science. 1998; 282: 1141-1144Crossref PubMed Scopus (438) Google Scholar, 17Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (557) Google Scholar, 18Fan Z. Makielski J.C. J. Gen. Physiol. 1999; 114: 251-269Crossref PubMed Scopus (76) Google Scholar). We recently localized the ATP-binding region to the COOH terminus in each of the three ATP-regulated channels: Kir1.1, Kir6.1, and Kir6.2 (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). We found that maltose-binding (MBP) fusion proteins containing Kir1.1 or Kir6.x COOH termini are efficiently produced in bacteria, are soluble without detergent, and directly bind TNP-ATP with a stoichiometry of at least one ATP per COOH-terminal monomer. In addition, we showed that phosphatidylinositol phospholipids competed for ATP binding to the COOH termini of Kir6.x and Kir1.1 channels (2MacGregor G.G. Dong K. Vanoye C.G. Tang L. Giebisch G. Hebert S.C. Proc. Natl. Acad. Sci., U. S. A. 2002; 99: 2726-2731Crossref PubMed Scopus (115) Google Scholar), providing a mechanism for PIP2 antagonism of ATP effects. Kir1.1 or ROMK, originally cloned from rat kidney outer medulla (20Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (831) Google Scholar), forms the small conductance ATP-regulated K+ channel involved in apical K+ recycling in thick ascending limb and in K+ secretion in principal cells of the mammalian kidney (11Wang W. Hebert S.C. Giebisch G. Ann. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (175) Google Scholar). Mutations in the human ROMK gene (Kcnj1) that cause loss of channel function give rise to Bartter's syndrome, which is characterized by volume depletion resulting from renal salt wasting (21Karolyil L. Konrad M. Kockerling A. Ziegler A. Zimmermann D.K. Roth B. Wieg C. Grzeschik K.-H. Koch M.C. Seyberth H.W. Vargus R. Forestier L. Jean G. Deschaux M. Rizzoni G.F. Niaudet P. Antignac C. Feldman D. Lorridon F. Cougoureux E. Laroze F. Alessandri J.-L. David L. Saunier P. Deschenes G. Hildebrandt F. Vollmer M. Proesmans W. Brandis M. van den Heuvell L.P.W.J. Lemmink H.H. Nillesen W. Monnens L.A.H. Knoers N.V.A.M. Guay-Woodford L.M. Wright C.J. Madrigal G. Hebert S.C. Hum. Mol. Genet. 1997; 6: 17-26Crossref PubMed Scopus (186) Google Scholar, 22Simon D.B. Karet F.E. Rodriguez-Soriano J. Hamdan J.H. DiPietro A. Trachtman H. Sanjad S.A. Lifton R.P. Nat. Genet. 1996; 14: 152-156Crossref PubMed Scopus (725) Google Scholar). Mice with deletion of the ROMK (Kir1.1) gene exhibit a phenotype consistent with Bartter's syndrome with polyuria, increased urinary Na+ loss, and extracellular fluid volume depletion (23Lu M. Wang T. Yan Q. Yang X. Dong K. Knepper M.A. Wang W. Giebisch G. Shull G.E. Hebert S.C. J. Biol. Chem. 2002; 277: 37881-37887Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The ROMK-deficient mice have no small conductance channel activity on apical membranes of thick ascending limb or principal cells, consistent with ROMK encoding the small conductance K+ recycling/secretory channel in kidney. In the present study, we further localized the ATP binding within the COOH terminus of Kir1.1. We assessed TNP-ATP binding and its antagonism by PIP2 to purified maltose-binding fusion proteins with deletions of the cytosolic COOH terminus of Kir1.1. The ATP-PIP2 binding domain was localized to the first 39 amino acids of the COOH terminus of Kir1.1. We also defined specific arginine residues within this 39-amino acid domain that are critical for TNP-ATP binding. Because these arginine residues are conserved in the Kir6.x channels, similar domains in these channels are likely to also form ATP-binding pockets. DISCUSSIONOur previous study provided direct evidence that TNP-ATP can bind to the COOH termini of Kir1.1 and the ATP-sensitive K+channels, Kir6.1 and Kir6.2 (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). Because TNP-ATP did not bind to the NH2 termini of Kir1.1 or Kir6.1, the COOH terminus of ATP-regulated K+ channels was both necessary and sufficient to bind nucleotides. The present observations support the suggestion that ATP inhibition of KATP channel activity is mediated by interaction of ATP with the COOH terminus of the Kir subunit. Here we have further localized the nucleotide binding segment of Kir1.1 to the initial 39 amino acid residues of the COOH terminus. In addition, we show that specific residues within this segment are critical to nucleotide binding. Finally, we demonstrate that this 39-residue segment also retains the critical sequences necessary for PIP2 competition of TNP-ATP binding.Several lines of evidence indicate that the initial 39 amino acid residues of the COOH terminus of Kir1.1 is the minimal segment necessary for the high affinity binding of TNP-ATP and for PIP2 competition and that this segment is the only significant adenine nucleotide-binding region along the COOH terminus of Kir1.1. First, MBP_1.1CΔC170 bound TNP-ATP with higher affinity than the entire COOH terminus (Figs. 1 and 2, MBP_1.1C-WT) or any of the other truncation constructs (Figs. Figure 1, Figure 2, Figure 3, Figure 4). In addition, TNP-ATP binding to the construct with a 10-residue greater truncation (MBP_1.1CΔC180) was dramatically reduced compared with MBP_1.1CΔC170 (Fig. 1). Moreover, MBP_1.1ΔC170 exhibited a significantly higher γ (Fig. 2 C) than for MBP_1.1C-WT, consistent with an improved protein environment for TNP-ATP binding. Second, the 170-amino acid segment of the COOH terminus beyond the initial 39 amino acid residues (MBP_1.1CΔN39) did not exhibit significant TNP-ATP binding (Fig. 3 D), demonstrating that nucleotide binding is limited to the first 39 amino acids of the COOH terminus. Third, truncations of as few as 5 amino acids from the NH2-terminal end of the COOH terminus (Fig. 3 C) virtually abolished TNP-ATP binding. Fourth, mutation of any one of three individual residues (Fig. 6, Arg188,Arg203, or Arg217) within the 39-amino acid MBP_1.1CΔC170 construct abolished TNP-ATP binding, indicating that they are directly involved in the formation of the TNP-ATP binding structure of Kir1.1. The observation that R212A did not alter TNP-ATP binding (Fig. 6 C) indicates that the effects of R188A (Fig. 6 A), R203A (Fig. 6 B), and R217A (Fig. 6 D) on TNP-ATP binding are unlikely to be nonspecific.All three arginine residues that affect TNP-ATP binding in Kir1.1 (Arg188, Arg203, andArg217) are conserved in Kir6.x channels (Fig.5) as well as most Kir channels, the only exception being that Arg203 is a histidine residue in Kir4.1. Mutation of any of these three residues in Kir6.2 (Arg177,Arg192, or Arg206; Fig.5) has been shown to alter channel gating by ATP and/or PIP2 (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar). Because ATP and PIP2 are competitive regulators of KATP channel gating and we have shown that TNP-ATP and PIP2 interactions occur within the same 39-amino acid segment (Fig. 7), this mutational study in Kir6.2 is fully consistent with our observations of the critical roles of Arg188 and Arg217 in nucleotide binding (Fig.6). Interestingly, the R201A mutation in Kir6.2 (Fig. 5 A), corresponding to R212A in Kir1.1, reduces Kir6.2 channel gating by ATP and PIP2, whereas this mutation in Kir1.1 has no effect on TNP-ATP binding (Fig. 6 C) and enhances competition of TNP-ATP binding by PIP2 (Fig. 7 B). This suggests that Arg201 in Kir6.2 functions differently than Arg212 in Kir1.1 for ATP binding or that Arg201in Kir6.2 affects channel gating by a mechanism other than modification of direct binding of ATP. Finally, a number of other residues in the first 39 amino acids of Kir6.2 have also been shown to affect ATP and/or PIP2 gating of channel activity (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar, 27Trapp S. Proks P. Tucker S.J. Ashcroft F.M. J. Gen. Physiol. 1998; 112: 333-349Crossref PubMed Scopus (149) Google Scholar, 28Tucker S.J. Gribble F.M. Proks P. Trapp S. Ryder T.J. Haug T. Reimann F. Ashcroft F.M. EMBO J. 1998; 17: 3290-3296Crossref PubMed Scopus (198) Google Scholar, 29Drain P. Li L. Wang J. Proc. Natl. Acad. Sci., U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (171) Google Scholar) or the binding of 8-azido-[γ32P]ATP (30Tanabe T. Tucker S.J. Matsuo M. Proks P. Ashcroft F.M. Seino S. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 3931-3933Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), consistent with the role of the initial region of the Kir6.2 COOH terminus, and by analogy the Kir1.1 COOH terminus, in ATP/PIP2 effects.We previously suggested that TNP-ATP binds to the COOH termini of Kir1.1 and the Kir6.x ATP-sensitive channels with a stoichiometry of at least one molecule of ATP binding per COOH-terminal protein (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). The TNP-ATP/protein stoichiometry of 0.78 for the entire Kir1.1 COOH terminus (MBP_1.1C-WT) confirms our previous observations of a single adenine nucleotide binding site for each of the four Kir subunits forming the kidney ATP-regulated K+ channel. The present results provide additional support for this model by demonstrating that TNP-ATP binding is confined to a single short segment in the COOH terminus of Kir1.1. Although the No for MBP_1.1CΔC170 (Fig. 2 B) was reduced compared with WT, the stoichiometry of 0.5 is still consistent with one binding site per protein molecule. The reduced stoichiometry was also observed in MBP_1.1CΔC84 and MBP_1.1CΔC160, suggesting that amino acids in the distal COOH terminus may be involved in the stabilization of the proximal 39-residue adenine nucleotide binding pocket.One of the major regulatory mechanisms of Kir1.1 and KATPchannels, as well as other Kir K+ channels, is channel gating (opening) by phosphatidylinositol phospholipids like PIP2. PIP2 competition of the inhibitory action of ATP provides a mechanism for opening KATP channels in intact cells in the presence of millimolar concentrations of cytosolic ATP. We have recently reported that PIP2 competed with TNP-ATP binding to the COOH terminus of Kir1.1 and Kir6.x channels. Our present results show that the 39-amino acid nucleotide binding domain in the COOH termini of Kir1.1 retains the ability of PIP2to compete off TNP-ATP. Thus, the initial 39-amino acid residue segment in the COOH terminus of Kir1.1 represents both the ATP binding domain and a PIP2 binding segment. The enhancement, rather than diminution, of PIP2 competition of TNP-ATP binding in the R212A mutant suggests that the effect of mutation of the similar residue in Kir6.2 (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar) on PIP2-ATP interactions may involve a mechanism other than ATP-PIP2 competition for binding. It should be noted, however, that although the initial COOH-terminal sequences of Kir1.1 and Kir6.2 are similar, they are not identical. Thus, it is possible that certain distinct amino acid residues in Kir6.2 may change the role of this arginine residue in PIP2binding compared with Kir1.1.In summary, we have defined a 39-amino acid segment in the Kir1.1 (ROMK) COOH terminus that forms an ATP-PIP2 binding domain and have identified three arginine residues that significantly alter TNP-ATP binding. Because this region is similar and the three critical arginine residues are conserved in Kir6.x channels (Fig.5 B), it seems likely that the initial COOH-terminal segment in KATP channels also forms an ATP-PIP2 binding domain. Our previous study provided direct evidence that TNP-ATP can bind to the COOH termini of Kir1.1 and the ATP-sensitive K+channels, Kir6.1 and Kir6.2 (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). Because TNP-ATP did not bind to the NH2 termini of Kir1.1 or Kir6.1, the COOH terminus of ATP-regulated K+ channels was both necessary and sufficient to bind nucleotides. The present observations support the suggestion that ATP inhibition of KATP channel activity is mediated by interaction of ATP with the COOH terminus of the Kir subunit. Here we have further localized the nucleotide binding segment of Kir1.1 to the initial 39 amino acid residues of the COOH terminus. In addition, we show that specific residues within this segment are critical to nucleotide binding. Finally, we demonstrate that this 39-residue segment also retains the critical sequences necessary for PIP2 competition of TNP-ATP binding. Several lines of evidence indicate that the initial 39 amino acid residues of the COOH terminus of Kir1.1 is the minimal segment necessary for the high affinity binding of TNP-ATP and for PIP2 competition and that this segment is the only significant adenine nucleotide-binding region along the COOH terminus of Kir1.1. First, MBP_1.1CΔC170 bound TNP-ATP with higher affinity than the entire COOH terminus (Figs. 1 and 2, MBP_1.1C-WT) or any of the other truncation constructs (Figs. Figure 1, Figure 2, Figure 3, Figure 4). In addition, TNP-ATP binding to the construct with a 10-residue greater truncation (MBP_1.1CΔC180) was dramatically reduced compared with MBP_1.1CΔC170 (Fig. 1). Moreover, MBP_1.1ΔC170 exhibited a significantly higher γ (Fig. 2 C) than for MBP_1.1C-WT, consistent with an improved protein environment for TNP-ATP binding. Second, the 170-amino acid segment of the COOH terminus beyond the initial 39 amino acid residues (MBP_1.1CΔN39) did not exhibit significant TNP-ATP binding (Fig. 3 D), demonstrating that nucleotide binding is limited to the first 39 amino acids of the COOH terminus. Third, truncations of as few as 5 amino acids from the NH2-terminal end of the COOH terminus (Fig. 3 C) virtually abolished TNP-ATP binding. Fourth, mutation of any one of three individual residues (Fig. 6, Arg188,Arg203, or Arg217) within the 39-amino acid MBP_1.1CΔC170 construct abolished TNP-ATP binding, indicating that they are directly involved in the formation of the TNP-ATP binding structure of Kir1.1. The observation that R212A did not alter TNP-ATP binding (Fig. 6 C) indicates that the effects of R188A (Fig. 6 A), R203A (Fig. 6 B), and R217A (Fig. 6 D) on TNP-ATP binding are unlikely to be nonspecific. All three arginine residues that affect TNP-ATP binding in Kir1.1 (Arg188, Arg203, andArg217) are conserved in Kir6.x channels (Fig.5) as well as most Kir channels, the only exception being that Arg203 is a histidine residue in Kir4.1. Mutation of any of these three residues in Kir6.2 (Arg177,Arg192, or Arg206; Fig.5) has been shown to alter channel gating by ATP and/or PIP2 (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar). Because ATP and PIP2 are competitive regulators of KATP channel gating and we have shown that TNP-ATP and PIP2 interactions occur within the same 39-amino acid segment (Fig. 7), this mutational study in Kir6.2 is fully consistent with our observations of the critical roles of Arg188 and Arg217 in nucleotide binding (Fig.6). Interestingly, the R201A mutation in Kir6.2 (Fig. 5 A), corresponding to R212A in Kir1.1, reduces Kir6.2 channel gating by ATP and PIP2, whereas this mutation in Kir1.1 has no effect on TNP-ATP binding (Fig. 6 C) and enhances competition of TNP-ATP binding by PIP2 (Fig. 7 B). This suggests that Arg201 in Kir6.2 functions differently than Arg212 in Kir1.1 for ATP binding or that Arg201in Kir6.2 affects channel gating by a mechanism other than modification of direct binding of ATP. Finally, a number of other residues in the first 39 amino acids of Kir6.2 have also been shown to affect ATP and/or PIP2 gating of channel activity (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar, 27Trapp S. Proks P. Tucker S.J. Ashcroft F.M. J. Gen. Physiol. 1998; 112: 333-349Crossref PubMed Scopus (149) Google Scholar, 28Tucker S.J. Gribble F.M. Proks P. Trapp S. Ryder T.J. Haug T. Reimann F. Ashcroft F.M. EMBO J. 1998; 17: 3290-3296Crossref PubMed Scopus (198) Google Scholar, 29Drain P. Li L. Wang J. Proc. Natl. Acad. Sci., U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (171) Google Scholar) or the binding of 8-azido-[γ32P]ATP (30Tanabe T. Tucker S.J. Matsuo M. Proks P. Ashcroft F.M. Seino S. Amachi T. Ueda K. J. Biol. Chem. 1999; 274: 3931-3933Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), consistent with the role of the initial region of the Kir6.2 COOH terminus, and by analogy the Kir1.1 COOH terminus, in ATP/PIP2 effects. We previously suggested that TNP-ATP binds to the COOH termini of Kir1.1 and the Kir6.x ATP-sensitive channels with a stoichiometry of at least one molecule of ATP binding per COOH-terminal protein (19Vanoye C.G. MacGregor G.G. Dong K. Tang L. Buschmann A.E. Hall A.E. Lu M. Giebisch G. Hebert S.C. J. Biol. Chem. 2002; 272: 23260-23270Abstract Full Text Full Text PDF Scopus (44) Google Scholar). The TNP-ATP/protein stoichiometry of 0.78 for the entire Kir1.1 COOH terminus (MBP_1.1C-WT) confirms our previous observations of a single adenine nucleotide binding site for each of the four Kir subunits forming the kidney ATP-regulated K+ channel. The present results provide additional support for this model by demonstrating that TNP-ATP binding is confined to a single short segment in the COOH terminus of Kir1.1. Although the No for MBP_1.1CΔC170 (Fig. 2 B) was reduced compared with WT, the stoichiometry of 0.5 is still consistent with one binding site per protein molecule. The reduced stoichiometry was also observed in MBP_1.1CΔC84 and MBP_1.1CΔC160, suggesting that amino acids in the distal COOH terminus may be involved in the stabilization of the proximal 39-residue adenine nucleotide binding pocket. One of the major regulatory mechanisms of Kir1.1 and KATPchannels, as well as other Kir K+ channels, is channel gating (opening) by phosphatidylinositol phospholipids like PIP2. PIP2 competition of the inhibitory action of ATP provides a mechanism for opening KATP channels in intact cells in the presence of millimolar concentrations of cytosolic ATP. We have recently reported that PIP2 competed with TNP-ATP binding to the COOH terminus of Kir1.1 and Kir6.x channels. Our present results show that the 39-amino acid nucleotide binding domain in the COOH termini of Kir1.1 retains the ability of PIP2to compete off TNP-ATP. Thus, the initial 39-amino acid residue segment in the COOH terminus of Kir1.1 represents both the ATP binding domain and a PIP2 binding segment. The enhancement, rather than diminution, of PIP2 competition of TNP-ATP binding in the R212A mutant suggests that the effect of mutation of the similar residue in Kir6.2 (14Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (171) Google Scholar) on PIP2-ATP interactions may involve a mechanism other than ATP-PIP2 competition for binding. It should be noted, however, that although the initial COOH-terminal sequences of Kir1.1 and Kir6.2 are similar, they are not identical. Thus, it is possible that certain distinct amino acid residues in Kir6.2 may change the role of this arginine residue in PIP2binding compared with Kir1.1. In summary, we have defined a 39-amino acid segment in the Kir1.1 (ROMK) COOH terminus that forms an ATP-PIP2 binding domain and have identified three arginine residues that significantly alter TNP-ATP binding. Because this region is similar and the three critical arginine residues are conserved in Kir6.x channels (Fig.5 B), it seems likely that the initial COOH-terminal segment in KATP channels also forms an ATP-PIP2 binding domain.
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