The Carboxyl Termini of KATP Channels Bind Nucleotides
2002; Elsevier BV; Volume: 277; Issue: 26 Linguagem: Inglês
10.1074/jbc.m112004200
ISSN1083-351X
AutoresCarlos G. Vanoye, Gordon G. MacGregor, Ke Dong, Lieqi Tang, Alexandra S. Buschmann, Amy Hall, Ming Lu, Gerhard Giebisch, Steven Hébert,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoATP-sensitive potassium (KATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of KATP channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5′-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH2- and COOH-terminal cytosolic regions of the three known KATP channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K+ channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known KATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of KATP channels because this nucleotide did not bind to the NH2 termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to KATP COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all KATP COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg2+ and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of KATPtetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer. ATP-sensitive potassium (KATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of KATP channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5′-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH2- and COOH-terminal cytosolic regions of the three known KATP channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K+ channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known KATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of KATP channels because this nucleotide did not bind to the NH2 termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to KATP COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all KATP COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg2+ and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of KATPtetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer. ATP-regulated potassium sulfonylurea receptor 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene) adenosine triphosphate maltose-binding protein 4-morpholineethanesulfonic acid β-mercaptoethanol confidence interval dithiothreitol small-conductance K channel ATP-sensitive or ATP-regulated potassium (KATP)1 channels couple metabolism to either cell excitability (Kir6.x) (1Ashcroft S.J.H. Ashcroft F.M. Cell. Signal. 1990; 2: 197-214Crossref PubMed Scopus (677) Google Scholar, 2Aguilar-Bryan L. Clement J.P. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (516) Google Scholar, 3Yokoshiki H. Sunagawa M. Seki T. Sperelakis N. Am. J. Physiol. 1998; 274: C25-C37Crossref PubMed Google Scholar, 4Ashcroft F.M. Annu. Rev. Neurosci. 1988; 11: 97-118Crossref PubMed Scopus (769) Google Scholar, 5Nichols C.G. Lederer W.J. Am. J. Physiol. 1991; 261: H1675-H1686PubMed Google Scholar, 6Quayle J.M. Nelson M.T. Standen N.B. Physiol. Rev. 1997; 77: 1165-1232Crossref PubMed Scopus (720) Google Scholar) or potassium secretion (Kir1.1 in kidney) (7Misler S. Giebisch G. Curr. Opin. Nephrol. Hypertens. 1992; 1: 21-33Crossref PubMed Scopus (43) Google Scholar, 8Wang W. Hebert S.C. Giebisch G. Annu. Rev. Physiol. 1997; 59: 413-436Crossref PubMed Scopus (176) Google Scholar) and provide therapeutic targets for diseases including tissue ischemia, diabetes, hypertension, and disorders of potassium homeostasis. KATPchannels are formed by an octameric complex of four pore-forming subunits (Kir6.x or Kir1.1) and four sulfonylurea receptors, SUR1 or SUR2 for Kir6.x (2Aguilar-Bryan L. Clement J.P. Gonzalez G. Kunjilwar K. Babenko A. Bryan J. Physiol. Rev. 1998; 78: 227-245Crossref PubMed Scopus (516) Google Scholar) or the cystic fibrosis transmembrane conductance regulator or SUR2b for Kir1.1 (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 (85) Google Scholar, 10Tanemoto M. Vanoye C.G. Dong K. Welch R. Abe T. Hebert S.C. Xu J.Z. Am. J. Physiol. 2000; 278: F659-F666Crossref PubMed Google Scholar). Although the SUR/fibrosis transmembrane conductance regulator subunits contain nucleotide-binding folds (11Clement J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 12Zerangue N. Schwappach B. Jan Y.N. Jan L.Y. Neuron. 1999; 22: 537-548Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar), this subunit is not required for ATP-mediated inhibition of K+ channel activity. For example, deletion of the last 36 amino acids from the COOH terminus of Kir6.2 (Kir6.2ΔC36) produces functional K+ channels in the absence of coexpressed SURs that are sensitive to ATP (13Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar). Nevertheless, SUR subunits are required for ADP-mediated activation of KATP channels (14Trapp S. Tucker S.J. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8872-8877Crossref PubMed Scopus (55) Google Scholar, 15Gribble F.M. Tucker S.J. Ashcroft F.M. EMBO J. 1997; 16: 1145-1152Crossref PubMed Scopus (311) Google Scholar, 16Gribble F.M. Tucker S.J. Haug T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7185-7190Crossref PubMed Scopus (150) Google Scholar). Thus, ATP inhibition of KATP channel activity is thought to involve direct interaction with Kir subunits despite the lack of identifiable nucleotide-binding motifs. The recent demonstration of the photoaffinity labeling of Kir6.2 channel by 8-azido-[γ-32P]ATP (17Tanabe 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, 18Tanabe K. Tucker S.J. Ashcroft F.M. Proks P. Kioka N. Amachi T. Ueda K. Biochem. Biophys. Res. Commun. 2000; 272: 316-319Crossref PubMed Scopus (34) Google Scholar) also supports the direct binding of ATP with the pore-forming subunit of KATPchannels. In addition, mutations in both the NH2- and COOH-terminal regions of the Kir6.2 (13Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar, 19Tucker 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 (200) Google Scholar, 20Trapp S. Proks P. Tucker S.J. Ashcroft F.M. J. Gen. Physiol. 1998; 112: 333-349Crossref PubMed Scopus (151) Google Scholar, 21Drain P. Li L. Wang J. Proc. Natl. Acad. Sci., U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (174) Google Scholar, 22Shyng S.L. Cukras C.A. Harwood J. Nichols C.G. J. Gen. Physiol. 2000; 116: 599-608Crossref PubMed Scopus (174) Google Scholar, 23Koster J.C. Sha Q. Shyng S. Nichols C.G. J. Physiol. (Lond.). 1999; 515: 19-30Crossref Scopus (87) Google Scholar) and Kir1.1 (24McNicholas C.M. Yang Y. Giebisch G. Hebert S.C. Am. J. Physiol. 1996; 40: F275-F285Google Scholar) subunits alter the EC50 for ATP-mediated channel gating. Because ATP-mediated inhibition of channel activity must be a complex process involving residues that form an ATP-binding pocket and others that may be required for linking ATP binding to channel closure, those mutational studies of channel gating by nucleotides do not provide unequivocal evidence for direct involvement of those residues in ATP binding. In the present study, we assessed the direct binding of fluorescent 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene) adenosine triphosphate (TNP-ATP) to purified maltose-binding fusion proteins of the cytosolic NH2 and COOH termini of the three known KATP channels and the COOH terminus of a ATP-insensitive inward rectifier K+ channel, Kir2.1 (25Collins A. German M.S. Jan Y.N. Jan L.Y. J. Neurosci. 1996; 16: 1-9Crossref PubMed Google Scholar). We provide herein what we believe to be the first evidence of direct binding of ATP to cytosolic domains of the pore-forming subunits of KATP channels and show that the COOH termini, but not the NH2 termini, of Kir subunits of KATP channels bind TNP-ATP. The kinetic analyses of TNP-ATP binding suggest that the COOH termini have a single nucleotide-binding site. Based on glutaraldehyde cross-linking studies, the COOH termini of these three ATP-sensitive channels also exhibit multimerization potential so that they may interact in these intact tetrameric channels. DNAs encoding the NH2 and COOH termini of Kir6.1 (encoding amino acids 1–73 and 178–424 (247 amino acids), respectively) and the COOH terminus of Kir6.2Δ36 (encoding amino acids 169–354 (186 amino acids)) were obtained by reverse transcription-PCR from rat kidney and brain, respectively. The NH2 and COOH termini of Kir1.1 (encoding amino acids 1–80 and 183–391 in ROMK2 (209 amino acids), respectively) were derived from the previously cloned rat Kir1.1 (26Zhou H. Tate S.S. Palmer L.G. Am. J. Physiol. 1994; 266: C809-C824Crossref PubMed Google Scholar,27Ho 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 (834) Google Scholar). The COOH terminus of mouse Kir2.1 encoded amino acids 179–428 (250 amino acids). The sequences of all of the constructs were confirmed using the cycle sequencing method (Keck Facility, Yale). All channel cDNA constructs were ligated into the pMBPT vector kindly provided by Dr. G. A. Altenberg (28Wang C. Castro A.F. Wilkes D.M. Altenberg G.A. Biochem. J. 1999; 338: 77-81Crossref PubMed Scopus (52) Google Scholar). The vector was derived from the MALTM-c2 vector (maltose-binding protein (MBP) fusion vector; New England Biolabs). We constructed MBP fusion proteins containing the NH2 (MBP_1.1N and MBP_6.1N) or the COOH (MBP_1.1C and MBP_6.1C) terminus of rat Kir1.1 and Kir6.1, respectively, and the COOH termini of mouse Kir2.1 (MBP_2.1C) and rat Kir6.2CΔ36 (MBP_6.2CΔ36) channels. We used the MBP_6.2CΔ36 construct for these studies because deletion of the last 36 amino acids from the end of the COOH terminus of Kir6.2 gives rise to functional and ATP-sensitive channel activity in cells in the absence of SUR1 (13Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar). Recombinant proteins were expressed using the pMBPT vector as per the manufacturer's instructions (New England Biolabs). Briefly, 1 liter of Luria-Bertani medium with 0.1 mg/ml ampicillin and 0.5% glucose was inoculated with 10 ml of an overnight culture of Epicurian coli® BL21-CodonPlusTM-RIL-competent cells (Stratagene) expressing the fusion vector and grown to anA 600 of ∼0.5 at 37 °C. Induction was performed with 0.3 mm isopropyl β-d-thiogalactoside at 37 °C for 2.5 h. The cells were harvested and centrifuged at 4,000 × g for 20 m at 4 °C. The cell pellet was resuspended in 50 ml of column buffer (20 mm Tris-Cl, 200 mm NaCl, 1 mm EDTA, pH 7.4) and frozen overnight at −20 °C. The sample was thawed in ice water and lysed with a probe sonicator (four times for 30 s, with 30-s intervals in an ice water bath. The sample was then centrifuged at 9,000 × g for 30 m at 4 °C. The supernatant was kept and diluted 1:5 with column buffer. The diluted extract was loaded into a 25-ml column containing 15 ml of amylose resin and washed with 12 column volumes of column buffer. The fusion protein was eluted with column buffer with 10 mm maltose, and 1.5-ml fractions were collected. The protein was detected by UV absorbance at 280 nm, dialyzed against 50 mm Tris-HCl, pH 7.5, and kept at −80 °C until the experiments were performed. The yields of purified recombinant fusion proteins were 15–25 mg/liter. To assess the binding of ATP to these recombinant fusion proteins, we used fluorescent TNP-ATP (Molecular Probes, Inc.) (29Hiratsuka T. Biochim. Biophys. Acta. 1976; 453: 293-297Crossref PubMed Scopus (44) Google Scholar, 30Hiratsuka T. Uchida K. Biochim. Biophys. Acta. 1973; 320: 635-647Crossref PubMed Scopus (143) Google Scholar), which has been widely employed to study nucleotide binding to enzymes and other proteins (31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar, 32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar, 33Divita G. Goody R.S. Gautheron D.C. Di Pietro A. J. Biol. Chem. 1993; 268: 13178-13186Abstract Full Text PDF PubMed Google Scholar, 34Moutin M.J. Cuillel M. Rapin C. Miras R. Anger M. Lompre A.M. Dupont Y. J. Biol. Chem. 1994; 269: 11147-11154Abstract Full Text PDF PubMed Google Scholar). The binding of TNP-ATP to recombinant proteins was performed generally as described by Faller (32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar). Briefly, 5 μm recombinant protein was dissolved in 50 mm Tris-Cl at pH 7.5 or 5 mmMES monohydrate (Sigma) at pH 6.5, and TNP-ATP binding was detected by the increase in fluorescence upon binding to recombinant protein using a SPEX Fluromax-3 spectrofluorometer (Jobin Yvon Inc., Edison, NJ). The fluorescence units reported here were scaled by 1000. Excitation wavelength (403 nm) and emission wavelength (546 nm) were determined for the Kir1.1 COOH terminus fusion protein and utilized for all recombinant proteins (slit widths, 5 nm) because they did not vary significantly among proteins examined. A typical 10-nm blue shift in emission wavelength was detected upon binding of TNP-ATP to proteins (32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar). The temperature was maintained at 22 ± 0.1 °C by a circulating water bath (Neslab, Newington, NH). Incremental additions of TNP-ATP were delivered to polystyrene cuvettes (Elkay Products Inc., Shrewsbury, MA) from stock solutions (0.2–1.0 mm). Total fluorescence was measured 30 s after the additions to allow for equilibration. All of the titrations were corrected for dilution. TNP-ATP fluorescence was also measured in the presence of 5 mm MgATP or by denaturing the protein with 4m urea. MgATP was added from a stock solution of 0.2m adjusted to pH 7.5 or 6.5, as indicated. Free TNP-ATP is weakly fluorescent in buffer, but upon binding to proteins fluorescence is enhanced severalfold with the absolute magnitude dependent on the specific protein environment within the nucleotide-binding pocket (31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar, 32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar). The fluorescence enhancement factor (γ), TNP-ATP to protein subunit stoichiometry (N o), and dissociation constant (K d (μm)) were determined by least squares fitting to a modified version of the binding equation derived by Faller (32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar) using GraphPad PRISMTM 3.0 software. The observed fluorescence intensity (F obs) in arbitrary units is given by the following equation.Fobs=(Q[TNPATP]+Q2[TNPATP]2)+Qc2(γ−1)([TNPATP]+N0P+Kd)−([TNPATP]+N0P+Kd)2−4N0P[TNPATP]1/2Equation 1 where P is the protein concentration (μm). Q and Q 2 are constants (fluorescence intensity/μm or μm2 of free TNP-ATP, respectively) derived independently from the concentration dependence of TNP-ATP fluorescence intensity in buffer alone (F Buffer) and account for the “inner filter” effect (32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar):F Buffer = Q[TNP-ATP] +Q 2[TNP-ATP]2.Q c is the slope of theF Buffer versus [TNP-ATP] curve in buffer alone.dFBufferd[TNPATP]=Q+Q2[TNPATP]Equation 2 Concentrations of TNP-ATP above 20 μm were not used to minimize inner filter effects. Protein light scatter intensity (F light scatter) was subtracted from allF obs values. The concentration dependence of light scattering of individual recombinant proteins was:F light scatter = RP +R 2 P 2, where Rand R 2 are constants (light intensity/μm and μm2 of protein, respectively). We independently determined the enhanced factor (γ) by measuring the increase in F obs with increasing protein concentration at a fixed concentration of TNP-ATP (5 μm). The F obs data were corrected for light scatter and were fit well by a single exponential.F obsmax was determined as F obs at infinite protein concentration when all TNP-ATP would be bound. The enhancement factor was then calculated as follows.γ=FobsmaxFBufferEquation 3 Using this enhancement factor we calculated the concentrations of free ([F]) and bound ([B]) TNP-ATP as described by Moczydlowski and Fortes (31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar) taking into account the inner filter effect.[B]=(Fobs−Qc[TNPATP])/Qc(γ−1)Equation 4 Free TNP-ATP is then the difference between total [TNP-ATP] and [B]. Bound versus free TNP-ATP plots were analyzed using a standard binding model that follows mass action.[B]=Bmax[F]Kd+[F]Equation 5 where B max is the maximal TNP-ATP binding. The data were also plotted for Scatchard or Hill analyses (36Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17809) Google Scholar) as described (31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar, 37Klingenberg M. Mayer I. Dahms A.S. Biochemistry. 1984; 23: 2442-2449Crossref PubMed Scopus (19) Google Scholar, 38Huang S.G. Weisshart K. Fanning E. Biochemistry. 1998; 37: 15336-15344Crossref PubMed Scopus (39) Google Scholar). For noncompetitive binding the Scatchard analysis is linear as described by Moczydlowski and Fortes (31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar).[B][F]P=1KdN−[B]PEquation 6 where N is the number of TNP-ATP binding sites in μmol/mg. For MgATP, NaATP, or MgCl2 competition of TNP-ATP binding, we used a two-site model as described by Faller (39Faller L.D. Biochemistry. 1989; 28: 6771-6778Crossref PubMed Scopus (30) Google Scholar).ΔFobsΔFobsmax=1−Sfrac[ATP][ATP]+K1+(1−Sfrac)[ATP][ATP]+K2Equation 7 where ΔF obs/ΔF obsmaxis the fractional change in fluorescence intensity,S frac is the fraction of binding sites in the first site, and K 1 and K 2are the apparent substrate affinities for the first and second sites, respectively. Photoaffinity labeling of recombinant proteins with 8-azido-[γ-32P]ATP was performed as described previously (40Csermely P. Kahn C.R. J. Biol. Chem. 1991; 266: 4943-4950Abstract Full Text PDF PubMed Google Scholar, 41Lacapere J.-J. Bennett N. Dupont Y. Guillain F. J. Biol. Chem. 1990; 265: 348-353Abstract Full Text PDF PubMed Google Scholar). 5 μg of the purified protein was added to solution A (50 mm HEPES, 10 mm Tris, pH 7.4, 10 mm CaCl2, 0.5 mm MgCl2, and 2 μCi of [γ-32P]azido-ATP; ICN Biochemicals, Inc.) and incubated for 15 min in the dark at 4 °C. The reaction mixture was irradiated with UV light at 350 nm for 1 min at room temperature to covalently link the azido-ATP to neighboring amino acid residues. The labeled protein was resolved by SDS-PAGE and visualized by autoradiography. Cross-linking of fusion proteins with glutaraldehyde was performed as described previously (42Lee S.F. Wang C.T. Liang J.Y. Hong S.L. Huang C.C. Chen S.S. J. Biol. Chem. 2000; 275: 15809-15819Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, 0.15 μg of purified MBP fusion proteins (total volume, 40 μl) were incubated with different concentrations (final concentrations, 0, 0.005, 0.01, 0.025, 0.05, 0.075, and 0.1%) of glutaraldehyde in phosphate-buffered saline on ice for 30 min. The cross-linking was quenched with the addition of 100 mm glycine, pH 8.0. The proteins were solubilized in Laemmli buffer with 5% β-ME and resolved by SDS-7.5% PAGE. The proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad), blocked with 5% milk in a shaker at room temperature for 1 h, incubated with rabbit anti-MBP antibody (1:10,000; New England Biolabs) overnight at 4 °C on a rocker, and then incubated with horseradish peroxidase-conjugated donkey anti-rabbit Ig (1:10,000; Amersham Biosciences) for 1 h at room temperature on a rocker. The proteins were visualized by ECL (Amersham Biosciences). Inside out patch-clamp experiments were performed at room temperature (22–24 °C) as described (−V p = −40 mV) (43Wang W. Giebisch G. J. Gen. Physiol. 1991; 98: 35-61Crossref PubMed Scopus (121) Google Scholar) to assess the effects of TNP-ATP on apical KATP channel activity in rat cortical collecting ducts principal cells. Briefly, Sprague-Dawley rats (80–100g) were obtained from Taconic Farms Inc. and kept on normal chow diet (PMI Nutrition International, Inc.) for 7–10 days before experiments. The animals were euthanized, their kidneys were removed, and coronary slices were cut and placed in ice-cold dissection solution. Individual cortical collecting ducts were dissected at room temperature, and the tubules were immobilized on a 5 × 5-mm cover glass coated with Cell Tac (Becton Dickinson) and then transferred to a perfusion chamber mounted on the stage of an inverted microscope (IMT-2; Olympus). The tubules were opened with a sharpened pipette to gain access to the apical membrane. The principal cells were identified by their hexagonal shape and large flat surface. The bath solution contained 140 mm NaCl, 5 mm KCl, 1 mm EGTA, 10 mm HEPES, 0.2 mm MgATP, pH 7.4. The pipette solution contained 140 mm KCl, 1.8 mm MgCl2, 10 mm HEPES, pH 7.4. TNP-ATP (0–1000 μm) was added to the bath solution where indicated. MgATP is required in the bath solution to keep the KATP channels in principal cells from running down (43Wang W. Giebisch G. J. Gen. Physiol. 1991; 98: 35-61Crossref PubMed Scopus (121) Google Scholar). All of the chemicals were research grade or better and were from Sigma unless otherwise stated. All MBP fusion proteins were efficiently expressed in bacteria and could be highly purified at milligram quantities (5–25 mg/liter of bacterial culture) without exposure to detergents or denaturing agents (28Wang C. Castro A.F. Wilkes D.M. Altenberg G.A. Biochem. J. 1999; 338: 77-81Crossref PubMed Scopus (52) Google Scholar). The recombinant MBP and the NH2-terminal (MBP_1.1N and MBP_6.1N) and COOH-terminal (MBP_1.1C, MBP_6.1C, MBP_6.2CΔ36, and MBP_2.1C) MBP fusion proteins ran at their expected molecular masses as shown in Fig. 1. MBP_6.2CΔ36 consistently produced the lowest yield of 5–10 mg/liter, whereas the yields of MBP_1.1C and MBP_6.1C were 15–25 mg/liter. Cleaving the MBP from the channel protein at the thrombin site resulted in insoluble protein under our current buffer conditions, probably because of the hydrophobicity of these cytosolic NH2 and COOH termini. Thus, all of the experiments were performed using the MBP fusion proteins. We used fluorescent TNP-ATP to assess the binding of ATP to the cytosolic domains of Kir channels (31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar, 32Faller L.D. Biochemistry. 1990; 29: 3179-3186Crossref PubMed Scopus (25) Google Scholar, 33Divita G. Goody R.S. Gautheron D.C. Di Pietro A. J. Biol. Chem. 1993; 268: 13178-13186Abstract Full Text PDF PubMed Google Scholar, 34Moutin M.J. Cuillel M. Rapin C. Miras R. Anger M. Lompre A.M. Dupont Y. J. Biol. Chem. 1994; 269: 11147-11154Abstract Full Text PDF PubMed Google Scholar). The concentration dependence relationships of TNP-ATP fluorescence with MBP_1.1C, MBP_1.1N, and MBP alone at pH 7.5 are shown in Fig.2. F obs for unbound TNP-ATP in buffer without protein was low and increased in a nonlinear, concentration-dependent manner (Fig. 2,A and B), consistent with the intrinsic fluorescence of this ATP analogue and the inner filter effect (29Hiratsuka T. Biochim. Biophys. Acta. 1976; 453: 293-297Crossref PubMed Scopus (44) Google Scholar, 30Hiratsuka T. Uchida K. Biochim. Biophys. Acta. 1973; 320: 635-647Crossref PubMed Scopus (143) Google Scholar,31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar). All of the buffer data were well fit using a second order polynomial that accounts for this inner filter effect (see “Materials and Methods”; r 2 ≥ 0.99). In contrast,F obs was significantly enhanced over the buffer control in the presence of MBP_1.1C (Fig. 2, A andB, F P ), consistent with binding of TNP-ATP to this fusion protein. F obs with MBP_1.1C saturated (Fig. 2 B) and was well fit by Equation 1(r 2 = 0.999) using a γ of 7.7 (see Fig. 5) and gave a K d of 2.64 ± 0.26 μm(n = 11). Denaturing MBP_1.1C protein with 4m urea (Fig. 2 A, F P Urea ;n = 15) or 0.1% SDS (Fig. 2 B,F P SDS ; n = 7) diminished the nucleotide concentration-dependent increase inF obs to values close to that of TNP-ATP in the urea or SDS buffers without protein, respectively. The increase inF obs with MBP_1.1C was not due to TNP-ATP interactions with MBP because the TNP-ATP concentration-dependent increase inF obs with MBP (Fig. 2 C,F P ; n = 5) was similar to the TNP-ATP curve in buffer alone (Fig. 2 A, buffer) and was not significantly different in the absence or presence of 5 mmMgATP (Fig 2 C; F P MgATP ) or 4m urea (Fig. 2 C; F P Urea ). The binding of TNP-ATP was specific for the COOH terminus of Kir1.1 because the increase in F obs with MBP_1.1N was small and unaffected by 5 mm MgATP or 4 m urea (Fig. 2 D; n = 10). Mixing of MBP_1.1N and MBP_1.1C (1:1) did not significantly affect the affinity for TNP-ATP binding (control K d = 1.84 ± 0.14, (n = 6); mixing K d = 1.63 ± 0.22; (n = 5); data not shown).Figure 5Enhancement factor (γ), affinity (K d ), and stoichiometry (N) of TNP-ATP binding to MBP_1.1C at pH 7.5. A, MBP_1.1C protein titration of 1 μm (triangles) and 5 μm (squares) TNP-ATP.F obs was corrected for protein light scatter. The lines were calculated using an exponential fit, and fluorescence at infinite protein concentration (P∞) was determined. γ was calculated asF obsBuffer/F P∞Buffer. The dashed line is the intrinsic fluorescence of 5 μm TNP-ATP in buffer. B, bound TNP-ATP ([B]) plotted against free TNP-ATP ([F]). Bound and free TNP-ATP concentrations were calculated using Equation 4, as described under “Materials and Methods” and Ref. 31Moczydlowski E.G. Fortes P.A.G. J. Biol. Chem. 1981; 256: 2346-2356Abstract Full Text PDF PubMed Google Scholar. The line was calculated according to Equation 5. C, Scatchard plot for TNP-ATP binding to MBP_1.1C. The line was calculated according to Equation 6.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further support for nucleotide binding to MBP_1.1C was obtained by photoaffinity labeling by 8-azido-[γ-32P]ATP as shown in Fig. 3 A. The 8-azido-[γ-32P]ATP labeling was competed with unlabeled MgATP consistent with specific labeling of MBP_1.1C with this nucleotide analogue. We also examined the ability of MgATP to compete the TNP-ATP binding to MBP_1.1C. The TNP-ATP concentration-dependent increase inF obs with MBP_1.1C was reduced by 5 mm MgATP (Fig. 3 B, triangles), and the K d for TNP-ATP binding affinity was significantly increased; K d increased from 3.0 ± 0.2 (F P) to 6.9 ± 1.9 (F P 5 mm MgATP; n= 13). Increasing MgATP concentration to 50 mm virtually abolished TNP-ATP fluorescence enhancement with MBP_1.1C (K d = 50.9 ± 14.7 μm; Fig.3 B; n = 5). We also assessed the competition of TNP-ATP binding to MBP_1.1C by MgATP (Fig. 3 C). Increasing concentrations of MgATP reduced ΔF obs/ΔF obsmaxin a concentration-dependent manner. The shape of the MgATP competition curve was complex, suggesting multiple binding interactions; the data were well fit, however, using the two-site model described by Equation 7 (r 2 = 0.99).K 1 and K 2 were 71 ± 5 and 3.8 ± 0.8 mm,
Referência(s)