Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and Pi alter access to the Na+-K+-ATPase transport site

Craig Gatto,1 Jeff B. Helms,1 Megan C. Prasse,1 Krista L. Arnett,2 and Mark A. Milanick2

1Division of Biomedical Sciences, Cell Biology & Physiology Section, Department of Biological Sciences, Illinois State University, Normal, Illinois; and 2Department of Medical Pharmacology and Physiology, School of Medicine, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri

Submitted 4 February 2005 ; accepted in final form 17 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Current models of the Na+-K+-ATPase reaction cycle have ATP binding with low affinity to the K+-occluded form and accelerating K+ deocclusion, presumably by opening the inside gate. Implicit in this situation is that ATP binds after closing the extracellular gate and thus predicts that ATP binding and extracellular cation binding to be mutually exclusive. We tested this hypothesis. Accordingly, we needed a cation that binds outside and not inside, and we determined that tetrapropylammonium (TPA) behaves as such. TPA competed with K+ (and not Na+) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low Na+, and TPA decreased the rate of EP hydrolysis in a K+-competitive manner. Having established that TPA binding is a measurement of extracellular access, we next determined that TPA and inorganic phosphate (Pi) were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when Pi is bound, the transport site has extracellular access. Surprisingly, we found that ATP and TPA also were not mutually exclusive inhibitors of pNPPase activity, implying that when the cation transport site has extracellular access, ATP can still bind. This is consistent with a model in which ATP speeds up the conformational changes that lead to intracellular or extracellular access, but that ATP binding is not, by itself, the trigger that causes opening of the cation site to the cytoplasm.

quaternary ammonium; Dixon plot; P-type adenosine triphosphatase; inorganic phosphate


P-TYPE ATPASES ARE ENZYMES responsible for maintaining electrolyte homeostasis, which is critical for cell function. This protein family includes the Na+-K+ pump, the H+-K+ pump, and sarcoendoplasmic reticulum Ca2+-ATPase, all of which share similar amino acid sequences. The Na+-K+-ATPase couples the energy of hydrolysis of ATP to the active exchange of three intracellular Na+ ions for two extracellular K+ ions to create the low intracellular levels of Na+ and the high intracellular levels of K+ of nearly all mammalian cells (for review, see Refs. 21, 22).

The ATPase cycle of the Na+-K+ pump must coordinate the movements of the cytoplasmic domains, which are involved in ATP hydrolysis, to the movements of the transmembrane domains, which are involved in ion translocation. The kinetics of the Na+-K+-ATPase are generally described in terms of the Post-Albers model (1, 31). This model considers two conformations of the enzyme, E1 and E2, both of which can be in either a phosphorylated or an unphosphorylated state. The formalism of E1/E2 is based on cation effects on phosphorylation events and on conformation of the enzyme as detected by fluorescence and susceptibility to protease digestion. It is not clear to what extent these properties reflect the transport states of the enzyme, because both cytoplasmic domains and membrane domains of the enzyme molecule must be moving. Focusing on the orientation of transport sites and of the properties of the cytoplasmic domains may make analysis more manageable.

At various points during the pump cycle, the transmembrane domains switch between three different states: the ion binding sites have cytoplasmic access; the ion binding sites have extracellular access; and the ions are occluded (access to neither aqueous solution). Mechanistically, significant details about how the enzyme couples ATP hydrolysis with cation translocation remain unclear. For example, does ligand binding to cytosolic domains cause the opening or closing of the transmembrane domain gates?

ATP is the energy source as well as an important ligand for these pumps. Two ATP effects are observed during the Na+-K+-ATPase catalytic cycle: a high-affinity (~0.1 µM) ATP effect that directly results in phosphoenzyme (EP) formation and a low-affinity (~100 µM) effect that accelerates the deocclusion of K+.

One approach to determine the status of the transmembrane domain is to make use of a transport site inhibitor that binds only to the extracellular site. Earlier studies have indicated that certain inhibitors may act exclusively from the inside or the outside (36), but these studies did not test whether the inhibitors bound to the transport sites. Schuurmans Stekhoven et al. (36) characterized the effect of several amine compounds on the phosphorylation of Na+-K+-ATPase. In their study, it is clear that some compounds were strict inhibitors of pump phosphorylation, whereas others stimulated pump phosphorylation. Subsequent studies demonstrated that the stimulatory effects of these compounds were mediated extracellularly (39). Although increases in steady-state EP levels could be achieved either by increased rate of formation or by decreased rate of breakdown, Van der Hijden et al. (39) favored the former as a hypothesis and did not explicitly consider the possibility that the amines bound to the extracellular transport site-stabilized covalent phosphointermediate (E-P) by decreasing its rate of breakdown.

In intact red cells, the extracellular effects of two quaternary amines [e.g., bretylium, Refs. 17, 38; and tetrapropylammonium (TPA), Ref. 24] were determined to be competitive inhibitors of K(Rb) influx, each with a Ki <5 mM in the presence of 1 mM K(Rb). In a recent study, Milanick and Arnett (28) showed that extracellular TPA mimics K in that it changes the negative log of the dissociation constant for extracellular protons in a manner similar to K+, yet different from that of Na+, thus providing important evidence that TPA does bind to the outside transport site. However, in none of these studies of TPA was there an examination of whether TPA bound to the inside site. TPA was also shown to decrease the rate of 86Rb deocclusion with roughly the same affinity as its ability to inhibit K(Rb) influx (14). Because this effect was observed only when deocclusion was measured in the presence of phosphate, the TPA effect was interpreted to occur at the extracellular face (14).

The conceptual framework of interpreting the occlusion data by Forbush (14) is an example of an overall framework in the field; namely, ATP binding opens the inside gate and phosphate binding opens the outside gate.1 A more recent example of this framework is the model used by Artigas and Gadsby (3) to guide their experiments on the study of palytoxin that converts the Na+-K+ pump into a channel (see Fig. 1 in Ref. 3). However, it is important to point out that the evidence in this and other studies is also consistent with ATP increasing the opening of not only the intracellular gate but also the extracellular gate (see DISCUSSION).



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Fig. 1. A simplified kinetic model of the Na+-K+ pump cycle. In this schema, the left and right parentheses represent closed inside and outside gates, respectively. The binding and release of cations and nucleotides do not imply an order, but rather are separated for easy interpretation of competitive inhibition with respect to cation-binding conformations. E-P, covalent phosphointermediate.

 
Important questions remain concerning the cause and effect between movements within the cytoplasmic domains and movements within the transmembrane domain. That is, in which circumstances does a conformational change within the cytoplasmic domains cause the inside or outside gate to open or close? At some steps, it is thought that ligand binding triggers specific conformational changes; for example, kinetic models have been posed that imply that ATP binding at the low-affinity site causes the inside gate to open and facilitate K+ deocclusion (3, 12, 13).

High-resolution structural determination of several of the intermediary structures of the enzyme cycle provides key information for understanding how the pump works. However, a crystalline structure locks the enzyme in only one conformational state, and there can be ambiguity about branches in the cycle and about cause and effect when a conformational change is observed between two different crystalline structures from separate ligand-bound pump states. Kinetic studies of partial reactions have been useful for deciphering the conformational stages involved in the P-type ATPase mechanism as well as for deducing the substrate-binding constraints within particular conformations and provide important information and constraints on the order of the reaction, whether branches occur during the cycle, and on possible cause-and-effect relationships.

The ability to hydrolyze organic phosphates (especially para-nitrophenylphosphate, pNPP) is one partial reaction that has been used extensively to study this protein family. Hydrolysis of pNPP has been used as a measure of K+-bound conformations (4, 5, 23). Specifically, Drapeau and Blostein (10) showed that the K+ dependence is an effect of cytoplasmic K+ and not extracellular K+.2 They considered the possibility that the K+-occluded form was the enzyme species that mediated this activity. If this occluded form, in the presence of pNPP, could open to the outside at the same rate as pNPP hydrolysis, then Drapeau and Blostein should have observed a strophathidin-sensitive, pNPP-activated 86Rb flux; but they did not. Likewise, Campos et al. (6) also were unable to observe K+ transport activity associated with para-nitrophenylphosphatase (pNPPase) activity. Consequently, the particular enzyme form that mediates pNPP hydrolysis remains unresolved. In addition, whether the external gate opens at all or periodically remains unknown. The inability to detect pNPP-mediated K(Rb) fluxes (6, 10) is consistent with the external gate either remaining closed or opening periodically at a rate much slower than the rate of pNPP hydrolysis. In any case, pNPPase activity is a direct measurement of a cytoplasmic K+ effect and provides a useful assay of the inwardly facing cation site. In addition, because inorganic phosphate (Pi) and ATP have been shown to inhibit pNPPase activity in the Na+-K+-ATPase, this protocol can be useful in answering questions that simultaneously address the relationship between cation site access with cytosolic domain orientation. Particularly interesting is the demonstration that Pi competes with pNPP for this reaction, whereas para-nitrophenol (pNP) does not, consistent with a sequential reaction mechanism whereby pNPP binds and is hydrolyzed and pNP is released before Pi (32). Whether E-P is formed during pNPPase remains controversial (18, 33), but there certainly appears to be a stable, noncovalent phosphointermediate form (E:P) (32).

In this study, we used TPA to determine the properties of the Na+-K+ pump when the transport site is accessible to the extracellular solution (i.e., when the extracellular transport gate is open). Our data provide information about the relationship between low-affinity ATP binding and transport site accessibility. Preliminary accounts of this work were presented previously to the Biophysical Society (27).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [32P]ATP and 86Rb were obtained from PerkinElmer Life Sciences (Boston, MA). Ammonium molybdate, hydrochloric acid, sodium phosphate, KCl, sucrose, choline chloride, ascorbic acid, {beta}-mercaptoethanol, EDTA, Folin's reagent, imidazole, MgCl, Na2ATP, NaHCO3, NaCl, antipain, pepstatin, leupeptin, phenylmethylsulfonyl fluoride, HEPES, and Trizma base were purchased from Sigma (St. Louis, MO). TPA and tetramethylammonium (TMA) were obtained from Fisher (Fair Lawn, NJ). Dog kidneys were salvaged from euthanized dogs that were used in other Institutional Animal Care and Use Committee-approved experiments and were the generous gift of Dr. James L. Cook (University of Missouri-Columbia).

Na+-K+-ATPase purification from dog kidney. Na+-K+-ATPase was purified from dog kidney as described by Jorgensen (19). Briefly, the outer medulla was dissected from five dog kidneys and then homogenized in a blender in medium containing (in mM) 250 sucrose, 25 imidazole, and 1 EDTA (pH 7.2). The homogenate was filtered through four layers of cheesecloth, and the filtrate was centrifuged at 10,000 g for 15 min. The supernatant liquid was collected and centrifuged once again at 10,000 g for 15 min. These low-speed centrifugation steps removed a significant portion of cellular debris from the microsomal fractions. The supernatant liquid was collected from the second spin, and the microsomal fraction was sedimented at 65,000 g for 1 h. The pellets were collected and resuspended in the above-described homogenizing medium with the addition of 3 mM ATP to a protein concentration of 6 mg/ml. The microsomal preparation was incubated with ATP present for 10 min and then diluted to a final concentration of 2 mg/ml with 0.4% SDS (wt/wt) (in ATP-containing 25 mM imidazole and 1 mM EDTA and incubated for an additional 10 min at room temperature. The detergent-treated microsomes were layered on top of a three-step sucrose gradient (all densities were prepared in homogenizing medium) of 15, 25, and 45% and centrifuged in a swinging bucket rotor (Beckman SW-28) at 100,000 g for 2.5 h. The membrane fractions collected at the 25–45% interface were used for these experiments. Protein concentration was determined using the method of Lowry et al. (25). The ouabain-sensitive enzyme activity of the Na+-K+-ATPase preparations used in this study ranged from 7 to 14 µmol ATP hydrolyzed·mg of protein–1·min–1.

Na+-K+-ATPase assay. ATP hydrolysis was measured as reported previously for the red cell Ca2+ pump (15), with minor modifications for Na+-K+-ATPase. Briefly, 0.1 mg of purified canine renal Na+ pump enzyme was diluted in 2.8 ml of 200 mM imidazole and titrated to pH 7.4 with diluted HCl. This mixture was warmed at 37°C for 10 min before being diluted 12-fold in 550 µl of an assay solution containing 50 mM imidazole, pH 7.4, 10 mM MgCl2, 0.5 mM Tris-ATP, 0.5 µCi [32P]ATP, and either 130 mM NaCl or 5 mM KCl, plus the indicated TPA concentrations. (Measurements at submillimolar ATP allow for a high ouabain-sensitive ATPase signal with low amounts of radioactivity; although 0.5 mM ATP is not saturating, the Na+-K+ pump is operating at >75% Vmax with respect to [ATP], and these conditions are sufficient to determine whether TPA competes with Na+ or K+.) Similar experiments were performed with TPA. (Concentrations are indicated in the figure legends.) The quadruplicate samples were then incubated for 10 min at 37°C. Ouabain (1 mM) was included in quadruplicate samples to determine ouabain-insensitive enzyme activity. The reactions were stopped with the addition of 1 ml of an ice-cold solution containing 12 mM trichloroacetic acid and 100 g/l activated charcoal. The activated C binds organophosphates and is removed from the suspension by centrifugation in a microfuge at 4,000 rpm for 2 min. A 0.5-ml volume of each supernatant containing unbound Pi was assayed with liquid scintillation spectroscopy.

pNPPase activity. All pNPPase assays were conducted essentially as described previously (10) using 2–4 µg of Na+-K+-ATPase from dog kidney preparations. It is important to note our observation that the pNPPase activity was sensitive to the total salt concentration of the reaction medium, especially <100 mM total salt (data not shown). Thus we were careful to match the concentration of inhibitor present with equimolar amounts of imidazole, Tris, MOPS, or choline. We detected no differences between the ionic strength equalizing cations. In brief, the assay buffer contained 50 mM MOPS-Tris and 3 mM MgCl2, pH 7.4, with final concentrations of 5 mM di-Tris-pNPP and 100 mM choline chloride (or other salt for ionic strength equilibration) for each reaction tube. For IC50 experiments, 2 mM K+ was used, while the concentration of TPA was varied over the range indicated in the figure legends. For K+ competition experiments, TPA concentration was fixed, while the concentration of K+ was varied as indicated in figure legends. Each reaction tube was incubated at 37°C for 15 min, the reaction was stopped by the addition of 200 µl of ice-cold 200 mM NaOH, and the reaction tubes were placed in an ice bath for 10 min. Absorbance at 410 nm was then recorded using a Beckman DU-530 spectrophotometer, and absorbance was converted to activity on the basis of a pNP standard curve. We were careful to ensure that these experiments measured initial rates, i.e., <5% of the pNPP was hydrolyzed. Thus free PO4 concentrations never rose >200 µM in these experiments and did not complicate conclusions based on inhibitor studies.

Phosphorylation by [32P]ATP. The amount of EP was determined similarly to methods reported previously (8). Three different types of phosphorylation experiments were performed. The first set dealt with whether TPA would prevent phosphorylation from Na+ and MgATP specifically from the cytoplasmic surface. For these experiments, chymotrypsin-treated enzyme was used. The chymotrypsin treatment was performed according to the method of Jorgensen and Farley (20). Briefly, the dog kidney enzyme was digested for 15 min at 37°C in a solution containing 1.7 mg/ml purified Na+-K+ pump, 0.1 mg/ml chymotrypsin, 0.85 mM NaCl, 13 mM Tris·HCl (pH 7.4), and 0.085 mM EDTA. The reaction was stopped by diluting the solution 23-fold into an ice-cold solution of 1 mM Na+ and 190 mM NMDG-HEPES (pH 7.4) containing 0.4 mg/ml aprotinin. Enzyme phosphorylation was then determined by diluting this chymotrypsin-treated enzyme stock to ~10 µg of protein with 144 mM N-methyl-D-glucamine, 1 mM NaCl, 3.5 mM MgCl2, 35 mM Tris·HCl, pH 7.5, and 10 µM [32P]ATP in the presence or absence of inhibitor or K+ for 30 s on ice. The reaction was quenched by adding 60% trichloroacetic acid, 3 mM ATP, and 6 mM Tris-phosphate, filtering, and washing three times in the same solution. Each condition was assayed via liquid scintillation spectroscopy (in quadruplicate).

The second set of experiments dealt with whether the inhibitors would prevent dephosphorylation of E2P. In these experiments, EP was first formed by incubating ~10 µg of protein in 144 mM N-methyl-D-glucamine, 1 mM NaCl, 3.5 mM MgCl2, 35 mM Tris·HCl, pH 7.5, and 10 µM [32P]ATP on ice for 30 s. Next, K+ and the indicated concentrations of inhibitor were added and incubated for an additional 30 s on ice. The reaction was stopped and washed as described above. Each condition was performed in quadruplicate.

The third set of experiments addressed the question of whether the inhibitors compete with K+ for dephosphorylation. These experiments were performed at 0°C. A 10-µg quantity of dog enzyme was mixed with 1 mM KCl or 10 mM NaCl and 25 µl of MgATP32 solution (145 µM MgCl2/130 µM Tris-ATP, ~15 µCi ATP32) for 30 s in a final volume of 100 µl of buffer containing (in mM) 100 NMDG and 200 HEPES (pH 7.4). To the K+ and Na+ controls then were added 50 mM choline chloride and 1 mM EDTA (no K+), and they were incubated for and additional 30 s on ice. To all other samples were added either 50 mM choline, TPA, or guanidine and 0.1 mM K+ and 1 mM EDTA, and these samples were incubated for an additional 30 s on ice. All reaction tubes were stopped and radioactivity was determined as described above.

Statistics. Differences between the ionic conditions during steady-state EP measurements (see Figs. 4 and 5) were analyzed with independent sample Student's t-tests using SPSS version 9.0 software (SPSS, Chicago, IL). In each case, Student's t-test values were computed, and the significance at the P = 0.01 level on the basis of two-tailed analysis was reported. Tests for equality of slopes (see Figs. 6 and 7) were conducted using the General Linear Model (GLM) procedure (Regress) with SAS version 9.1 software. The slopes of the lines for the Dixon plots were found to be significantly different from one another and thus were not parallel (see Fig. 6, F1,28 = 231.37, P < 0.0001; see Fig. 7A, F1,35 = 112.10 P < 0.0001; see Fig. 7B, F1,36 = 134.18, P < 0.001).



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Fig. 4. Effect of TPA on phosphoenzyme (EP) levels of chymotrypsin-treated Na+-K+-ATPase. Purified dog kidney enzyme was mildly treated with chymotrypsin to prevent major conformation between inside and outside facing. After chymotrypsin cleavage, the Na+-K+-ATPase was phosphorylated in the presence of 10 µM MgATP32 and the indicated cations (27°C). Protein was precipitated with 5% TCA and trapped on a 0.45-µm filter, and radioactivity was determined. Tetramethylammonium (TMA) was used as a control cation condition for TPA because TMA does not inhibit the Na+-K+ pump. In the first bar at left, TXA indicates that experiments using either TMA or TPA gave similar results when EDTA was present. The data show that 100 mM TPA was unable to reduce the level of EP compared with control (i.e., 10 mM Na+, 100 mM TMA), consistent with the inability of TPA to bind to the inside cation site. The last bar at right shows that the addition of K+ did not facilitate dephosphorylation, confirming that the chymotrypsin treatment worked. Data are from four separate experiments of quadruplicate determinations for each experiment and were normalized to the level of EP in the presence of 10 mM Na+ and 100 TMA. Bars represent means ± SE. *P < 0.01 vs. other 3 bars.

 


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Fig. 5. Effect of organic cations on the stability of EP. A: after EP steady state was established (see, e.g., Fig. 6), 10 mM EDTA and the indicated organic cation were added. The chelation of Mg2+ by EDTA prevented further EP formation, and thus these experiments measured the effect of cations on the rate of spontaneous dephosphorylation. Our data demonstrate the ability of the TPA to stabilize EP. *P < 0.01, EP levels differed significantly from 1 mM Na+ with no additional cations added. B: after establishing an EP steady state (see, e.g., Fig. 6), 10 mM EDTA and the indicated organic cations were added. The addition of 0.1 mM K+ produced a rapid dephosphorylation (compare middle two bars). This accelerated dephosphorylation was retarded by 50 mM TPA (compare second bar from left with last bar at right). These data are consistent with TPA binding to the extracellularly facing phosphorylated pump. Data shown in both graphs are from three separate experiments of quadruplicate determinations for each experiment and were normalized to the level of EP in the presence of TPA. Bars represent means ± SE. NS, no significant difference from 10 mM Na+, 50 mM choline chloride, and no K+ (i.e., P > 0.1).

 


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Fig. 6. TPA IC50 curve with or without inorganic phosphate (Pi). The concentration dependence of TPA inhibition was measured in the presence or absence of Pi. The hydrolysis of para-nitrophenylphosphate (pNPP) was measured in the presence of the indicated concentration of TPA either without ({circ}) or with ({square}) 1 mM Pi. The data are displayed in a Dixon plot for easy determination of whether the inhibitors bound in a mutually exclusive manner. Clearly, the lines are not parallel, thus suggesting that both TPA and Pi can bind simultaneously to the enzyme. The data are from a single experiment, representative of three separate experiments. Points are means and bars indicate SE of quadruplicate determinations. Data were normalized to the para-nitrophenylphosphatase (pNPPase) activity in the absence of either inhibitor. Lines are the best fit linear regression to the mean values.

 


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Fig. 7. TPA IC50 curve with or without nucleotides. The concentration dependence of TPA inhibition was measured in the presence or absence of ATP (A) or ADP (B). These experiments were performed as in Fig. 6 either without ({circ}) or with either ({square}) 0.1 mM ATP (A) or ({square}) 2 mM ADP (B). Again, the nonparallel lines are consistent with TPA and adenine nucleotides binding simultaneously to the enzyme. The data in A and B are from single experiments and are representative of three separate experiments with each nucleotide. Points are means and bars indicate SE of quadruplicate determinations. Data were normalized to the pNPPase activity in the absence of inhibitor. Lines are the best fit linear regression to the mean values.

 

    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Initially, the focus of our study was to determine whether TPA bound to the inward-facing cation site, the outward-facing cation site, or both, to block Na+-K+ pump function. A simple kinetic model (Fig. 1) predicts the behavior of alkali metal inhibitors that mimic binding to either the inside or the outside monovalent cation sites. For example, an inhibitor that bound to the transport site solely from the inside (i.e., X in Fig. 1) would compete with Na+, but not with K+, for Na+-K+-ATPase activity. In contrast, an inhibitor that bound to the transport site solely from the outside (i.e., Y in Fig. 1) would compete with K+, but not with Na+, for Na+-K+-ATPase activity. An additional prediction is that inhibitor X would compete with Na+ to block pump phosphorylation from Na+, Mg2+, and ATP, whereas inhibitor Y would have no effect on E-P formation under these conditions. Also, the model predicts that Y should compete with K+ for dephosphorylation. Such an approach provides a clear delineation between inside and outside effects even in the absence of a preparation that affords physical sidedness.

Na+-K+-ATPase measurements. To determine whether inhibition by TPA was competitive with K+, the K+ dependence of ATPase activity was measured in the presence of 50 mM Na+ and in the presence and absence of TPA (Fig. 2). The inhibition produced by 5 mM TPA was substantially reversed by increasing K+ concentration ([K+]), and the extrapolated maximal velocities were the same in the presence and absence of TPA, whereas 5 mM TPA raised the for K+ 20-fold as predicted for a competitive inhibitor. The fractional activity (i.e., TPA activity/control activity for each [K+] level) dramatically increased as the [K+] increased (Fig. 2), consistent with competitive inhibition.



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Fig. 2. K+ concentration ([K+]) dependence of inhibition of ATPase activity. K+ activation of ATPase activity was measured in the presence of saturating substrates. A: in contrast to the effects on Na+ (cf. Fig. 3), 5 mM tetrapropylammonium (TPA; {lozenge}) increased the for K+, while Vmax was not significantly different from control ({circ}), consistent with competition. Control Vmax = 0.98 ± 0.05, = 0.25 ± 0.07 mM, TPA Vmax = 0.91 ± 0.04, and = 5.0 ± 0.6 mM. B: fractional activity clearly reveals that TPA inhibition decreases as [K+] increases. The data from at least three different experiments of quadruplicate determinations were normalized to the maximal velocity of ATP hydrolysis in the absence of inhibitor. Points represent means ± SE. Data were fit to the Michaelis-Menten equation with a S2 term to represent that more than one K+ ion binds, producing the sigmoidicity observed.

 
To determine whether inhibition by TPA was competitive with Na+, the Na+ dependence of ATPase activity was measured in the presence of 2 mM K+ and saturating concentrations of ATP and Mg2+ and in the presence and absence of TPA (Fig. 3). In contrast to the results shown in Fig. 2, TPA caused no significant change in the for Na+ activation, but it did lower the maximal velocity of the enzyme (Fig. 3A). The fractional activity does not increase with increasing Na+ (Fig. 3B). Indeed, the slope appears to be negative, consistent with uncompetitive inhibition. Clearly, TPA inhibition is not reduced by increasing Na+ concentration ([Na+]), and thus TPA does not compete with Na+ for Na+-K+-ATPase.



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Fig. 3. Na+ concentration ([Na+]) dependence of inhibition of ATPase activity. Na+ activation of ATPase activity was measured in the presence of 3 mM MgCl2, 3 mM ATP, and 2 mM KCl. A: increasing concentrations of Na+ in the presence of 10 mM TPA ({square}) was unable to displace TPA and attain control Vmax levels. Control Vmax = 1.04 ± 0.02, = 11.5 ± 0.98 mM, TPA Vmax = 0.72 ± 0.03, = 7.8 ± 1.3 mM. B: fractional activity (TPA/control) that does not decrease with increasing Na+ as would be expected if the two competed for the same enzyme form; it may actually increase, consistent with uncompetitive inhibition. However, the slope is relatively shallow; thus we hesitate to conclude that it differs from a horizontal line. The data from at least three different experiments of quadruplicate determinations were normalized to maximal velocity of ATP hydrolysis in the absence of inhibitor. Points represent means ± SE. Data were fit to the same equation as in Fig. 2.

 
Phosphoenzyme measurements. In the Na+-K+-ATPase, Na+ ions binding from the inside in the presence of Mg2+ and ATP results in the covalent transfer of the {gamma}-phosphate from ATP to the enzyme, forming an acyl phosphate intermediate with Asp369. This reaction is inhibited by organic cations that bind to the intracellular cation site. We measured the EP levels with 1 mM Na+ in the presence and absence of 100 mM TPA. In native pump, in the absence of substrates, the pumps are probably distributed in several different conformational states, including inside and outside facing binding sites. To avoid this distribution of pumps, we used chymotrypsin-digested enzyme. It has been shown that mild chymotrypsin digestion of the Na+-K+-ATPase prevents the major conformational change between inside and outside (19). Figure 4 shows that TPA is indistinguishable from TMA (which does not inhibit the Na+-K+ pump and was used as an ionic strength control), consistent with TPA being an exclusive extracellular cation site probe.

The EP levels measured in Fig. 4 are in a steady state, because the presence of Na+, Mg2+, and ATP produces EP at the same rate at which it is being hydrolyzed in the forward direction. One can measure the stability of EP by the addition of EDTA after the phosphorylation reaction has reached the steady state. The EDTA chelates the necessary Mg2+ and prevents further EP formation. Figure 5 shows the results of experiments to determine the effect of TPA on the rate of spontaneous dephosphorylation; TPA was able to stabilize the EP form (Fig. 5A). Thus TPA appears to bind to the extracellular cation site and slows the rate of spontaneous dephosphorylation. Although this is clearly a form of the enzyme that prefers K+ over Na+, TPA cannot mimic K+ closely enough to induce the conformational change that allows EP hydrolysis. Rather, the binding of TPA directly antagonized the binding of K+ and prevented the conformational change that promotes hydrolysis (Fig. 5B) as predicted for the inhibitor Y in Fig. 1.

ATP, Pi, and TPA. The results described above led to the conclusion that TPA binds to outwardly facing transport sites but not to inwardly facing sites. We next asked the following questions. 1) Are TPA binding and Pi binding mutually exclusive? 2) Are TPA binding and ATP binding mutually exclusive? Traditionally, there are two approaches to determining whether two compounds are mutually exclusive. If one compound is a substrate, then it is possible to determine whether the inhibitor is competitive, in which case the two compounds are mutually exclusive. However, if the inhibitor is not competitive, this does not necessarily imply that both the inhibitor and the substrate are bound at the same time, but merely that they do not bind to the same enzyme intermediate. Thus we preferred to examine the interactions under conditions in which all the compounds were inhibitors, that is, on pNPPase activity. We determined that TPA cannot mimic K+ to stimulate pNPPase activity (data not shown). This analysis allows the use of a Dixon plot to determine whether the inhibitors are mutually exclusive. If the two inhibitors are mutually exclusive, then the Dixon plot lines are parallel. Importantly, if the Dixon lines are not parallel, this implies that both inhibitors are able to bind at the same time.

Figure 6 shows the concentration dependence of TPA inhibition of pNPPase activity (stimulated by 2 mM K+) in the absence and presence of 1 mM Pi. The plot shows that the lines are not parallel. Therefore, TPA and Pi are not mutually exclusive inhibitors; that is, both Pi and TPA can bind simultaneously to the Na+-K+-ATPase.

Similarly, we wanted to test whether TPA and ATP bind at the same time, so we measured velocity vs. TPA in the presence and absence of ATP. Figure 7A shows the concentration dependence of TPA inhibition of pNPPase activity in the absence and presence of ATP (0.1 mM). The plot shows that the lines are not parallel; therefore, TPA and ATP are not mutually exclusive inhibitors, consistent with both ATP and TPA binding simultaneously to the Na+-K+-ATPase. However, using flame photometry, we discovered that some TPA supplies contained minor amounts of contaminating Na+ (Na+ contamination ranged from 0 to 1%), which would allow TPA to bind to the outside-facing EP conformation. To determine whether this was the case, we also measured whether TPA and ADP bind at the same time to inhibit pNPPase activity. ADP has also been shown to bind at the low-affinity nucleotide site to facilitate Rb+ deocclusion and speed up the outward-facing to the inward-facing conformational change, albeit with much lower affinity than ATP (12, 13). These experiments revealed that TPA and ADP also were not mutually exclusive (Fig. 7B, slopes differ significantly; P < 0.001), thus confirming that the nucleotide binding site is accessible while the outside-facing cation site is accessible to TPA.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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For more than three decades, quaternary ammonium compounds have been known to inhibit Na+-K+-ATPase activity (35), and they remain critical tools for deciphering structural and functional information about cation coordination (30). In this study, we have shown that TPA binds to the extracellular transport site and is excluded from the intracellular transport site using SDS-solubilized Na+-K+-ATPase in which the membrane sidedness of sites is disrupted and TPA competes with K+ (an extracellular effect) for activation of ATPase and does not compete for the intracellular effect of K+ (for activation of pNPPase) or of Na+ (for activation of ATPase or for formation of the phosphointermediate from ATP). Furthermore, while TPA binds to the extracellular site, it does not mimic K+ in promoting dephosphorylation; indeed, TPA protects the pump from K+-stimulated dephosphorylation. These properties, in combination with the findings of Forbush (14) that TPA is not occluded and those of Milanick and Arnett (28) that extracellular protons affect K+ and TPA binding similarly, allowed us to use TPA to determine the properties of the pump when the transport site is accessible to the extracellular solution with the transport gate open. By studying the influence of Pi and ATP on the TPA inhibition of pNPPase, we have demonstrated that Pi as well as ATP can bind to an enzyme conformation with the transport site facing outside.

In this discussion, most of our work deals with nucleotide inhibition of pNPPase. This is a low-affinity effect observed in the presence of K+. This is distinct from the high-affinity ATP binding that occurs in the presence of Na+ and results in EP formation.

Pi and TPA. We have found that a Dixon plot of pNPPase inhibition by TPA alone, compared with TPA and Pi together, produces slopes that are not parallel lines (Fig. 6). This implies that there is a term in the velocity equation that includes both TPA and Pi as factors. Because both TPA and Pi are dead end inhibitors of pNPPase, this can occur only if both TPA and Pi can be bound at the same time; i.e., they are not mutually exclusive. We do not know whether the phosphate is bound covalently under these conditions, but others have shown that in the presence of K+, there is a distribution of pump species, with some pumps having phosphate bound noncovalently and some with phosphate bound covalently (7, 11). The simplest explanation of our results is that in the presence of phosphate, at least occasionally, the outside gate opens and TPA can bind. Indeed, it is known that the Na+-K+ pump can perform K+/K+ exchange in the presence of Mg2+ and Pi, which is consistent with both gates alternatively opening and closing in this mode of operation. Do our results support a model in which the binding of phosphate (either noncovalently or covalently, or both) causes the extracellular gate to open? It is a possibility. Our results are consistent with such a model, but the experiments were not designed to examine whether some of the pumps bound with phosphate had the inside gate open. Thus we think it is premature to conclude that the binding of phosphate causes the extracellular gate to open.

Nucleotide site and cation site accessibility. We have found that a Dixon plot of pNPPase inhibition by TPA alone vs. TPA and ATP produces slopes that are not parallel lines; likewise, our findings were similar when we used ADP instead of ATP. Both ATP and ADP have been shown to speed up the rate-limiting deocclusion of K+ (Fig. 7, A and B). With the use of the same reasoning used for the TPA and Pi case, this implies that both TPA and ATP (or ADP) can be bound at the same time; i.e., they are not mutually exclusive. The simplest explanation of our results is that in the presence of ATP, at least occasionally, the outside gate opens and TPA can bind. How do we reconcile this observation with the currently accepted model that ATP binding leads to deocclusion by opening of the inside gate? All direct deocclusion experiments (see, e.g., Refs. 12, 13, 16, 34) have been performed with unsided preparations, i.e., in experiments in which K+ was released directly into the medium, whether from extracellularly or intracellularly facing transport sites. The motivation for considering ATP-induced K+ release as being from intracellularly facing transport sites is derived from consideration of ATP stimulation of the forward pump cycle. ATP stimulation of the forward pump cycle as well as K+ deocclusion can also occur, however, in a model in which ATP increases the probability that either one of the gates (inside or outside) opens. In the forward ATPase cycle, if ATP occasionally opened the outside gate, K+ might be released to the outside, but because all of these studies were performed in the presence of K+, another K+ would bind, the outside gate would close, and either the outside gate would reopen or the inside gate would open quickly. In the latter case, the forward cycle would continue. The sputtering of the outside gate might slightly slow the overall pump cycle rate (depending on its rate compared with the slow steps in the cycle) compared with a mechanism in which ATP opened only the inside gate. An ATPase measurement cannot be used to determine whether the outside gate sputters during the cycle.

Forbush (14) found that TPA had an effect on the rate of Rb+ deocclusion when Pi was present but not when ATP was present. We can suggest a model that reconciles his results with ours. ATP binding increases the probability that one of the gates opens, and it is more likely that the inside gate opens. In the Forbush experiments, as soon as K+ is deoccluded from a particular pump, that pump is no longer able to provide a signal, because the K+ has been released into a very diluted medium. In our experiments, when ATP opens the inside gate, K+ falls off, another K+ comes on, and the pump can contribute further to pNPPase hydrolysis. However, occasionally, ATP opens the outside gate, the K+ falls off, and TPA can bind, and this pump momentarily cannot hydrolyze pNPP.

Does TPA bind exclusively to the extracellular site? Our conclusion that ATP binds to the pump with the outside gate open relies on the finding that TPA binds exclusively to the extracellular site. As mentioned in the introduction herein, previous studies of TPA using sided preparations examined inhibition only from the extracellular medium and thus did not address the issue of TPA binding to the inside. The data of Forbush (14) are consistent with TPA binding only to the outside but do not eliminate the possibility that TPA can also bind to the inside site, particularly considering that there is no conclusive evidence that ATP opens only the inside gate and never the outside gate and that Pi opens only the outside gate and never the inside gate.

While the present studies were in progress, Artigas and Gadsby (2) reported that TPA inhibited the palytoxin-treated Na+-K+ pump only from the outside. All of our results regarding ATPase and EP are consistent with TPA inhibition at the extracellular site. TPA and K+ compete for ATPase (Fig. 2), and TPA prevented K+ from stimulating EP hydrolysis (Fig. 5B). If TPA bound to the inside transport site, then it should have been competitive with intracellular Na+; but it was not (Fig. 3). Also, TPA should have inhibited EP formation from Na+ and MgATP, but it did not (Figs. 4 and 5). Thus the simplest explanation for these data, as well as the data reported previously in the literature, is that TPA binds exclusively to the outside site.

However, the results of TPA inhibition of pNPPase are not so clear. Previous work on pNPPase is consistent with either the outside gate remaining closed or opening periodically at a rate that is much slower than the pNPPase hydrolysis rate. If the outside gate remained closed during all conformations involved in pNPPase, then TPA should not have inhibited pNPPase; yet it did (Figs. 6 and 7). We considered the possibility that TPA could inhibit pumps that were not directly involved in pNPPase activity by examining the pNPP dependence of TPA inhibition. If TPA inhibited the phosphatase reaction by binding to pumps not in the pNPP-bound state (and that had the outside gate open), then an increase in pNPP concentration should have decreased TPA inhibition; in fact, the inhibition increased (data not shown). Thus we conclude that during the pNPPase reaction, the outside gate opens periodically, but at a rate that is much slower than the rate of pNPPase hydrolysis.

Because TPA and K+ compete at the outside site, and because our preparation allowed K+ access to both the inside and outside sites, it may seem puzzling that K+ and TPA did not compete for pNPPase. There are two simple models that could explain this result. In model 1, K+ release from the extracellular site might be a product of the reaction; increasing the product would not be able to overcome the inhibition by TPA. In model 2, for K+ and TPA not to compete, there must be a term in the denominator of the velocity equation that includes the factors K*K*TPA (because two K+ ions bind for pNPPase activity). For most enzyme kinetic models, this K2*TPA term is achieved for a dead end inhibitor only when there are two K+ ions and one TPA ion bound. This seems unlikely for the Na+-K+ pump on the basis of Forbush's (14) data and our results that K+ and TPA compete. However, for enzyme models in which there are branched steps that include slow steps, it is possible to generate a term with K2*TPA when TPA binds to pump with only one K+ ion bound. The generation of a squared substrate term, even though only one substrate is bound at a given time, is the basis of the model of Moczydlowski and Fortes (29) that explains the low- and high-affinity ATP effects observed with only one ATP bound per cycle. Moreover, the observation that TPA slows the rate of Rb+ deocclusion (14) strongly supports the existence of an E-K+-TPA form of the pump, and our pNPPase data are also consistent with such an enzyme form. A few possible branch points exist that may account for a K2*TPA term in the pNPPase equation: 1) whether the outside gate opens during pNPPase, 2) whether one or two K+ ions dissociate, and 3) whether TPA binds to empty pumps or pumps with one K+ bound. Furthermore, Forbush's (14) flickering gate model postulates a slow step between the individual K+ release (or binding) steps, thus satisfying the requirement for a slow step in the second model. Consequently, one can construct several branched models with slow steps that, in theory, would generate K2*TPA terms and therefore in which TPA and K+ would not compete for pNPPase activity. Note that these models would not apply to ATPase measurements because, in the ATPase reaction, K+ binding to the extracellular site is a substrate binding reaction going in the catalytic direction for ATPase. In the pNPPase reaction, while intracellular K+ is a substrate, extracellular K+ is not a substrate and would be an inhibitor or product inhibitor.

Previously, the effects of several organic amines on the phosphorylation of Na+-K+-ATPase were studied, but TPA was not included in that study (36). One particular observation was that many compounds had an apparent stimulatory effect on EP (36). Subsequent studies by these investigators of a sided preparation of reconstituted Na+-K+-ATPase revealed that these stimulatory effects were produced by amines binding to the extracellular side of the membrane (39). There were two possibilities proposed for these observations: 1) inhibition of the dephosphorylation reaction or 2) stimulation of the phosphorylation reaction (39). Although never experimentally tested, those authors concluded that extracellular amines stabilize a "pre-E1" conformational state that in the presence of Na+, Mg2+, and ATP is readily recruited to the genuine E1 state from the cytoplasmic side and is phosphorylated (39). In contrast, our interpretation of TPA stabilization of EP (Fig. 5A) is that this extracellular amine exerts its potentiation of EP by substantially reducing dephosphorylation (i.e., possibility 1 above). An additional prediction of our interpretation is that TPA should antagonize the stimulatory effect of K+ on EP hydrolysis; Fig. 5B confirms this prediction and strongly suggests that TPA preferentially binds to the outside-facing, and phosphorylated, conformation of the Na+-K+-ATPase. Whether the amines studied by Schuurmans Stekhoven et al. (37) act by the same mechanism remains to be tested.

TPA and K+. TPA binds to K+ sites on many proteins (e.g., K+ channels, Ref. 26; K+-HCO3 cotransporters, Ref. 9); yet, we found that TPA did not compete with inside K+ for pNPPase activity. Indeed, to our knowledge, this is the first demonstration in a membrane transport protein that a quaternary ammonium compound does not behave as a K+ competitor to a transporter conformation that can bind K+. Because of the novelty of this finding, we tested the ability of TPA to inhibit EP formation from Na+, Mg2+, and ATP, a reaction that depends solely on the cytoplasm-facing cation site. As predicted, TPA was unable to inhibit EP formation, even at 20-fold concentrations compared with the Ki for ATPase inhibition (Fig. 4), convincingly demonstrating an inability of TPA to bind from the inside. More intriguing is that when we measured the stability of EP (i.e., Mg2+ chelation to prevent rephosphorylation), TPA significantly reduced the level of spontaneous dephosphorylation (Fig. 5A). Given the EP stabilizing effect of TPA (Fig. 5) and in light of Forbush's observation (13) that TPA blocks Rb+ deocclusion in a competitive manner but that TPA itself is not occluded, a strong argument can be made for TPA binding to the K+ transport site with the external gate stuck open. Taken together, these data are consistent with an ordered reaction schema in which the closing of the extracellular gate facilitates EP hydrolysis rather than promotion of gate closure by Pi release.

An additional advantage to TPA stabilization is that it will allow us to lock the enzyme in a phosphorylated conformation with the outside transport gate open. This ability to lock the enzyme in a specific state with an inhibitor will aid future structural studies (e.g., using fluorescence resonance energy transfer) to characterize the relative positions of the different cytoplasmic domains as the enzyme undergoes conformational changes (e.g., after dilution to remove TPA).

In conclusion, we have shown that TPA inhibition occurs from the extracellular side of the protein and cannot bind from the cytoplasmic side, which is consistent with inferences from earlier studies of TPA effects on Rb+ occlusion by the Na+-K+-ATPase (14). These findings were achieved by methodically testing inherent competitive predictions on the basis of the Post-Albers model (1, 31) of the Na+-K+ pump. An added advantage to this approach is that investigators can easily exploit it by using recombinant enzyme. Using the side-specific inhibition of TPA, we have demonstrated that indeed both ATP and Pi can bind to the Na+-K+-ATPase while the transport site is facing the extracellular space with the occlusion gate open. Therefore, kinetic models that show that the low-affinity ATP site becomes available only after K+ occlusion should be adjusted to accommodate the possibility that the nucleotide-binding domain is accessible before closing of the extracellular gate.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by (listed alphabetically) American Heart Association (AHA) Grant 030161N (to C. Gatto), AHA Predoctoral Fellowship 0315236Z (to J. B. Helms), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37512 (to M. A. Milanick), and National Institute of General Medical Sciences Grant GM-061583 (to C. Gatto).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Charles J. Costa (Eastern Illinois University), Dr. John S. Willis (University of Georgia), and Dr. Pablo Artigas (Rockefeller University) for helpful comments on the manuscript. In addition, we thank Drs. Victoria Borowicz and Steven Juliano (Illinois State University) for statistical expertise used in Figs. 47.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. Gatto, Division of Biomedical Sciences, Dept. of Biological Sciences, Illinois State Univ., 210 Julian Hall, Campus Box 4120, Normal, IL 61790-4120 (e-mail: cgatto{at}ilstu.edu)

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.

1 The term "phosphointermediate" generally refers to a covalent bond between the phosphate and the aspartic acid residue, but it is also clear that many of the properties of some of the phosphointermediates are similar to the properties of the enzyme with phosphate bound noncovalently. In many studies, phosphate is added and there is an equilibrium between E:P and E-P that depends not only on the ionic conditions but also on the source of the enzyme (7, 11). "Gate" refers to a conformational change that allows access to the respective solution. This could include twisting of the transmembrane helices relative to each other or a physical capping of an access channel. Back

2 The nomenclature of E1/E2 for pNPPase can be confusing. In many reports citing this result, pNPPase is identified as occurring in an E2 state, and in other (usually non-pNPP) studies, E2 is sometimes identified as a conformation in which the transport site has extracellular access. Back


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