Article pubs.acs.org/biochemistry
Modulation of the Na,K-ATPase by Magnesium Ions Hans-Jürgen Apell,* Tanja Hitzler, and Grischa Schreiber Department of Biology, University of Konstanz, 78464 Konstanz, Germany S Supporting Information *
ABSTRACT: Since the beginning of investigations of the Na,K-ATPase, it has been well-known that Mg2+ is an essential cofactor for activation of enzymatic ATP hydrolysis without being transported through the cell membrane. Moreover, experimental evidence has been collected through the years that shows that Mg2+ ions have a regulatory effect on ion transport by interacting with the cytoplasmic side of the ion pump. Our experiments allowed us to reveal the underlying mechanism. Mg2+ is able to bind to a site outside the membrane domain of the protein’s α subunit, close to the entrance of the access channel to the ion-binding sites, thus modifying the local concentration of the ions in the electrolyte, of which Na+, K+, and H+ are of physiological interest. The decrease in the concentration of these cations can be explained by electrostatic interaction and estimated by the Debye−Hückel theory. This effect provokes the observed apparent reduction of the binding affinity of the binding sites of the Na,K-ATPase in the presence of various Mg2+ concentrations. The presence of the bound Mg2+, however, does not affect the reaction kinetics of the transport function of the ion pump. Therefore, stopped-flow experiments could be performed to gain the first insight into the Na+ binding kinetics on the cytoplasmic side by Mg2+ concentration jump experiments.
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of enzyme phosphorylation and dephosphorylation, respectively.17,18 This will not necessarily occur under physiological conditions, but the ionic substrates with physiologically high extracellular Na+ and high intracellular K+ concentrations are well-known to have detectable inhibitory effects on the pump rate that is produced by back binding of the respective ions.19−21 It is also well-known that the proton concentration can modulate pump function and activity, although cells have strict pH control mechanisms and high buffer capacity that secure only minor fluctuations of its value. Another ion needed by the Na,K-ATPase is Mg2+. This cation is not transported but essential for enzymatic activity. As in the case of all other P-type ATPase, ATP binds as a Mg2+ complex to the ATP-binding site at the cytoplasmic N domain of the ion pump, and only the presence of Mg2+ permits phosphorylation of the specific aspartate at the P domain.22 It is known that in cells free Mg2+ concentrations of ≤2 mM are found. Therefore, additional and direct interactions of Mg2+ with the Na,K-ATPase were also assumed and studied. The first effect reported was that Mg2+ decreases the apparent affinity for K+ binding in the E1 conformation, but it had no effect on the rate constant of the subsequent conformation transition (E1K → E2K).23 Analogously, Mg2+ also reduces the apparent binding affinity for Na+ in the E1 conformation. The equilibrium dissociation constant, Kd, of 0.7 mM in the absence of Mg2+ increased to 8 mM at 10 mM Mg2+,24 and the relation between Mg2+ and Kd(Na+) is almost
iving cells fuel numerous metabolic functions that involve the cell membrane by using the electrochemical potential gradients of Na+ and K+ ions across this membrane. To counteract the depletion of these gradients, the Na,K-ATPase is located in the membrane and extracts three Na+ ions from the cytoplasm and takes up two K+ ions upon expenditure of the free energy released by the hydrolysis of one ATP molecule. Because of its crucial role in cell survival, the structure− function relation of the Na,K-ATPase has been studied now for more than 50 years.1−3 This active ion transporter is a so-called P-type ATPase,4 and its reaction scheme is represented by the well-accepted Post−Albers cycle.5,6 Because metabolic demands may vary widely, short- and long-term regulation of pump function and capacity is needed, and for this purpose, a variety of mechanisms have been detected. Regulation is controlled by expression and isoform selection of the pump.7 In several tissues, a third regulatory subunit that is a member of the FXYD family was identified.8,9 Its effect on pump activity again is changed by their phosphorylation and/or dephosphorylation.10 Furthermore, well-known is the regulation by cardiotonic steroids,11 of which ouabain is the most famous. These compounds are not only administered as therapeutical drugs but also present as endogenous compounds that act as signaling compounds.12,13 In addition, Na,K-ATPase pump activity is also modulated by the membrane potential 14,15 in a physiologically relevant manner.16 The transport rate is, however, affected not only by regulatory interactions but also by the substrates involved in the function of the Na,K-ATPase. Therefore, it is known that elevated concentrations of ADP and inorganic phosphate reduce the turnover rate by generation of considerable reversal © 2017 American Chemical Society
Received: December 9, 2016 Revised: January 13, 2017 Published: January 26, 2017 1005
DOI: 10.1021/acs.biochem.6b01243 Biochemistry 2017, 56, 1005−1016
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10 μg/mL membrane fragments containing Na,K-ATPase were added to the cuvette and equilibrated until a steady fluorescence signal, F0, was obtained. To allow a comparison between different titration experiments, relative fluorescence changes, ΔF/F0 = (F − F0)/F0, were calculated with respect to F0. Specific fluorescence levels could be assigned to defined states in the pump cycle of the Na,K-ATPase.24 Unless mentioned otherwise, all experiments were performed at 20 ± 0.5 °C. The steady-state fluorescence levels obtained upon addition of substrates allow the discrimination between differently occupied binding sites of the enzyme.34 Lipid vesicles used in control experiments were produced from dioleoylphosphatidylcholine (PC 18:1) by dialysis as described previously.35 The concentration dependencies were fitted with a single binding isotherm or the sum of two binding isotherms c c Fnorm(c) = ΔFn,1 + ΔFn,2 K m,1 + c K m,2 + c (1)
linear.25 Mg2+ can reverse Na+ binding, and an antagonistic effect between both ions was observed.26,27 It has been proposed that cytoplasmic loop L67 of the pump’s α subunit that contains three negatively charged amino acids (E818, E821, and D823) may form a binding site for divalent cations that may be occupied by Mg2+ and protects against dissociation of the M5/M6 fragment from the chymotrypsin-digested enzyme.28 The Mg2+ bound to the L67 loop that contributes to the entrance port to the ion-binding sites may be the origin of the observed effects. Recently, the first structures in the E1 conformation with three Na+ ions bound became available.29,30 In one structure, a Na+ is located in the cytoplasmic region30 but no Mg2+ could be found associated with the L67 loop. Scrutinizing the experimental conditions under which the protein crystals were performed revealed that the experiments were performed in a Mg2+-depleted solution. From Na,K-ATPase crystallized in the E2 conformation with the inhibitor ouabain bound, it can be seen that a Mg2+ ion is located in the ion-binding sites.31 This state stands in contrast to the situation in the E1 conformation where no electrogenic binding of Mg2+ or Ca2+ to the ionbinding sites could be observed under any condition.26 A systematic investigation has been performed to detect in a comprehensive manner the mutual effects of Mg2+ and ions that bind to the Na,K-ATPase in its E1 conformation, to uncover the underlying mechanism, and to test how this may be used to elucidate details about the Na+ binding kinetics of the E1 conformation.
where Fnorm is the normalized fluorescence of the experiment, c is the ion concentration, Km,1 and Km,2 are dissociation constants, and ΔFn,1 and ΔFn,2 are the respective normalized maximal fluorescence contributions. In the case of pH titrations, the ion concentration was replaced by 10−pH. In the case of pH titrations, it turned out that cooperative binding of H+ takes place and the concentration pH dependence, therefore, had to be fitted the sum of two Hill functions 10−pH × nH,1 + 10−pH × nH,1 + 10−pK1nH,1 10−pH × nH,2 ΔF2 −pH × n H,2 10 + 10−pK 2nH,2
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Fnorm(pH) = ΔF1
MATERIALS AND METHODS Materials. The fluorescent styryl dye RH421 was obtained from Molecular Probes (Eugene, OR) and added from a 200 μM stock solution in ethanol to give a final concentration of 200 nM. ATP (disodium salt, special quality) was purchased from Roche Life Science. Dioleoylphosphatidylcholine was obtained from Avanti Polar Lipids (Alabaster, AL). All other reagents were purchased from Merck or Sigma-Aldrich at the highest quality available. Na,K-ATPase was prepared from the outer medulla of rabbit kidneys using procedure C of Jørgensen.32 The specific ATPase activity was measured by the pyruvate kinase/lactate dehydrogenase assay, and the protein concentration was determined by the Lowry method, using bovine serum albumin as a standard. The specific activity of the used preparations was in the range from 1700 to 2200 μmol of Pi mg−1 h−1 at 37 °C. For experiments at pH 8.0, the enzyme activity was determined by the malachite green assay.33 Free Mg2+ concentrations were calculated from the total Mg2+ concentration and the buffer composition using the program winmaxc32 (C. Patton, Stanford University, Stanford, CA). Fluorescence measurements with a low time resolution were performed with a homemade setup as described previously.34 In short, the thermostated cell holder was equipped with a magnetic stirrer. RH421 was excited with a HeNe laser working at 594 nm. The emitted light was collected by a photomultiplier (model R2066, Hamamatsu Photonics). An interference filter with transmission at 663 ± 18 nm selected the emitted light of the styryl dye before it entered the photomultiplier. Equilibrium titration experiments were performed in standard buffer containing 25 mM histidine with 1 mM EDTA (pH ≤7.2) or 25 mM imidazole with 1 mM EDTA (pH >7.2) and the indicated concentrations of MgCl2, NaCl, and KCl at the indicated pH. Unless otherwise noted, 200 nM RH421 and 8−
(2)
Fluorescence experiments with a high time resolution were performed in a homemade setup as described previously.36 The light source and fluorescence detection system were identical to that used in the experiments with a low time resolution. The thermostated stopped-flow machine had a 60 μL cuvette with a 2 cm pathway. Approximately 100−200 μL was exchanged to start the measurement, and the dead time was approximately 3−5 ms. The temperature was set to 10 °C, at which the signalto-noise ratio was maximal and the detected protein-dependent reaction steps could be detected reliably. With one filling of both supply syringes, we could execute 8−10 identical experiments that were subsequently averaged to obtain the fluorescence signal that was analyzed. The standard buffer contained 25 mM histidine and 0.1 mM EDTA (pH 7.2), unless described otherwise. One syringe was filled with standard buffer, 300 nM RH421, 20−30 μg/mL protein in membrane fragments, and the desired concentration of NaCl. The second syringe contained the identical standard buffer with the same NaCl concentration and a MgCl2 concentration appropriate for the experiment, 20 mM unless described otherwise. As a control experiment, the second syringe was also filled with standard buffer, 5 mM MgCl2, and 200 μM Na2ATP. The experiments were performed after thermal equilibration. The fluorescence data were collected with a sample rate of 100 kHz and stored on a computer for further analysis.
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RESULTS To find indications of the mechanisms of interaction of Mg2+ with the Na,K-ATPase besides that in the nucleotide-binding site, titration experiments were performed in which the occupation of the binding sites for the ions to be transported 1006
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Biochemistry inside the α subunit was investigated. This is conveniently possible because of the use of the electrochromic styryl dye RH421 whose fluorescence is affected by the local electric field generated by the number of ions bound.37,38 Mg2+ Titrations. When the interaction of divalent cations with the Na,K-ATPase was studied in the E1 conformation ∼15 years ago, it was found that, in the absence of other cations, addition of 5 mM Mg2+ produced a significant increase in RH421 fluorescence, addition of 5 mM Ca2+ produced a minor increase, and addition of Ba2+ and Sr2+ caused a fluorescence decrease. 26 To reveal the underlying mechanism, Mg 2+ titrations were performed up to 40 mM when no Na+ or K+ was present. Because it is known that in the absence of transported cations the ion-binding sites are occupied by H+ in a pH-dependent manner,39 initial experiments were performed at pH 7.2 and 6.0 (Figure 1). At pH 7.2, an initial increase in
Because no one ever reported electrogenic binding of Mg2+ ions, the fluorescence decrease at higher Mg2+ concentrations has to be produced by a different mechanism. A systematic study with related styryl dyes38 suggests that unspecific binding of Mg2+ to the polar headgroups of the lipid molecules may affect the fluorescence of the membrane-inserted fluorescence dye. To test this hypothesis, experiments with pure lipid vesicles were performed. The vesicles were made from dioleoylphosphatidylcholine, a zwitterionic lipid. Although it is known that the membrane fragments have a complex lipid composition with a considerable fraction of negatively charged phosphatidylserine lipid on the cytoplasmic side, in principle, a Mg2+-induced RH421 fluorescence decrease could be demonstrated (Figure 1). When the Mg2+ concentration dependence was approximated by a binding isotherm, the Km values were 132 mM (pH 7.2) and >1 M (pH 6.0). To visualize the influence of the unspecific effect of Mg2+, the differences in the respective fluorescence signals (membranes minus vesicles) were included in Figure 1 (empty symbols, dashed lines). In these difference signals, the Km value of the rising phase was the same within the error range, while the falling phase in the higher concentration range was significantly reduced (pH 7.2) or disappeared (pH 6.0). Supplemental Figure 1 provides information about the variations in the Mg2+ concentration dependence between different enzyme preparations. These findings indicate that the effect of higher Mg2+ concentrations on the RH421 fluorescence may be neglected as being artificial with respect to the properties of the Na,K-ATPase. In a second series of control experiments, the Mg2+ dependence of the enzyme activity was compared with the RH421 fluorescence. These experiments were performed at pH 7.2 and 8.0. The measurement of the enzyme activity comprises the whole pump cycle and is controlled by the rate-limiting reaction step(s) of the pump cycle. During the Mg2+ titration experiments that were performed in the absence of ATP, the ion pumps are confined to the E1 conformation in which the ion-binding sites are accessible from the cytoplasmic side of the membrane. Each data point shown in Figure 2 is the average of at least three independent experiments. At pH 7.2, the concentration dependence of both types of experiments overlaps. At pH 8.0, the enzyme activity is also reduced at Mg2+ concentrations above the maximum at ∼3 mM but not as explicitly as the RH421 fluorescence. This indicates that enhanced Mg2+ concentrations also affect the rate-limiting reaction step of the pump cycle, which has been found to be the conformational transition [(Na3)E1P → P-E2Na3] under the chosen saturating concentrations of substrates.24 This step is not part of those reaction steps that contribute to the RH421 experiments. Therefore, it may be stated that free Mg2+ has an inhibitory effect on the E1−E2 conformational transition of Na,K-ATPase or on the E2−E1 transition, as has been reported previously.23 The experiments were performed with Na2ATP; however, because the equilibrium dissociation constant for MgATP was on the order of 2 × 10−5 M40 and the ATP concentration was 150 μM, at Mg2+ concentrations above 500 μM the supply of MgATP for the enzyme was not limiting and the increase in enzyme activity had to be generated by an interaction of Mg2+ with the Na,K-ATPase different from that of MgATP. These experiments indicate that only in the Mg 2+ concentration range of 0−10 mM can the RH421 methods be used to obtain unambiguous information about the interaction of this ion with the Na,K-ATPase.
Figure 1. Mg2+ titrations of the Na,K-ATPase in isolated open membrane fragments monitored by RH421 at pH 7.2 and 6.0. At pH 7.2 (●), a biphasic behavior was found with a maximal fluorescence increase at ∼5 mM Mg2+. At pH 6.0, a biphasic behavior is hardly indicated (filled gray circles). The experiments were repeated with pure lipid vesicles under otherwise identical conditions (black and gray squares). The fluorescence difference (Fmembranes − Fvesicles) has been amended (empty black and gray circles). The lines drawn through the data represent fits with one binding isotherm or the sum of two binding isotherms (see the text).
the fluorescence amplitude up to 30% at 5 mM Mg2+ was followed by an almost linear decrease. The concentration dependence can be fitted by the sum of two binding isotherms (eq 1) with a Km,1 of 1.6 ± 0.2 mM for the rise (and a Km,2 of 80 ± 23 mM for the falling phase). At pH 6.0, the fluorescence increase was smaller (∼15%), reached its maximum in the range of 20−30 mM Mg2+ with a Km,1 of 8.1 ± 1.4 mM, and is followed by a moderate decrease at higher concentrations (data not shown). An increase in RH421 fluorescence under the chosen experimental condition indicates a displacement of positive charge from the ion-binding sites inside the Na,KATPase, which can be explained by a removal of protons.34 Accordingly, it may be expected that at a lower pH, where the binding equilibrium is shifted to a higher and stronger occupation of the ion sites by protons, higher Mg 2+ concentrations are needed to reverse H+ binding and do so with less efficiency in the concentration range covered by the experiments. 1007
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Figure 2. Comparison of enzyme activity and Mg2+ titrations in the E1 conformation by RH421 experiments at (A) pH 7.2 and (B) pH 8.0. Scales were chosen to visualize the degree of conformity of both types of experiments, measurement of the Na,K-ATPase turnover (black symbols), and local electric-field-dependent fluorescence level (gray symbols). The lines drawn through the data are fits with the sum of two binding isotherms (eq 1). For the rising phase, the Km values were 2.6 ± 0.9 and 2.8 ± 0.9 mM for the enzyme activity and RH421 fluorescence, respectively, at pH 7.2 and 1.2 ± 0.8 and 3.0 ± 1 mM for the enzyme activity and RH421 fluorescence, respectively, at pH 8. Error bars were omitted for the sake of clarity.
Figure 3. Mg2+ titrations at different pHs. (A) In the pH range of 6.0− 7.2, the experiments were performed in 25 mM histidine and 1 mM EDTA at the indicated pH. At pH 8, the buffer consisted of 25 mM imidazole and 0.1 mM EDTA. All error bars were omitted for the sake of clarity. The concentration dependencies were fitted with the sum of two binding isotherms (eq 1). Error bars were omitted for the sake of clarity. (B) As explained in the text, the fluorescence increase at low Mg2+ concentrations is considered to be specific for the Na,K-ATPase, and the respective Km,1 value of the fit is plotted vs pH. The concentration dependence shows that the binding affinity at high pH is ∼3 times larger than at low pH.
Mg2+/H+ Competition. When Mg2+ binds to the proposed specific site (different from the MgATP site at the N domain), the binding kinetics may be affected by pH. To investigate such possibly mutual interference, two series of experiments have been performed, Mg2+ binding at different pHs and pH titrations at various Mg2+ concentrations. In both cases, the impact on RH421 fluorescence has been recorded. In Figure 3, an overview of the Mg2+ tritrations at pH values between 6.0 and 8.0 is shown. The data taken at pH 8.0 are the same as those shown in Figure 2. They were measured with a membrane preparation different from that of the other data sets presented in Figure 3, and imidazole buffer was used instead of histidine. However, as Supplemental Figure 2 demonstrates, differences between preparations should not lead to significant changes. At all pH values, a biphasic behavior of the Mg2+-induced fluorescence changes was found. The slope of the initial rising phase became steeper with an increase in pH, as did the slope of the falling phase at high Mg2+ concentrations (Figure 3A). As discussed above, the initial rise can be assigned to a Na,K-ATPase specific process. The almost perfect fit of the concentration-dependent fluorescence changes by eq 1 indicates that the fluorescence changes were caused by binding of Mg2+ to pH-dependent sites. The fluorescence increase upon Mg2+ binding indicates a reduction
in the positive charge in the binding sites for the transported ions, and an analysis of the pH dependence of the respective Km,1 values reveals that binding of Mg2+ is affected by competing H+ binding at this site (Figure 3B). In the presence of high concentrations of H+ in the buffer, the Km,1 was 6.4 ± 0.5 mM, and at low concentrations, it was 1.7 ± 0.4 mM. The pH dependence of Km,1 has been fitted with the Hill equation that resulted in a pK of 6.7 ± 0.1 and a Hill coefficient, nH, of 3.5 ± 1.5. This number indicates (cooperative) binding of multiple H+ ions that are competing with Mg2+ binding. Conversely, it may be expected that Mg2+ affects the interaction of H+ with the Na,K-ATPase. It has been shown that protons bind as congeners of K+ to the ion-binding sites.34,39,41,42 Therefore, pH titration experiments were performed in the absence and presence of ≤5 mM Mg2+. These are concentrations at which the unspecific binding of Mg2+ to the membrane still does not play a significant role (Figure 1). All experiments were started in a buffer containing 25 mM histidine and 1 mM EDTA (pH 7.2), without other cations. The resulting fluorescence was used as a reference level for normalization. The desired amount of MgCl2 was added, and then pH titrations were performed. All experiments were performed in at least triplicate, and with different enzyme 1008
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Biochemistry preparations. In Figure 4, one set of experiments is shown that was measured with the same enzyme preparation. In the
Figure 5. Mg2+ concentration dependence of the (A) pK values and (B) fluorescence amplitudes ΔF of the pH titration experiments (Figure 4). The data points are connected by regression lines to visualize the trend. Both pK1 and ΔF1 of the fits to the low-pH range were independent of Mg2+. The initial fluorescence increase upon the addition of MgCl2 to the electrolyte, ΔF+Mg, has been included in panel B for comparison (○, dashed line). The finding that ΔF+Mg is smaller than the related ΔF2 can be explained by the fact that the fluorescence increase is not at its saturation level at pH 7.2.
Figure 4. pH titrations in the absence and presence of various Mg2+ concentrations. All error bars were omitted for the sake of clarity. The concentration dependencies were fitted with the sum of two Hill functions (eq 2) in the Mg2+-containing experiments; at 0 M Mg2+, a single isotherm was sufficient. The fluorescence increase (with a decrease in pH) in the lower-pH range was characterized by pK1, which was 5.6 ± 0.1 independent of the Mg2+ concentration. The fitted pK2 of the fluorescence changes in the higher-pH range varied with Mg2+ concentration. Error bars were omitted for the sake of clarity.
remaining Mg2+-dependent fluorescence signal can be fitted with a single Hill function, and the resulting fit parameters, ΔF, pK, and nH, do not significantly differ from those shown in Figure 5. The fluorescence levels in the higher-pH range exhibit Mg2+dependent modulations. The pK values decrease linearly from 7.4 ± 0.2 (0.5 mM Mg2+) to 6.6 ± 0.1 (5 mM Mg2+), and the modulus of the maximal fluorescence decrease grows from 0.11 ± 0.05 (0.5 mM Mg2+) to 0.22 ± 0.02 (5 mM Mg2+). The Mg2+ concentration dependence can be fitted with a single binding isotherm with a Km of 0.9 ± 0.3 mM and a ΔF2,max of 0.23 ± 0.03. This value is significantly smaller than the Km of 1.7 mM for the Mg2+ titration at pH 7.2, obtained from the dashed line in Figure 5. These results are compatible with the concept that (1) at higher Mg2+ concentrations higher H+ concentrations are needed to produce a half-maximal fluorescence decrease and that (2) larger fluorescence decreases are obtained because more protons can rebind. Mg2+/Na+ and Mg2+/K+ Competition. The capability of Mg2+ to displace Na+ ions from their binding sites in the Na,KATPase was reported 15 years ago.26 Therefore, the mutual interference was investigated and analyzed in more detail in the framework of this study. RH421 experiments that examined the Mg2+ concentration dependence of the fluorescence signal as shown in Figure 3 were repeated at pH 6.9 and in the absence and presence of various Na+ concentrations of ≤50 mM. The results obtained from one set of experiments are shown in Figure 6. All measurements were started in buffer containing 25 mM histidine and 1 mM EDTA (pH 6.9), without other cations. After addition of NaCl to obtain the desired Na+ concentration, MgCl2 was added in aliquots up to a concentration of 80 mM. The apparent half-binding concentration for Na+ in the absence of other cations is on the order of 0.7 mM. Therefore, after addition of ≥7 mM Na+, the Na+binding sites were almost completely occupied. This is reflected in the decrease in the RH421 fluorescence to a level of approximately −0.25 at 0 M Mg2+. At Na+ concentrations of ≤14 mM, again a biphasic fluorescence change was detected in the Mg2+ concentration range covered by the experiments. The higher the Na+ concentration, the smaller the maximal
absence of Mg2+, the pH titration led to a fluorescence increase in below pH 6.5. The concentration dependence could be fitted with a single Hill function and a pK of 5.6 ± 0.1; the Hill coefficient was 1.5 ± 0.2. This behavior is obviously unrelated to Mg2+. When Mg2+ was added at pH 7.2, a concentrationdependent fluorescence increase occurred as presented in Figure 3, which may be explained by the displacement of H+ from the ion-binding sites inside the α subunit. The subsequent pH titration led in a first phase to a fluorescence decrease that can be explained by H+ rebinding in the transport sites of the Na,K-ATPase. Below pH 6.5 (and depending on the Mg2+ concentration), the fluorescence increased again as has been observed in the absence of Mg2+. At Mg2+ concentrations above 0.5 mM, the falling and rising phases partly overlapped. The pH-dependent fluorescence amplitudes could be fitted with the sum of two Hill functions (eq 2). In the fitting routine, the concentration dependence in the low-pH range was parametrized by ΔF1, pK1, and nH,1; in the high-pH range, the parameters were ΔF2, pK2, and nH,2, respectively. To reduce the number of fitting parameters, the Hill coefficient, nH,1, was fixed at its mean value of 1.5. In the high-pH range, nH,2 could be confidently determined only at ≥1 mM Mg2+. It decreased from 2.5 ± 0.3 at 1 mM Mg2+ to 1.3 ± 0.1 at 5 mM Mg2+. The Mg2+ dependence of the other fit parameters is shown in Figure 5. The first important result is that the pH-induced fluorescence increase in the low-pH range is completely independent of the Mg2+ concentration. Both fit parameters of the corresponding Hill function, pK1 and ΔF1, remained unaltered for all Mg2+ concentrations. The fluorescence signal of this process may be assumed to overlay the Mg2+-dependent fluorescence changes that control the high-pH range. In Supplemental Figure 2, the fitted fluorescence of the experiment at 0 M Mg2+ has been subtracted from the total fluorescence signal and the remaining fluorescence fraction confirms this idea. The fit of the 1009
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Figure 7. pH dependence of Na+ binding affinity in the absence and presence of 25 mM Mg2+. From Na+ titration experiments, the apparent half-saturating Na+ concentrations, Km(Na+), have been determined and are plotted vs the solution pH. The fit through the data at 0 M Mg2+ () has been shifted upward to guide the eye in the case of the Km(Na+) in the presence of 25 mM Mg2+. It leads to the suggestion that a similar effect of the H+ occurs in the presence of Mg2+. In the presence of Mg2+ in the high-pH range, an increase in Km(Na+) can be seen.
Figure 6. Mg2+ titrations in the presence of the indicated Na+ concentrations. The fluorescence was normalized to the steady-state level before the addition of Na+ and Mg2+. The concentration dependencies were fitted with one binding isotherm (20 and 50 mM) or the sum of two binding isotherms (eq 1). All data were obtained from the same membrane preparation.
fluorescence increase that could be induced by Mg2+. The experimental data could be fitted satisfactorily with one Hill function or the sum of two Hill functions. At 7 mM Na+, the half-saturating Mg2+ concentration was 6.9 ± 1.4 mM, and at 50 mM Na + , it was 46 ± 12 mM. The higher the Na + concentration, the more Mg2+ is needed and the less effectively Mg2+ is able to dislocate Na+ ions from their binding sites within the membrane domain of the α subunit of the Na,KATPase. Na+ titrations were performed in the absence and presence of 25 mM Mg2+ between pH 8.0 and 5.9. The buffer contained 25 mM imidazole and 0.5 mM EDTA, and the pH was adjusted with HCl. The RH421 fluorescence was recorded while increasing concentrations of NaCl were added. The corresponding fluorescence levels indicate the occupation of the third binding site by Na+.24 The Na+ dependence of the fluorescence decrease (not shown) could be fitted by a Hill function, and the half-saturating Na+ concentration, Km(Na+), is plotted against the corresponding pH in Figure 7. In the absence of Mg2+, the apparent affinity of Na+ binding is diminished by an increased H+ concentration. In the low-pH range, the same effect has been observed also in the presence of 25 mM Mg2+. At pH ≥7.5, however, the Na+ binding affinity is also reduced, indicating that the reduced H+ concentrations strengthen Mg2+ binding and, as a consequence, apparently also strengthen the Na+ dislocating effect. Mg2+ also can displace K+ from their binding sites. RH421 experiments that aimed to examine the Mg2+ concentration dependence of the fluorescence signal as shown in Figure 3 were repeated at pH 7.2 in the absence and presence of 0.1 and 20 mM K+. Because in the E1 conformation of the Na,KATPase in the absence of other cations and at pH 7 the halfsaturating K+ concentration is on the order of 30 μM,39 at the chosen K+ concentrations 75 and ∼100%, respectively, of the binding sites were occupied before the Mg2+ titrations started. With respect to the experiments with Na+ present in the binding sites, additions of Mg2+ provoked a fluorescence increase (cf. Supplemental Figure 3) in the concentration range of 0−10 mM. As in the case of H+ and Na+, this behavior may be assigned to a displacement of K+ from its binding sites.
Mg2+ Concentration Jump Experiments. Using stoppedflow experiments, the Mg2+-induced fluorescence increase could be studied in a time-resolved manner to obtain additional information about the underlying processes. The experiments were performed at 10 °C to decelerate the reaction kinetics and obtain reliably resolvable signals above the dead time of the equipment of 3−5 ms. Na,K-ATPase equilibrated in buffers of 25 mM histidine at pH 7.2 with various concentrations of NaCl was mixed in a 1:1 ratio with buffer of the same Na+ concentration with 20 mM MgCl2, and the thus triggered RH421 fluorescence increase, as predicted from Figure 1, was recorded and analyzed. In Figure 8A, two fluorescence traces obtained in the presence of 1 and 3 mM Na+ are shown. The traces are the means of eight identical experiments each. The time course in these (and all other) experiments has been fitted by the sum of two exponentials F(t ) = F0 − F(τ1) × exp( −t /τ1) − F(τ2) × exp(−t /τ2) (3)
where F(τ1) and F(τ2) are the maximal fluorescence changes of the fast and slow components with time constants τ1 and τ2 respectively. F0 is the steady-state fluorescence at the end of the experiment. For the analysis of the reaction kinetics, the time constants and fluorescence changes were plotted versus the respective Na+ concentrations (Figure 8B,C). It was found that the faster process with a time constant τ1 of 4.1 ± 0.2 ms was not significantly dependent on Na+ concentration. The fluorescence amplitude representing this process, F(τ1), reveals also no Na+ dependence; therefore, it seems safe to claim that this process represents an interaction of Mg2+ with the membrane fragments, independent of Na+ ions. This proposal is supported by the fact that F(τ1) is the same even at a subsaturating Na+ concentration of 0.5 mM. The slower process is linearly decelerated with the Na+ concentration; such behavior is in disagreement with a Na+ binding and/or release reaction kinetics, which would require a decrease in τ2. The fluorescence amplitude change, F(τ2), shows a complex concentration dependence. From 0.5 to 6 mM Na+, the fluorescence change increases. The increase indicates that the 1010
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sample had been mixed with MgCl2-containing buffer, the Mg2+ concentration was 10 mM. Stopped-flow experiments were performed between 8.7 and 20.3 °C, and the time course of the RH421 fluorescence signals, obtained as averages of eight identical experiments, was fitted with eq 3. The Arrhenius plot for both time constants is shown in Figure 9. (One data point
Figure 9. Arrhenius plot of the rate constants obtained from the Mg2+ concentration jump-induced fluorescence changes. The Na+ concentration was 10 mM; the final Mg2+ concentration was 10 mM, and the temperature ranged from 8.7 to 20.3 °C. The time course of the RH421 fluorescence has been fitted with eq 3, and the reciprocal of both time constants is plotted vs the reciprocal of the absolute temperature, 1/T. Regression lines through the data were used to determine the activation energies of both reaction components: Ea(τ1) = 22.2 kJ/mol, and Ea(τ2) = 63.4 kJ/mol. Error bars were omitted for the sake of clarity.
Figure 8. Mg2+ concentration jump experiments were performed using the stopped-flow technique. When the Mg2+ concentration was increased from 0 to 10 mM, the RH421 fluorescence increased as expected from Figure 1. (A) Time-resolved fluorescence signal of experiments performed in buffer with 1 or 3 mM NaCl. The traces are the average of eight single experiments. The time course was fitted with the function of eq 3, which is added as a dashed line. (B) The time constants of the fast (τ1) and slow (τ2) components were determined and plotted vs Na+ concentration. While the fast process was independent of Na+ concentration, in the slow process the time constant increased with Na+ by a factor of ∼2 between 0.5 and 20 mM Na+. (C) The amplitude of the fast process, F(τ1), was independent of the Na+ concentration. The slower process showed a biphasic dependence on Na+ concentration, indicating that two different mechanisms control the fluorescence change. Error bars were omitted for the sake of clarity.
obtained at 15.2 °C with time constants that deviated extremely has been discarded.) The activation energy obtained from the τ values of the faster process, Ea(τ1), was 22.2 ± 11.7 kJ/mol with low precision because of the wide scattering of the data. For the slower process, activation energy Ea(τ2) was 63.4 ± 8.5 kJ/mol. The high activation energy of the slower process clearly indicates a conformational rearrangement of the protein as a rate-limiting process. The faster process would be compatible with binding of a Mg2+ ion to a binding site.
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DISCUSSION The viability of cells depends crucially on the fact that its enzymes and functional proteins are strictly controlled and are geared appropriately to maintain the metabolic network that is adapted to the cellular needs at all times. To regulate the required condition, a variety of different mechanisms have been established. One of these is the use of specific “messengers”, substrates, inhibitors, or activators to modify protein functions. In the case of the Na,K-ATPase, several approaches were found to control its activity under physiological conditions.43 The pump current may, however, also be affected by the concentrations of the substrates of the pump, i.e., MgATP, Na+, and K+ concentrations, and indirectly by pH and the Mg2+ concentration, as well as by the products of ATP hydrolysis, ADP and Pi. The concentration of these compounds does not change over a wider range in the cytoplasm under nonpathological conditions. Variations of their concentrations have, however, been used extensively to reveal numerous important details about the molecular mechanism of ion transport. This approach is also used here to reveal the role of Mg2+ in the performance of the Na,K-ATPase.
added 10 mM Mg2+ can displace more Na+ bound to the binding sites with an increasing Na+ concentration in the buffer at least up to the half-saturating Na+ concentration, Km(Na+), which is an obvious response. Above Km(Na+), which is ∼4 mM in the presence of 10 mM Mg2+,26 Mg2+ might be expected to become less effective in displacing the Na+ ions, and accordingly, the induced fluorescence change becomes smaller with higher Na+ concentrations. Another approach to gaining insight into the nature of the mechanism is the analysis of the temperature dependence of the rate constants. From the determined time constants, τ, the rate constants, k, of the controlling rate-limiting reaction can be calculated as k = 1/τ. When k is plotted in an Arrhenius diagram, the activation energy of the rate-limiting process can be obtained, and its magnitude provides clues about the kind of process being monitored. Experiments were performed in histidine buffer containing 10 mM NaCl (pH 7.2); after the 1011
DOI: 10.1021/acs.biochem.6b01243 Biochemistry 2017, 56, 1005−1016
Article
Biochemistry
To apply this theory to the data reported here, significant but inevitable simplifications must be made. The effect of the charge of an attached Mg2+ on the mobile ions is crucially dependent on the distance between them. A structure of the Na,K-ATPase in the open E1 conformation at atomic resolution is, however, still not available, and in addition, the location of the bound Mg2+ is not known beyond a putative binding site formed by the L67 loop.28 Before making hardly justifiable assumptions about spatial arrangements and the location of the bound Mg2+, we restrict ourselves, therefore, to a very simple model (Figure 10A) to test whether the experimental results
Since the early days of the investigation of the sodium pump, it has been clear that Mg2+ is an essential cofactor of the enzyme’s activity because ATP is hydrolyzed only as a complex with Mg2+.44 Is has been proven that Mg2+ is not transported by the Na,K-ATPase under any conditions and cannot enter the binding sites of the transported ions. The closest it can get to these sites is in the E2 conformation in which Mg2+ may enter the wide access channel connecting the binding sites with the extracellular aqueous phase.31 This ion well is so spacious that it can hold more than 60 water molecules and even bulky ions such as the benzyltriethylammonium ion.45 Nevertheless, as reported in the introductory section, Mg2+ ions affect ion binding in the E1 conformation on the cytoplasmic side of the protein and, as a consequence, also the pump current. The experimental results presented here prove a mutual influence of Mg2+ and Na+ (or K+) and also H+ in their interaction with the Na,K-ATPase. The Mg2+-induced modifications are not related to interactions with the ion-binding sites inside the transmembrane domain of the ion pump; therefore, an alternative mechanism must be proposed and discussed. Titration Experiments. The occupation of the ion-binding sites in the membrane domain of the Na,K-ATPase can be substantiated and quantified with high precision by the RH421 method that was introduced more than 20 years ago, and in the interim, it has been used and refined in numerous publications.24,26,28,34,39,45−49 However, detecting interactions of Mg2+ ions with the Na,K-ATPase, which are processes that probably occur mostly on the cytoplasmic surface of the protein, is difficult, to say the least, because no direct detection assay is known so far. Therefore, an indirect approach was introduced in which the effect of Mg2+ on binding of the monovalent cations to the binding sites was studied. One possible approach to explain the observed effects is based on the fact that ions interact with each other by the electric field that they create. Charge-induced Coulomb forces lead to a repulsion of charges of the same sign, and in electrolytes where ions are mobile, these forces cause ion movements until an equilibrium is maintained in which all ions of the same sign have an optimized distance to each other. In the specific case in which one ion is fixed in its position, e.g., if it is bound to some surface, the spatial distribution of the mobile ions in the neighborhood of this ion can be calculated by a theory introduced by Debye and Hückel almost 100 years ago.50 This theory allows the description of the essential impact of a Mg2+ ion attached to the surface close to the entrance of an access channel on the ion-binding sites of the Na,K-ATPase, which causes locally a decrease in the concentration of free cations in the electrolyte. This locally reduced concentration of Na+, K+, or H+ modifies, as a consequence, the equilibrium between the ions bound in sites inside the Na,K-ATPase in its E1 conformation and the free ions in the aqueous phase outside (nX+ + E1 ⇄ XnE1). The presence of an immobilized Mg2+ generates thus apparently increased equilibrium dissociation constants of the ion-binding sites of the Na,K-ATPase. The theory predicts, vice versa, that the increase in the concentration of monovalent cations in the electrolyte reduces the impact of the fixed Mg2+ ion on the local ion concentration at the access channel entrance. In addition, experiments revealed that H+ ions reduce the level of binding of Mg2+ ions to its proposed site in the vicinity of the access channel, probably by protonation of amino acid side chains. All these observations must be taken into account to explain the experiments presented here.
Figure 10. Effect of the Debye−Hü c kel theory on the Na + concentration at the entrance of the access channel. (A) For the sake of simplicity, the protein environment around the access channel to the ion-binding sites has been reduced to a plane. The Mg2+ ion is represented by the black circle in the distance r to the access channel. (B) The actual Na+ concentration at the channel entrance has been calculated according to the theory presented in the Supporting Information (eq S6) using the indicated Mg2+ concentrations. The distance from the Mg2+ ion to the channel entrance was varied between 0.1 and 10 nm. In this model, no significant effects of Mg2+ could be detected at distances of >5 nm.
can be reproduced qualitatively this way. If such a simple model can describe the basics of the experimental results, any specifications of structural details (that may become available in the future) will introduce additional information and parameters that will allow refinements of the model and eventually even numerical fits to the presented data. A formal adoption of the Debye−Hückel theory by the model presented in Figure 10A is provided in the Supporting Information. It shows how to calculate the electric potential around the Mg2+ ion, the concentration of the free ions (e.g., 1012
DOI: 10.1021/acs.biochem.6b01243 Biochemistry 2017, 56, 1005−1016
Article
Biochemistry Scheme 1
Scheme 2
Na+, K+, or pH) at the mouth of the access channel, the occupation of the ion-binding sites in the Na,K-ATPase as a function of the ion concentration at the entrance of the access channel, and, finally, the dependence of the RH421 fluorescence signals on ion concentrations as presented in the Results. The parameters needed to do so are the composition of the electrolyte to calculate its ionic strength, the temperature, and the dielectric coefficient of water. With this information, the actual ion concentrations at the access channel entrance can be calculated. Figure 10B shows results for the electrolyte used in the experiments with a bulk Na+ concentration of 10 mM, pH 7.2, and the indicated Mg2+ concentrations as a function of the distance, r, between the fixed Mg2+ ion and the access channel entrance. As expected, the shorter the distance and the higher the Mg2+ concentration, the more the local Na+ concentration decreased. For the simulations of the RH421 fluorescence changes as a function of Mg2+, Na+, and H+ concentrations, a distance r of 0.3 nm was used throughout. The electrolyte composition was that of the standard buffer (see Materials and Methods), and the needed equilibrium dissociation constants of Na+ and Mg2+ and the pK of ion transport sites and the Mg2+-binding site were taken from the experiments. The simple model can reproduce the experiments in the concentration range of Mg2+ (
