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J. Biol. Chem., Vol. 277, Issue 27, 24427-24434, July 5, 2002

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Coupling of Cell Energetics with Membrane Metabolic Sensing

INTEGRATIVE SIGNALING THROUGH CREATINE KINASE PHOSPHOTRANSFER DISRUPTED BY M-CK GENE KNOCK-OUT^*

M. Roselle Abraham, Vitaliy A. Selivanov, Denice M. Hodgson, Darko Pucar, Leonid V. Zingman, Be Wieringa, Petras P. Dzeja, Alexey E. Alekseev, and Andre Terzic^§

From the Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology, and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905 and the  Center for Molecular Life Sciences, University Medical Center, University of Nijmegen, Nijmegen 6500, The Netherlands

Received for publication, February 21, 2002, and in revised form, April 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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⁺ (K_ATP) 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 K_ATP channel activity with cell metabolism. Knock-out of the creatine kinase M-CK gene disrupted signal delivery to K_ATP 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Maintenance of cellular homeostasis critically depends on the ability of the cell to adjust diverse energy-dependent processes in response to metabolic challenge (1-3). This requires efficient monitoring of cellular metabolism, secure delivery of information to energetic sensors, and accurate translation of metabolic signals into cellular response (4-10). Advances have been made in resolving the molecular identity and regulatory properties of energetic signal transducers (11, 12), yet mechanisms that integrate and synchronize metabolic sensors with cell metabolism are only partially understood (13-15).

ATP-sensitive K⁺ (K_ATP) channels are membrane metabolic sensors, which act as alarm systems to adjust cell electrical activity and regulate vital functions as diverse as hormone secretion, neurotransmitter release, or cytoprotection (6, 9, 11, 16-20). K_ATP 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 (21-23). While ATP closes K_ATP channels by interacting with Kir6.2, metabolic sensing seems to proceed through interactions of ATP/ADP with nucleotide-binding domains of SUR (9, 16, 24-27). 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 (15, 28-31). However, such independent nucleotide fluctuations within a particular cell compartment (15, 31) would hamper proper recognition of cellular signals rendering K_ATP channels ineffective metabolic sensors.

In response to metabolic alterations, the membrane content of polyphosphoinositides has been implicated in defining the ATP-sensitivity of cardiac K_ATP channels (32). Yet, altered K_ATP 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 (33). Rather, coordination of membrane sensor function with the cellular metabolic status mandates effective transfer of energetic signals between intracellular compartments (2, 15). 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 (3, 5). Emerging evidence suggests that phosphotransfer networks can process metabolic information for delivery to metabolic sensors, thereby serving a critical role in cellular homeostasis (2, 3, 34, 35). In this way, the phosphotransfer enzyme adenylate kinase physically associates with K_ATP channel proteins to facilitate communication of mitochondrial signals and promote channel opening in stress (34). A related signal delivery function has been suggested for the most active phosphotransfer enzyme in the myocardium, creatine kinase, which could control K_ATP channel closure and prevent accidental channel opening (3, 36, 37). Isoforms of creatine kinase are found in distinct intracellular compartments, including membranes where K_ATP channels reside (13, 38). Substrates of creatine kinase regulate nucleotide-dependent K_ATP channel gating and can overcome potassium channel opener-induced channel activation (36, 37, 39). In fact, in opener-primed cardiomyocytes inhibition of creatine kinase reduces the effect of mitochondrial uncoupling on K_ATP channel activity (40). Although an intimate relationship between phosphotransfer enzymes and the channel itself has been suggested (3, 36, 37, 40, 41), 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 K_ATP 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 K_ATP channels and generated a phenotype with increased electrical vulnerability.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Creatine Kinase Knock-out-- 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 (1). 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 the M-CK gene. Homozygous M-CK-knock-out mice were compared with age-matched wild-type controls.

Channel Recording-- Cardiomyocytes, isolated from wild-type and M-CK knock-out mice or guinea pig ventricles (34), were bathed in (mM) KCl, 140; MgCl₂, 1; EGTA, 5; HEPES-KOH, 5 (pH 7.3). Patch electrodes (7-10 M) 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 (36, 37). Channel activity was measured at 60 mV.

Phosphotransfer Scanned by NMR Spectroscopy-- ¹⁸O-assisted ³¹P NMR is based on incorporation of ¹⁸O, provided from ¹⁸O-water, into cellular phosphates proportionally to the rate of enzymatic reactions involved (42, 43).

The 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% of ¹⁸O-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 mM HClO₄ 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 (42, 43) and phosphotransfer fluxes calculated as described (38).

Enzymatic Activity-- 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 (38).

Action Potentials-- 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.

Allosteric Model of Channel Gating-- Nucleotide-dependent K_ATP 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 K_ATP channel complex (16, 22). Binding of ATP to the pore-forming Kir6.2 subunit inhibits channel opening (24, 25), whereas binding of ADP to the regulatory SUR subunit antagonizes ATP-binding to Kir6.2 (6, 26, 44). Distribution of channel species (D_i; i = 0 to 4) with 0-4 ADP bound molecules was as follows,

(Eq. 1)

with the percentage of D_i species expressed as a function of ADP concentration,

(Eq. 2)

where

(Eq. 3)

and k_ADP the dissociation constant of ADP from SUR, independent from ATP binding. Analogously to Equations 1-3, the distribution of channel species (T_i) with 0-4 ATP bound molecules was derived as follows,

(Eq. 4)

with k₀ and k₁ 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 K_ATP channel inhibition in the absence of ADP, at saturating ADP and at below saturating ADP revealed, respectively, the values for k₀, k₁, and k_ADP, with more than one ATP required to close the channel octamer.

Computation of Diffusional Restriction between Cell Compartments-- Diffusional restriction was estimated by integrating membrane ATPase activities and diffusional nucleotide fluxes (Fig. 1C). Membrane ATP consumption (J_ATPase) was simulated as a Michaelis-Menten reaction with the Michaelis constant at 0.05 mM (45). Sarcolemmal ATPase activity (1,800 nmol/min/g wet weight) was derived from total ATPase activity in working hearts measured by ¹⁸O-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 (46). 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,

(Eq. 5)

where J_ATP(x) is ATP flux at distance x. At steady-state, with J_ATP constant, C = DS/x defining,

(Eq. 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. 1D), were defined from the following equation.

(Eq. 7)

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Metabolic Sensing in the Submembrane Compartment-- The defining property of cardiac K_ATP channels as membrane metabolic sensors is their overt inhibition by ATP, which can be antagonized by MgADP (Fig. 1A). K_ATP 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. 1A). The regulatory SUR2A subunit harbors an intrinsic ATPase activity (36, 44) that facilitates conformational transitions imparting low or high ATP sensitivity to the K_ATP channel complex (37). MgADP prolongs the lifetime of the conformation associated with reduced sensitivity to ATP (37). Allosteric modeling, which integrated K_ATP channel stoichiometry and channel-nucleotide interactions (Fig. 1B), demonstrated that on saturating ADP-binding sites (at >100 µM ADP) no further reduction in ATP sensitivity can be achieved (Fig. 1, A and B), in accord with the efficacy of ADP to antagonize ATP-induced channel inhibition (47). For MgADP to open at least 1% of K_ATP channels, required for significant action potential shortening at 6-10 mM cytosolic ATP (47-50), 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 (30, 51), including ATP hydrolysis by the K_ATP channel itself (36, 37, 44), provided that nucleotide mobility between the cytosol and submembrane is limited (31) (Fig. 1C). 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 coefficient D = 2.3·10¹¹ cm²/s.² This value, 5 orders of magnitude lower than values for nucleotide diffusion in the cytosol (52), is in line with the restricted diffusion of molecules previously observed in the structurally crowded submembrane space of living cells (51, 53). Such a diffusional barrier implies virtual confinement of the metabolic sensor within the submembrane zone, impeding direct exposure to cytosolic signals. Thus, integration of K_ATP channel activity with cell metabolism requires efficient mechanisms able to shunt diffusional restrictions for proper delivery of energetic signals.

View larger version (56K): [in this window] [in a new window]   Fig. 1.   Nucleotide-dependent gating of KATP channels. A and B, MgADP antagonizes ATP-induced inhibition of cardiac KATP channels (inset). In excised patches, the ATP sensitivity of KATP channels was defined by an IC50 of 27 ± 5 µM in the absence (open triangles) versus 270 ± 19 µM in the presence (closed circles) of 100 µM ADP (n = 3-9). Relative channel activity (curves in A) constructed based on an allosteric model of nucleotide-dependent KATP channel gating (B) and expressed as a probability for the channel to be in an open state (columns T0-T1). In the absence of ADP, channels adopt the highest sensitivity to ATP (row D0) defined solely by the microscopic dissociation constant k0 = 45 µM (curve 1 in A). At saturating ADP concentrations, KATP channels convert to channel species with the lowest ATP sensitivity (row D4) defined solely by k1 = 450 µM (curve 4 in A). kADP (12.5 µM) was determined at different concentrations of ADP (curve 1, 0; curve 2, 10 µM ADP; curve 3, 50 µM ADP; curve 4, 100 µM ADP; curve 5, 500 µM ADP; curve 6, 1,000 µM ADP). C, membrane ATPases and diffusional restrictions generate nucleotide gradients between cytosol and subsarcolemma, generating lower ATP and higher MgADP at the channel site. D, Relationships between bulk and subsarcolemmal adenine nucleotide levels when nucleotide diffusional fluxes are at steady state (J = J). Subsarcolemmal ATP (ATPsub) and ADP (ADPsub) were calculated from Equation 7 (see "Experimental Procedures"). At bulk ATP (ATPb) between 4 and 10 mM, ATPsub follows ATPb, while ADPsub remains constant due to saturation of ATPase activity. At ATPb = 7 mM, ATPsub = 3 mM, sufficient to activate 1% of KATP channels at saturating ADPb (inset, curves from A expressed as percent of open KATP channels).

Creatine Kinase Phosphotransfer Synchronized with Cellular Energy Turnover Sets Nucleotide-dependent K_ATP Channel Gating-- Creatine kinase molecules are spatially arranged between cellular sites of ATP production and utilization, providing an integrated network for high energy phosphoryl conduction (1, 3, 38, 54-56). Here, net phosphotransfer flux through the creatine kinase system was captured in intact heart using ¹⁸O-assisted ³¹P NMR and was found to be tightly synchronized with cellular ATP turnover (Fig. 2A). Labeling of phosphoryl oxygens in creatine phosphate, which reflects net flux through creatine kinase, paralleled that of -phosphate in ATP, an indicator of total cellular high energy flux (Fig. 2A). On average, the percentage of ¹⁸O in creatine phosphate was comparable with that of -ATP, i.e. 55 ± 4% (n = 5) versus 61 ± 3% (n = 5), respectively (Fig. 2B). Such a vigorous creatine kinase-catalyzed phosphotransfer would rapidly dissipate local nucleotide gradients and could therefore transmit cellular metabolic signals to membrane metabolic sensors. In fact, creatine kinase catalysis was found high in the cardiac membrane fraction, i.e. 6.2 ± 0.6 µmol/min/mg (n = 3), and was sensitive to the conventional creatine kinase inhibitor 2,4-dinitrofluorobenzene (DNFB), which reduced such activity to 0.5 ± 0.1 µmol/min/mg (n = 3). To assess the role of creatine kinase flux in regulating K_ATP channels as metabolic sensors, channel behavior was measured in the absence and presence of creatine kinase phosphotransfer. To maintain a relative integrity of the cellular infrastructure, cardiomyocytes were permeabilized by local and brief application of digitonin to the region of the cell distal from the patched area (Fig. 2C). In such open cell-attached patch configuration, removal of creatine phosphate inactivated creatine kinase phosphotransfer and induced an aberrant sensitivity of K_ATP channels toward ATP (Fig. 2D). Indeed, ATP at 100 µM failed to inhibit K_ATP channels (Fig. 2D), a concentration that keeps channels closed in excised membrane patches (Fig. 2E). The reduced ATP sensitivity indicates that, despite clamped bulk nucleotide concentrations by continuous cell perfusion, local levels of ATP are decreased and ADP increased in an environment of active membrane ATPases. Activation of creatine kinase phosphotranfer, by addition of creatine phosphate, restored the K_ATP channel responsiveness to ATP (Fig. 2D) presumably through scavenging ADP and dissipating the membrane ATPase-induced nucleotide gradient. Creatine phosphate had no significant effect on its own, but secured K_ATP channel closure in open cell-attached patches in the presence of low ATP concentrations, which in inside-out patches produced only partial channel inhibition (Fig. 2, E and F). In the presence of creatine kinase substrates, inhibition of creatine kinase phosphotransfer by DNFB in permeabilized cells uncoupled K_ATP channels from phosphotransfer regulation (Fig. 2F). Coupling was restored by providing purified creatine kinase to bypass the irreversible inhibition of endogenous creatine kinase by DNFB (Fig. 2F). Thus, the creatine phosphate/creatine kinase system is a determinant of the K_ATP channel sensitivity to ATP. In permeabilized cells, in the absence of creatine kinase flux (J_CK = 0), the IC₅₀ for ATP-induced inhibition was 270 ± 2 µM (n = 4; Fig. 2E), close to the value measured in the presence of saturating MgADP in excised patches (Fig. 1A). Active creatine kinase flux (J_CK 0) significantly reduced the IC₅₀ to 7 ± 1 µM (n = 4), even below the sensitivity of the channel toward ATP seen in excised patches (Fig. 2E). Thus, creatine kinase flux can shunt nucleotide gradients between the bulk and subsarcolemmal space and facilitate delivery of metabolic signals that translate into K_ATP channel-dependent sensing of the cellular energetic state.

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Creatine kinase phosphotransfer determines nucleotide-dependent KATP channel gating. A, spectra of 18O-labeled -ATP and CrP captured by 18O-assisted 31P NMR. Incorporation of 18O induces an isotope shift in the 31P spectra of ATP and CrP. 16O, 18O1, 18O2, and 18O3 designate -ATP and CrP phosphoryls containing 0-3 atoms of 18O. -ATP is recorded as a doublet due to homonuclear scalar coupling between - and -phosphates. 18O labeling of CrP and ATP are comparable in magnitude, indicating that creatine kinase is responsible for transfer of the majority of newly synthesized ATP. B, average incorporation of 18O into phosphoryls of CrP reflects creatine kinase-catalyzed phosphotransfer and was ~90% of 18O incorporation into -ATP, which measures total cellular energy turnover (n = 5). C, permeabilization of cardiomyocytes for open cell-attached patch formation. i, transmitted light image of the initial step showing a patch-pipette (box 1) attached to the proximal edge of a cardiac cell and a perfusion pipette (box 2) approaching the distal edge of the same cell. ii-iv, fluorescent images showing flow of a digitonin-containing solution (visualized with rhodamine (ii)) and staining of the nucleus (visualized with propidium iodide (iii)) upon cell permabilization; immediate withdrawal of the perfusion pipette (iv) following open cell-attached patch formation secures channel recording within a fairly intact intracellular architecture. D, KATP channel activity, in an open cell-attached patch, was vigorous in the absence of ATP and was inhibited by 2 mM, but not 0.1 mM ATPb. Yet, 0.1 mM ATPb inhibited channel activity following application of CrP. E, concentration-response curves defining ATP-induced KATP channel inhibition in excised (open circles) and in open cell-attached patches in the absence (open triangles) and presence (closed triangles) of 1 mM CrP. Solid curves were constructed based on the allosteric model of channel regulation (with a recalculated k0 = 10 µM) using the highest ATP sensitivity in the presence of CrP), in conjunction with nucleotide diffusion (J, J), ATPase (JATPase), and creatine kinase (JCK) fluxes. F, by itself CrP had no significant effect on KATP channels. CrP-induced channel inhibition, in the presence of ATP, was irreversibly antagonized by DNFB and partially restored by purified creatine kinase (CK). Current was recorded at 21 °C.

Creatine Kinase Phosphotransfer Dynamics Transduce Metabolic Stress-induced Signals into K_ATP Channel-driven Membrane Electrical Events-- In normoxia, when cardiac K_ATP channels are closed, vigorous incorporation of ¹⁸O atoms into creatine phosphate reflected the high creatine kinase phosphotransfer rate of the myocardium (Fig. 3A). Hypoxia markedly reduced creatine kinase flux, from 279 ± 8 nmol CrP/min/mg of protein (n = 3) to 64 ± 17 nmol CrP/min/mg of protein (n = 3; p < 0.05), indicating a ~75% decrease in creatine kinase phosphotransfer (Fig. 3A). This reduction in creatine kinase phosphotransfer reflects a 4-fold drop, from 36 ± 1 to 9 ± 2 nmol/mg of protein, in creatine phosphate levels following hypoxia. Thus, under hypoxic stress, creatine kinase has a reduced ability to equilibrate nucleotide levels between the cytosol and subsarcolemma. Estimated subsarcolemmal concentrations of ATP ([ATP]_sub) and ADP ([ADP]_sub) were deduced by integrating diffusional restriction (C), bulk concentrations ([ATP]_b, [ADP]_b), and membrane ATPase activity (J_ATPase; see Equation 7) with creatine kinase flux (J_CK).

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Creatine kinase phosphotransfer dynamics translate cellular energetic status into membrane electrical events. A, hypoxia reduces creatine kinase (CK) phosphotransfer rate measured by 31P NMR in 18O-labeled hearts. B and C, simulation of nucleotide-dependent KATP channel gating, based on the allosteric model of channel regulation integrated with nucleotide diffusional fluxes and membrane ATPase activity (JATPase), in the presence of vigorous creatine kinase flux (JATPase = JCK) in normoxia (B) versus reduced (by 75%) creatine kinase flux (JATPase JCK) in hypoxia (C). While in normoxia [ATP]b = [ATP]sub ([ADP]sub = 10 µM), in hypoxia ATPsub<ATPb ([ADP]sub = 3 mM). Solid curves depict ATP-induced KATP channel inhibition at defined ATPsub and ADPsub. D, percent of open KATP channels at different creatine kinase (CK) flux (180, 300, 450, and 900 nmol/min/g wet weight), expressed relative to creatine kinase flux in normoxia (JCK = JATPase = 1,800 nmol/min/g wet weight). Each curve was constructed based on the allosteric model of channel gating, integrated with the model for nucleotide diffusion and ATPase activity (Equation 7 under "Experimental Procedures"), at different creatine kinase flux. E, shortening of action potential duration (APD), in hypoxia recorded by the monophasic action potential electrode. APD was corrected to heart rate using the modified Bazett's formula: APD = APD90/T1/2, where APD is corrected APD; APD90, APD measured at 90% repolarization; and T, cardiac cycle length.

(Eq. 8)

In normoxia, with creatine kinase flux compensating for membrane ATPase activity (J_ATPase = J_CK), nucleotide gradients were dissipated with [ATP]_b = [ATP]_sub and [ADP]_b = [ADP]_sub (Fig. 3B). With equilibrated [ATP]_b and [ATP]_sub, at 6-10 mM, K_ATP channels remained closed as the concentration-response curve for ATP-induced channel inhibition is far below the actual intracellular ATP levels (Fig 3B). In hypoxia, reduced creatine kinase flux (J_ATPase J_CK) unmasked the membrane ATPase-induced drop in ATP ([ATP]_b > [ATP]_sub) and increase in ADP ([ADP]_b < [ADP]_sub) in the subsarcolemmal space (Fig. 3C). In this way, metabolic challenge resulting in altered creatine kinase phosphotransfer could bring subsarcolemmal nucleotide levels to a range that now lies in the steeper portion defining ATP-dependent channel gating, securing effective signal delivery to K_ATP channels (Fig. 3C). A drop of 75% in creatine kinase phosphotransfer (J_CK:4), observed in hypoxia (Fig. 3A) at cytosolic ATP <6 mM, would translate into activation of >1% of K_ATP channels sufficient for significant action potential shortening in a cardiac cell (50), as computed from the allosteric model of nucleotide-dependent channel gating (Fig. 1B), taking into account nucleotide diffusion, membrane ATPase, and creatine kinase fluxes (Fig. 3D). Indeed, cardiac action potential duration was significantly decreased from 230 ± 4 ms prior to 130 ± 7 ms following hypoxic stress (Fig. 3E). Thus, the dynamics of creatine kinase phosphotransfer, governed by the cellular metabolic condition, determine the percentage of open K_ATP channels and provide a mediator translating cellular energetic signals into membrane electrical events.

Knock-out of Creatine Kinase Disrupts Signal Delivery to the Metabolic Sensor-- Cytosolic creatine kinase (M-CK) is the major creatine kinase isoform in the heart (1). Deletion of the M-CK gene blunted creatine kinase phosphotransfer and essentially eliminated creatine kinase activity in the sarcolemma (Fig. 4A). In wild-type cardiomyocytes, K_ATP channel activity was highly sensitive to creatine kinase-mediated channel inhibition, revealed from titration with creatine phosphate (IC₅₀ = 94 ± 5 µM, n = 5; Fig. 4, B and C). This indicates tight integration of creatine kinase-catalyzed energetic signaling with K_ATP channel activity. In contrast, in cells lacking M-CK, K_ATP channels were uncoupled from the cellular energetic infrastructure and were no longer sensitive to creatine phosphate regulation (Fig. 4, B-D). Thus, cells lacking the creatine kinase phosphotransfer system display a defective regulation of nucleotide-gated membrane functions.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Knock-out of M-CK disrupts creatine kinase-dependent control of KATP channel gating. A, spectrophotometric recordings of creatine kinase (CK) activity in sarcolemmal fraction from wild-type (WT) and creatine kinase knock-out (M-CK KO) hearts. Creatine kinase activity was diminished from 2.9 ± 0.2 (n = 3) in wild type to 0.1 ± 0.02 (n = 3) µmol/min/mg of protein in M-CK knock-out. B, concentration-response curves for CrP-induced KATP channel inhibition, at 100 µM ATP, in open cell-attached patches from WT and M-CK knock-out cells. Channel activity expressed relative to that measured in the absence of CrP. C and D, KATP channel recordings in WT (C) and M-CK knock-out (D). While in WT CrP enhanced KATP channel inhibition by 100 µM ATP, in the M-CK knock-out the creatine kinase substrate was virtually deprived of any significant effect. Temperature was 31 °C.

Fidelity in Membrane Metabolic Sensing Lost with Deletion of the M-CK Gene-- Besides creatine kinase, additional phosphotransfer pathways, such as the glycolytic system, have been identified as interrelated components of the cellular energetic network (2, 3, 5). Here, in wild-type cardiomyocytes, inhibition of phosphoryl delivery through the glycolytic system by deoxyglucose did not elicit a K_ATP channel response (Fig. 5A), suggesting privileged control of this metabolic sensor by creatine kinase. In contrast, in cells lacking M-CK, metabolic stress induced with deoxyglucose triggered K_ATP channel opening (Fig. 5, B and C). In fact, aberrant coupling of K_ATP channels with cellular metabolism in creatine kinase knock-out hearts generated a phenotype with electrical instability manifested by premature shortening of action potentials in response to deoxyglucose (Fig. 5, D and E). The rate of stress-induced action potential shortening was significantly faster in creatine kinase knock-out (0.042 ± 0.005 min¹, n = 4) than wild-type (0.016 ± 0.004 min¹, n = 4) hearts (p < 0.05; Fig. 5D). Accordingly, action potential duration, measured at 90% of repolarization, was essentially unchanged (94 ± 4% of control value, n = 4) in wild type, but was reduced, by 39 ± 9% (n = 4), in the M-CK knock-out following a 7-min long application of deoxyglucose (Fig. 5E). Thus, creatine kinase phosphotransfer is required for proper linkage of cellular energetics with membrane excitability.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Electrical instability under stress in M-CK knock-out hearts. A, prolonged (>20 min) metabolic challenge induced by deoxyglucose (DOG), an inhibitor of glycolysis, did not trigger electrical events in WT cardiomyocytes, which remain sensitive to an uncoupler of mitochondrial oxidative phosphorylation, dinitrophenol (DNP). B, M-CK knock-out (M-CK KO) hearts are highly susceptible to DOG, which triggers early opening of KATP channels. C, time course of DOG effect in WT (n = 3; open squares) and M-CK knock-out (n = 3; closed circles). While in WT there was no effect over the 17-min-long DOG application, in M-CK knock-out the time course of DOG-induced KATP channel opening was characterized by a half-maximal activation time of 16.1 ± 0.3 min (slope: 0.8 ± 0.1 min) obtained by the best fit of experimental data with Boltzman's function. In A-C, channel recording was in the open cell-attached mode at 31 °C. D, rates of action potential shortening in WT (closed circles) and M-CK knock-out (open squares) hearts, measured throughout DOG application fitted by linear regressions (solid lines). E, action potential in WT (left) and M-CK knock-out (right) hearts prior to (solid line) and following a 7-min long application of 5 mM DOG (dashed line).

	DISCUSSION

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Metabolic signal transduction governs vital functions that enable cells to respond to metabolic challenges, but how the operation of metabolic sensors is orchestrated to accurately sense the cellular energetic status has remained a long-standing enigma. Using cardiac K_ATP channels as prototypic membrane metabolic sensors, we demonstrate that phosphotransfer enzyme-catalyzed metabolic signal delivery synchronizes channel gating with cell energetics. Genetic disruption of the creatine kinase pathway generated a phenotype with increased electrical vulnerability, underscoring the significance of an intact intracellular phosphotransfer network in integrating metabolic signaling.

The extremely low diffusional flux of nucleotides, estimated here in the subsarcolemmal space, indicates that K_ATP channels are virtually secluded from cellular bulk nucleotide oscillations. Restricted metabolite mobility in the premembrane area could be due to molecular crowding and reduction in the free diffusional space as previously suggested for different cellular compartments (13-15, 53). In fact due to the "fuzzy space" in the submembrane (31), channel gating would be relegated to local fluctuations of nucleotides ("metabolic background noise"), independent of the cellular metabolic status. Instead of random fluctuations in adenine nucleotides, which would distort energetic signaling, we provide direct evidence that the creatine kinase phosphotransfer system controls exchange of nucleotides securing signal processing between the subsarcolemmal space and cytosolic compartment.

Indeed, the dynamics of creatine kinase flux closely followed total ATP turnover, indicating tight coupling between creatine kinase phosphotransfer and the cellular metabolic state (38, 43). Present throughout the cell, creatine kinase reactions form a phosphotransfer relay able to respond to changes in the cellular metabolic state and propagate metabolic waves between cellular compartments (3, 57, 58). Such catalyzed phosphotransfer can deliver metabolic signals at a rate exceeding simple diffusion (38, 58). Accordingly, here, under the normal status of cell metabolism, vigorous creatine kinase phosphotransfer dissipated local nucleotide gradients created by membrane ATPases and diffusional restrictions in the channel environment, keeping K_ATP channels predominantly closed. Under metabolic stress, however, reduced creatine kinase phosphotransfer unmasked nucleotide changes in the channel vicinity alerting K_ATP channels to adjust membrane excitability. Reduced creatine kinase flux under metabolic insult is known to be associated with concomitant up-regulation of adenylate kinase phosphotransfer, which catalyzes the conversion of ATP to ADP at the channel site (34, 57). Such interplay between phosphotransfer pathways effectively amplifies the metabolic signal translating into K_ATP channel opening and ultimately shortening of the cardiac action potential under stress (19, 59). Thus, membrane metabolic sensors respond to the dynamics of cellular phosphotransfer flux, reflecting with high fidelity the energetic status of a cell.

Integration of phosphotransfer with K_ATP channels appears critical in supporting metabolic signaling. Knock-out of the dominant creatine kinase isoform, M-CK, disrupted K_ATP channel regulation by creatine phosphate. This produced a cellular phenotype characterized by increased electrical instability, in line with observations that muscles lacking creatine kinase genes display abnormal contractile response and reduced energetic efficiency (1, 4, 54, 55). Moreover, coupling of creatine kinase with Ca²⁺-ATPases of the sarcoplasmic reticulum, which is essential in securing Ca²⁺ handling and proper kinetics of intracellular Ca²⁺ signals, is compromised following deletion of creatine kinase genes (60). In this regard, creatine kinase can also functionally couple with K_ATP channels through direct creatine kinase-dependent regulation of the ATPase catalytic cycle harbored within SUR, the channel regulatory subunit (36, 37). The ATP hydrolysis cycle at SUR drives conformational transitions associated with distinct outcomes on channel behavior, with creatine kinase promoting disengagement of the MgADP-bound state and K_ATP channel closure (37). Thus, nucleotide exchange between cellular phosphotransfer catalyzed by creatine kinase and membrane ATPases, including the channel's own ATPase, provides a mechanistic basis for coupling cell energetics with metabolic signal transduction.

Along with creatine kinase, distinct phosphotransfer systems can also efficiently communicate energetic signals to metabolic signal transducers and regulate ATP-sensitive cellular components (3, 34, 61). The interrelationship between intracellular energetic pathways is revealed upon deletion of the M-CK gene, which translated into redistribution of metabolic flux through glycolytic enzymes (56, 62). In accord with the adaptive potential of phosphotransfer pathways (1, 5, 42), such energetic remodeling was sufficient to maintain apparently normal K_ATP channel gating in the absence of metabolic challenge in the M-CK knock-out heart. However, knock-out of M-CK did produce increased electrical vulnerability manifested by premature action potential shortening in hearts stressed by inhibition of glycolytic enzymes. Thus, an intact phosphotransfer network is a prerequisite for optimal decoding of energetic signals securing adequate function of a metabolic sensor. The significance of these findings is underscored in human disease where compromised creatine kinase phosphotransfer has been associated with cardiac electrical instability (63) and extrapyramidal movement disorders (64).

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL-64822 and HL-07111 and by the American Heart Association, the Guidant Foundation, the Marriott Foundation, the Miami Heart Research Institute, the Bruce and Ruth Rappaport Program in Vascular Biology and Gene Delivery, the American Physicians Fellowship for Medicine in Israel, and the CR Program at the Mayo Clinic.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Division of Cardiovascular Diseases, Depts. of Medicine, Molecular Pharmacology, and Experimental Therapeutics, Mayo Clinic, Guggenheim 7, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-2747; Fax: 507-284-9111; E-mail: terzic.andre@mayo.edu.

Published, JBC Papers in Press, April 19, 2002, DOI 10.1074/jbc.M201777200

² D = C·x/S was derived from C = 0.45 cm³/min/g wet weight (Equation 7, Fig. 1D), assuming a subsarcolemmal space width (x) of 10⁵ 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.

	ABBREVIATIONS

The abbreviations used are: M-CK, M-creatine kinase; DNFB, 2,4-dinitrofluorobenzene; CrP, creatine phosphate; WT, wild-type; DOG, deoxyglucose.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES