Coupling of Cell Energetics with Membrane Metabolic Sensing
2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês
10.1074/jbc.m201777200
ISSN1083-351X
AutoresM. Roselle Abraham, Vitaly A. Selivanov, Denice M. Hodgson, Darko Pucar, Leonid V. Zingman, Bé Wieringa, Petras P. Dzeja, Alexey E. Alekseev, André Terzic,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoTransduction of metabolic signals is essential in preserving cellular homeostasis. Yet, principles governing integration and synchronization of membrane metabolic sensors with cell metabolism remain elusive. Here, analysis of cellular nucleotide fluxes and nucleotide-dependent gating of the ATP-sensitive K+ (KATP) channel, a prototypic metabolic sensor, revealed a diffusional barrier within the submembrane space, preventing direct reception of cytosolic signals. Creatine kinase phosphotransfer, captured by 18O-assisted 31P NMR, coordinated tightly with ATP turnover, reflecting the cellular energetic status. The dynamics of high energy phosphoryl transfer through the creatine kinase relay permitted a high fidelity transmission of energetic signals into the submembrane compartment synchronizing KATP channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to KATP channels and generated a cellular phenotype with increased electrical vulnerability. Thus, in the compartmentalized cell environment, phosphotransfer systems shunt diffusional barriers and secure regimented signal transduction integrating metabolic sensors with the cellular energetic network. Transduction of metabolic signals is essential in preserving cellular homeostasis. Yet, principles governing integration and synchronization of membrane metabolic sensors with cell metabolism remain elusive. Here, analysis of cellular nucleotide fluxes and nucleotide-dependent gating of the ATP-sensitive K+ (KATP) channel, a prototypic metabolic sensor, revealed a diffusional barrier within the submembrane space, preventing direct reception of cytosolic signals. Creatine kinase phosphotransfer, captured by 18O-assisted 31P NMR, coordinated tightly with ATP turnover, reflecting the cellular energetic status. The dynamics of high energy phosphoryl transfer through the creatine kinase relay permitted a high fidelity transmission of energetic signals into the submembrane compartment synchronizing KATP channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to KATP channels and generated a cellular phenotype with increased electrical vulnerability. Thus, in the compartmentalized cell environment, phosphotransfer systems shunt diffusional barriers and secure regimented signal transduction integrating metabolic sensors with the cellular energetic network. M-creatine kinase 2,4-dinitrofluorobenzene creatine phosphate wild-type deoxyglucose Maintenance of cellular homeostasis critically depends on the ability of the cell to adjust diverse energy-dependent processes in response to metabolic challenge (1van Deursen J. Heerschap A. Oerlemans F. Ruitenbeek W. Jap P. ter Laak H. Wieringa B. 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Terzic A. Circulation. 1998; 98: 1548-1555Crossref PubMed Scopus (112) Google Scholar). KATP channels are expressed in high density in metabolically active tissues, in particular heart muscle, where the pore-forming Kir6.2 protein assembles with the regulatory sulfonylurea receptor SUR2A subunit to form functional hetero-octameric complexes (21Inagaki N. Gonoi T. Clement J.P. 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, 22Clement 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, 23Lorenz E. Terzic A. J. Mol. Cell. Cardiol. 1999; 31: 425-434Abstract Full Text PDF PubMed Scopus (71) Google Scholar). While ATP closes KATP channels by interacting with Kir6.2, metabolic sensing seems to proceed through interactions of ATP/ADP with nucleotide-binding domains of SUR (9Aguilar-Bryan L. Bryan J. Nakazaki M. Recent Prog. Horm. Res. 2001; 56: 47-68Crossref PubMed Scopus (68) Google Scholar, 16Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 24Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar, 25Drain P., Li, L. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (174) Google Scholar, 26Shyng S.L. Ferrigni T. Nichols C.G. J. Gen. Physiol. 1997; 110: 643-654Crossref PubMed Scopus (246) Google Scholar, 27Zingman L.V. Hodgson D.M. Bienengraeber M. Karger A.B. Kathmann E.C. Alekseev A.E. Terzic A. J. Biol. Chem. 2002; 277: 14206-14210Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In this regard, active membrane ATPases constantly reduce the local ATP concentration setting the submembrane ATP/ADP ratio distinct from that of the "bulk" cytosol (15Weiss J.N. Korge P. Circ. Res. 2001; 89: 108-110Crossref PubMed Scopus (41) Google Scholar, 28Weiss J.N. Lamp S.T. Science. 1988; 238: 67-69Crossref Scopus (311) Google Scholar, 29Lederer W.J. Nichols C.G. J. Physiol. (Lond.). 1989; 419: 193-211Crossref Scopus (252) Google Scholar, 30Weiss J.N. Venkatesh N. Cardiovasc. Drugs Ther. 1993; 7: 499-505Crossref PubMed Scopus (78) Google Scholar, 31Lederer W.J. Niggli E. Hadley R.W. Science. 1990; 248: 283Crossref PubMed Scopus (266) Google Scholar). However, such independent nucleotide fluctuations within a particular cell compartment (15Weiss J.N. Korge P. Circ. Res. 2001; 89: 108-110Crossref PubMed Scopus (41) Google Scholar, 31Lederer W.J. Niggli E. Hadley R.W. Science. 1990; 248: 283Crossref PubMed Scopus (266) Google Scholar) would hamper proper recognition of cellular signals rendering KATP channels ineffective metabolic sensors. In response to metabolic alterations, the membrane content of polyphosphoinositides has been implicated in defining the ATP-sensitivity of cardiac KATP channels (32Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (487) Google Scholar). Yet, altered KATP channel sensitivity per se may not provide an efficient mechanism of metabolic signal transduction as drastic reduction in the channel responsiveness to ATP, induced by mutation of Kir6.2, has no apparent consequences on channel behavior and/or membrane electrical activity in metabolically competent cardiac cells (33Koster J.C. Knopp A. Flagg T.P. Markova K.P. Sha Q. Enkvetchakul D. Betsuyaku T. Yamada K.A. Nichols C.G. Circ. Res. 2001; 89: 1022-1029Crossref PubMed Scopus (62) Google Scholar). Rather, coordination of membrane sensor function with the cellular metabolic status mandates effective transfer of energetic signals between intracellular compartments (2O'Rourke B. Ramza B.M. Marban E. Science. 1994; 265: 962-966Crossref PubMed Scopus (228) Google Scholar, 15Weiss J.N. Korge P. Circ. Res. 2001; 89: 108-110Crossref PubMed Scopus (41) Google Scholar). Cells with high and fluctuating energy demands, such as cardiomyocytes, possess catalyzed phosphotransfer circuits that facilitate energetic signaling between sites of ATP production and utilization (3Dzeja P.P. Terzic A. FASEB J. 1998; 12: 523-529Crossref PubMed Scopus (132) Google Scholar, 5Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (129) Google Scholar). Emerging evidence suggests that phosphotransfer networks can process metabolic information for delivery to metabolic sensors, thereby serving a critical role in cellular homeostasis (2O'Rourke B. Ramza B.M. Marban E. Science. 1994; 265: 962-966Crossref PubMed Scopus (228) Google Scholar, 3Dzeja P.P. Terzic A. FASEB J. 1998; 12: 523-529Crossref PubMed Scopus (132) Google Scholar, 34Carassco A.J. Dzeja P.P. Alekseev A.E. Pucar D. Zingman L.V. Abraham M.R. Hodgson D. Bienengraeber M. Puceat M. Janssen E. Wieringa B. Terzic A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7623-7628Crossref PubMed Scopus (218) Google Scholar, 35Elvir-Mairena J.R. Jovanovic A. Gomez L.A. Alekseev A.E. Terzic A. J. Biol. Chem. 1996; 271: 31903-31908Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In this way, the phosphotransfer enzyme adenylate kinase physically associates with KATP channel proteins to facilitate communication of mitochondrial signals and promote channel opening in stress (34Carassco A.J. Dzeja P.P. Alekseev A.E. Pucar D. Zingman L.V. Abraham M.R. Hodgson D. Bienengraeber M. Puceat M. Janssen E. Wieringa B. Terzic A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7623-7628Crossref PubMed Scopus (218) Google Scholar). A related signal delivery function has been suggested for the most active phosphotransfer enzyme in the myocardium, creatine kinase, which could control KATP channel closure and prevent accidental channel opening (3Dzeja P.P. Terzic A. FASEB J. 1998; 12: 523-529Crossref PubMed Scopus (132) Google Scholar, 36Bienengraeber M. Alekseev A.E. Abraham M.R. Carrasco A.J. Moreau C. Vivaudou M. Dzeja P.P. Terzic A. FASEB J. 2000; 14: 1943-1952Crossref PubMed Scopus (124) Google Scholar, 37Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Isoforms of creatine kinase are found in distinct intracellular compartments, including membranes where KATPchannels reside (13Kaasik A. Veksler V. Boehm E. Novotova M. Minajeva A. Ventura-Clapier R. Circ. Res. 2001; 89: 153-159Crossref PubMed Scopus (218) Google Scholar, 38Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (177) Google Scholar). Substrates of creatine kinase regulate nucleotide-dependent KATP channel gating and can overcome potassium channel opener-induced channel activation (36Bienengraeber M. Alekseev A.E. Abraham M.R. Carrasco A.J. Moreau C. Vivaudou M. Dzeja P.P. Terzic A. FASEB J. 2000; 14: 1943-1952Crossref PubMed Scopus (124) Google Scholar,37Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. 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Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 40Sasaki N. Sato T. Marban E. O'Rourke B. Am. J. Physiol. 2001; 280: H1882-H1888Crossref PubMed Google Scholar, 41Crawford R.M. Ranki H.J. Botting C.H. Budas G.R. Jovanovic A. FASEB J. 2002; 16: 102-104Crossref PubMed Scopus (122) Google Scholar), the requirement for creatine kinase phosphotransfer in synchronizing metabolic sensor function in response to fluctuations in the cellular metabolic state has not been defined. Here, we demonstrate that the dynamics of high energy phosphoryl transfer through the creatine kinase system coordinates KATP channel activity with cellular metabolism contributing to an integrative mechanism for delivery of energetic signals to the membrane sensor. Deletion of the M-CK gene, which encodes the major creatine kinase isoform, disrupted creatine kinase-dependent signal delivery to KATPchannels and generated a phenotype with increased electrical vulnerability. Mice lacking the M-creatine kinase (M-CK)1 isoform were derived from embryonic stem cells carrying a replacement mutation in the M-CK gene (1van Deursen J. Heerschap A. Oerlemans F. Ruitenbeek W. Jap P. ter Laak H. Wieringa B. Cell. 1993; 74: 621-631Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Inactivation of M-CK expression was achieved by homologous DNA recombination with a HygroB cassette vector used to replace exon 2 and parts of introns 1 and 2 in theM-CK gene. Homozygous M-CK-knock-out mice were compared with age-matched wild-type controls. Cardiomyocytes, isolated from wild-type and M-CK knock-out mice or guinea pig ventricles (34Carassco A.J. Dzeja P.P. Alekseev A.E. Pucar D. Zingman L.V. Abraham M.R. Hodgson D. Bienengraeber M. Puceat M. Janssen E. Wieringa B. Terzic A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7623-7628Crossref PubMed Scopus (218) Google Scholar), were bathed in (mm) KCl, 140; MgCl2, 1; EGTA, 5; HEPES-KOH, 5 (pH 7.3). Patch electrodes (7–10 MΩ) were filled with (mm) KCl, 140; CaCl2, 1; MgCl2, 1; HEPES-KOH, 5 (pH 7.3). For the open cell-attached patch, bath solution was supplemented with glucose (1 g/liter), malic acid (5 mm), and pyruvic acid (5 mm). Following seal formation with the patch pipette, cell permeabilization was achieved by digitonin (5–8 μg/ml) applied through a second pipette (filled with 5 μg/ml propidium iodide and 0.5 μg/ml rhodamine). Under ultraviolet light, rhodamine served for visualization of solution flow, and propidium iodide staining of the cell nucleus indicated formation of the open cell-attached patch configuration (36Bienengraeber M. Alekseev A.E. Abraham M.R. Carrasco A.J. Moreau C. Vivaudou M. Dzeja P.P. Terzic A. FASEB J. 2000; 14: 1943-1952Crossref PubMed Scopus (124) Google Scholar, 37Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Channel activity was measured at −60 mV. 18O-assisted 31P NMR is based on incorporation of 18O, provided from18O-water, into cellular phosphates proportionally to the rate of enzymatic reactions involved (42Pucar D. Janssen E. Dzeja P.P. Juranic N. Macura S. Wieringa B. Terzic A. J. Biol. Chem. 2000; 275: 41424-41429Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 43Pucar D. Dzeja P.P. Bast P. Juranic N. Macura S. Terzic A. J. Biol. Chem. 2001; 276: 44812-44819Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). γATP→[18O]H2O[18O]Pi+ADPATP HYDROLYSIS REACTION[18O]Pi+ADP→[18O]γATPATP SYNTHESIS REACTION[18O]γATP+Cr→[18O]CrP+ADPCREATINE KINASE PHOSPHOTRANSFER REACTIONThe 18O-phosphoryl labeling procedure detects only newly generated molecules containing 18O-labeled phosphoryls reflecting net flux through an individual phosphotransfer pathway. Hearts were perfused at 37 °C with 95% O2, 5% CO2 saturated buffer (in mm: 123 NaCl, 6 KCl, 2.5 CaCl2, 0.5 EDTA, 19 NaHCO3, 1.2 MgSO4, 11 glucose, and 20 units/liter insulin). Hypoxia was induced with 95%N2, 5% CO2 gassed buffer, to reduce partial oxygen pressure to 20–30 mm Hg. 18O labeling was achieved using the buffer supplemented with 40% of18O-H2O (Isotec) for 30 s. During this time 18O labeling is still within the initial pseudolinear phase of the labeling kinetic curve, which reaches full saturation only after 2 min following application of 18O-H2O. Hearts were freeze-clamped and extracted in 600 mmHClO4 and 1 mm EDTA. 18O-induced shifts in 31P NMR spectra of ATP and creatine phosphate were recorded at 242.9 MHz in a Bruker 14 T spectrometer (42Pucar D. Janssen E. Dzeja P.P. Juranic N. Macura S. Wieringa B. Terzic A. J. Biol. Chem. 2000; 275: 41424-41429Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 43Pucar D. Dzeja P.P. Bast P. Juranic N. Macura S. Terzic A. J. Biol. Chem. 2001; 276: 44812-44819Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and phosphotransfer fluxes calculated as described (38Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (177) Google Scholar). Hearts homogenized in (in mm) 10 HEPES, 1 EGTA, 1 dithiothreitol, 1 aprotinin, 0.2 phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptin (pH 7.4) were spun at 5,000 × g. Supernatant was centrifuged at 100,000 × g and membrane pellets suspended by sonication in (in mm) 20 HEPES (pH 7.4), 140 NaCl, 5 KCl, 2 MgCl2, 0.5 dithiothreitol, 1 aprotinin, 0.2 phenylmethylsulfonyl fluoride, and 2 μg/ml leupeptin. Creatine kinase activity was determined with a coupled enzyme assay (38Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (177) Google Scholar). Mouse hearts were perfused at 90 mm Hg with (in mm) NaCl, 108; KCl, 5; HEPES, 5; glucose or deoxyglucose, 5; sodium acetate, 20; MgCl2, 1; CaCl2, 2; malate, 1; pyruvate, 5; and insulin, 5 units/liter (pH 7.4, 37 °C). Monophasic action potentials were recorded from the left ventricular epicardial surface using a probe (EP Technologies) while pacing at 130-ms cycle length and 10-ms pulse width (Accupulser, World Precision Instruments). In guinea pig hearts, action potentials were measured without pacing. Nucleotide-dependent KATP channel gating was simulated by an allosteric model where four identical binding sites for ATP and ADP co-exist within the octameric stoichiometry of the KATP channel complex (16Ashcroft F.M. Gribble F.M. Trends Neurosci. 1998; 21: 288-294Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 22Clement 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). Binding of ATP to the pore-forming Kir6.2 subunit inhibits channel opening (24Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (683) Google Scholar,25Drain P., Li, L. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (174) Google Scholar), whereas binding of ADP to the regulatory SUR subunit antagonizes ATP-binding to Kir6.2 (6Nichols C.G. Shyng S.L. Nestorowicz A. Glaser B. Clement J.P. Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (471) Google Scholar, 26Shyng S.L. Ferrigni T. Nichols C.G. J. Gen. Physiol. 1997; 110: 643-654Crossref PubMed Scopus (246) Google Scholar, 44Matsuo M. Tanabe K. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 28757-28763Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Distribution of channel species (D i ; i = 0 to 4) with 0–4 ADP bound molecules was as follows, D1D0=4·[ADP]kADP;D2D1=3·[ADP]2kADP;D3D2=2·[ADP]3kADP;D4D3=[ADP]4kADP;∑j=14Dj=1Equation 1 with the percentage of D i species expressed as a function of ADP concentration,D1=4·D0[ADP]kADP;D2=6·D0[ADP]kADP2;D3=4·D0[ADP]kADP3;Equation 2 D4=D0[ADP]kADP4whereD0=1+4·[ADP]kADP+6·[ADP]kADP2+4·[ADP]kADP3+[ADP]kADP4−1Equation 3 and k ADP the dissociation constant of ADP from SUR, independent from ATP binding. Analogously to EquationsEquation 1, Equation 2, Equation 3, the distribution of channel species (T i ) with 0–4 ATP bound molecules was derived as follows,∑i=14TiDj=Dj,j=0–4Equation 4 with k 0 andk 1 representing dissociation constants for ATP binding to Kir6.2 in the absence and presence of ADP at the associated SUR (Fig. 1, A and B). The best fits of experimental data from ATP-induced KATP channel inhibition in the absence of ADP, at saturating ADP and at below saturating ADP revealed, respectively, the values for k 0,k 1, and k ADP, with more than one ATP required to close the channel octamer. Diffusional restriction was estimated by integrating membrane ATPase activities and diffusional nucleotide fluxes (Fig.1 C). Membrane ATP consumption (J ATPase) was simulated as a Michaelis-Menten reaction with the Michaelis constant at 0.05 mm (45Chazov E.I. Smirnov V.N. Saks V.A. Rosenshtraukh L.V. Lipina N.V. Levitsky D.O. Adv. Myocardiol. 1980; 1: 139-153PubMed Google Scholar). Sarcolemmal ATPase activity (1,800 nmol/min/g wet weight) was derived from total ATPase activity in working hearts measured by18O-assisted 31P NMR (300 nmol/min/mg of protein), assuming that 120 mg of protein (with 1 mg of sarcolemmal protein) is contained in 1 g of tissue and that ∼5% of total energy is consumed by sarcolemmal ATPases (46Golhaber J.I. Langer G.A. The Myocardium. Academic Press, San Diego, CA1997: 325-383Crossref Google Scholar). Nucleotide diffusion (with a coefficient D) was calculated according to Fick's law as one-dimensional flux (through total cell area in 1 g of tissue, S) perpendicular to the membrane. Diffusional flux for ATP was as follows, JATPdiff(x)=−DS∂[ATP(x)]∂xEquation 5 where J ATP(x) is ATP flux at distance x. At steady-state, withJ ATP constant, C = −DS/Δxdefining,JATPdiff=C([ATP]b−[ATP]sub)Equation 6 where [ATP]b and [ATP]sub are cytosolic bulk and subsarcolemmal ATP concentration, respectively. Diffusional flux for ADP was described analogously as in Equation 6. [ATP]sub and [ADP]sub, as a function of [ATP]b (Fig. 1 D), were defined from the following equation.C([ATP]b−[ATP]sub)−JATPase=0C([ADP]b−[ADP]sub)+JATPase=0Equation 7 The defining property of cardiac KATP channels as membrane metabolic sensors is their overt inhibition by ATP, which can be antagonized by MgADP (Fig. 1 A). KATP channels adopt their highest sensitivity to ATP in the absence of MgADP and convert to a range of lower ATP sensitivities with increasing concentrations of MgADP (Fig. 1 A). The regulatory SUR2A subunit harbors an intrinsic ATPase activity (36Bienengraeber M. Alekseev A.E. Abraham M.R. Carrasco A.J. Moreau C. Vivaudou M. Dzeja P.P. Terzic A. FASEB J. 2000; 14: 1943-1952Crossref PubMed Scopus (124) Google Scholar, 44Matsuo M. Tanabe K. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 28757-28763Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) that facilitates conformational transitions imparting low or high ATP sensitivity to the KATP channel complex (37Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). MgADP prolongs the lifetime of the conformation associated with reduced sensitivity to ATP (37Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Allosteric modeling, which integrated KATP channel stoichiometry and channel-nucleotide interactions (Fig. 1 B), demonstrated that on saturating ADP-binding sites (at >100 μm ADP) no further reduction in ATP sensitivity can be achieved (Fig. 1, A andB), in accord with the efficacy of ADP to antagonize ATP-induced channel inhibition (47Weiss J.N. Venkatesh N. Lamp S.T. J. Physiol. (Lond.). 1992; 447: 649-673Crossref Scopus (156) Google Scholar). For MgADP to open at least 1% of KATP channels, required for significant action potential shortening at 6–10 mm cytosolic ATP (47Weiss J.N. Venkatesh N. Lamp S.T. J. Physiol. (Lond.). 1992; 447: 649-673Crossref Scopus (156) Google Scholar, 48Bittl J.A. DeLayre J. Ingwall J.S. Biochemistry. 1987; 26: 6083-6090Crossref PubMed Scopus (119) Google Scholar, 49Decking U.K. Reffelmann T. Schrader J. Kammermeier H. Am. J. Physiol. 1995; 269: H734-H742PubMed Google Scholar, 50Nichols C.G. Lederer W.J. Am. J. Physiol. 1991; 261: H1675-H1686PubMed Google Scholar), ATP at the channel site needs to be reduced to <3 mm (Fig. 1,C and D). Local drop in ATP could be generated by membrane ATPases (30Weiss J.N. Venkatesh N. Cardiovasc. Drugs Ther. 1993; 7: 499-505Crossref PubMed Scopus (78) Google Scholar, 51Kabakov A.Y. Biophys. J. 1998; 75: 2858-2867Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), including ATP hydrolysis by the KATP channel itself (36Bienengraeber M. Alekseev A.E. Abraham M.R. Carrasco A.J. Moreau C. Vivaudou M. Dzeja P.P. Terzic A. FASEB J. 2000; 14: 1943-1952Crossref PubMed Scopus (124) Google Scholar, 37Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 44Matsuo M. Tanabe K. Kioka N. Amachi T. Ueda K. J. Biol. Chem. 2000; 275: 28757-28763Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), provided that nucleotide mobility between the cytosol and submembrane is limited (31Lederer W.J. Niggli E. Hadley R.W. Science. 1990; 248: 283Crossref PubMed Scopus (266) Google Scholar) (Fig. 1 C). Calculations, based on membrane ATPase activity and nucleotide gradients between the cytosolic bulk and subsarcolemmal space, revealed a strong diffusional hindrance with an apparent diffusion coefficientD = 2.3·10−11cm 2D =C·Δx/S was derived from C = 0.45 cm3/min/g wet weight (Equation 7, Fig.1 D), assuming a subsarcolemmal space width (Δx) of 10−5 cm and a total surface of cells in 1 g of tissue (1012 μm3) S = 3,200 cm2/g wet weight for an average cardiac cell (10 × 20 × 100 μm) with a volume of 20,000 μm3 and a surface of 6,400 μm2./s.2 This value, 5 orders of magnitude lower than values for nucleotide diffusion in the cytosol (52Kinsey S.T. Locke B.R. Penke B. Moerland T.S. NMR Biomed. 1999; 12: 1-7Crossref PubMed Scopus (72) Google Scholar), is in line with the restricted diffusion of molecules previously observed in the structurally crowded submembrane space of living cells (51Kabakov A.Y. Biophys. J. 1998; 75: 2858-2867Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 53Rich T.C. Fagan K.A. Nakata H. Schaack J. Cooper D.M. Karpen J.W. J. Gen. Physiol. 2000; 116: 147-161Crossref PubMed Scopus (233) Google Scholar). Such a diffusional barrier implies virtual confinement of the metabolic sensor within the submembrane zone, impeding direc
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