Binding of a Single Rb+ Increases Na+/K+-ATPase, Activating Dephosphorylation without Stoichiometric Occlusion

We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 μm ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained. We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 μm ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained. During its physiological operation, the plasma membrane Na+/K+-ATPase couples the hydrolysis of ATP to the transport of three intracellular sodium ions in exchange for two extracellular potassium ions. According to the currently accepted scheme of ATP hydrolysis, this takes place in a series of steps that include at least (Fig. 1) (i) the (Na+ + Mg2+)-dependent phosphorylation by ATP of the E1 conformer of the enzyme to form the E1P phosphoenzyme (phosphorylation requires the binding of three Na+ ions that are taken up from the cytosol and trapped in E1P), (ii) the E1P → E2P transition of the phosphoenzyme (this is accompanied by the release of Na+ to the extracellular medium), (iii) the activation by extracellular K+ of the dephosphorylation of E2P (it is generally accepted that this requires the binding of two K+ ions that are taken up from the extracellular medium and trapped in E2), and finally (iv), the return of E2 to E1 with the release K+ into the cytosol. The binding of ATP to a noncatalytic site in E2 whose Km (≈200 μm) is at least 103 times higher than the Km of the active site of the ATPase (≈0.2 μm) leads to a 100-fold increase in the rate of the E2(K2)→E1+ 2K+ reaction. Hence, as the concentration of ATP tends to zero the release of K+ becomes the slowest step of the Na+/K+-ATPase cycle (see Refs. 1Skou J.C. Esmann M. J. Bioenerg. Biomembr. 1992; 24: 249-281PubMed Google Scholar and 2Glynn I.M. Karlish S.J.D. Annu. Rev. Biochem. 1990; 59: 171-205Crossref PubMed Scopus (177) Google Scholar). The Na+ and K+ taken up by the enzyme in steps (i) and (iii) are in a state in which they exchange slowly with the incubation media. This is usually called “occlusion” and is considered to be the expression of an intermediate step in the movement of Na+ and K+ across the ATPase from one surface of the membrane to the other. No direct structural information with enough resolution is yet available to identify the occlusion domains in the enzyme. Phosphorylation is absolutely dependent on Na+, whereas for activation of dephosphorylation K+ can be replaced with varying degrees of effectiveness by Rb+, Cs+, Li+,NH4+, or Tl+. At least Rb+ (3Beaugé L.A. Glynn I.M. Nature. 1979; 280: 510-512Crossref PubMed Scopus (80) Google Scholar) or Tl+ (4Rossi R.C. Nørby J.G. J. Biol. Chem. 1993; 268: 12579-12590Abstract Full Text PDF PubMed Google Scholar) also replaces K+ for occlusion. Dephosphorylation persists in the absence of K+, albeit at a rate that is 100× slower than in the presence of K+. This gives rise to an ATPase activity, called the Na+-ATPase, which requires Na+ and Mg2+ and is coupled to the transport of Na+. Na+-ATPase is switched off as K+ or its congeners drive dephosphorylation toward the pathway that leads to occlusion. The model in Fig. 1 has three branching points at the E2P states that lead to three pathways. The velocity of dephosphorylation in the absence of products (vdephos) will be the sum of the contributions of the three pathways, i.e. vdephos=k40[E2P]+k41[E2PK]+k42[E2PK2] (Eq. 1) The relative abundance of each state of E2P will depend on the concentration of K+. Assuming rapid equilibrium for the addition of K+, this dependence will be [E2P]=E2P01+[K+]KK1+[K+]2KK1KK2 (Eq. 2) [E2PK]=E2P01+KK1[K+]+[K+]KK2 (Eq. 3) [E2PK2]=E2P01+KK1[K+]+KK1KK2[K+]2 (Eq. 4) where KK1 and KK2 are equilibrium dissociation constants and E2P0 = [E2P] + [E2PK] + [E2PK2]. E2P0 will be equal to the concentration of E2P in the absence of K+ when all the enzyme is catalyzing Na+-ATPase activity. Equations 2, 3, 4 illustrate the more general fact that, as [K+] goes from zero to infinity, the following will occur. (i) [E2P] will decrease continuously toward zero (Equation 2), and the same will happen with Na+-ATPase activity. (ii) [E2PK] will start at zero, pass through a maximum, and then tend to zero (Equation 3). If we accept the general view that k40 = k41, then this process, which is usually not explicitly included in the reaction schemes of the Na+/K+-ATPase, will have no expression on vdephos. (iii) [E2PK2] will start at zero and tend to saturation at a value equal to the total amount of E2P along an S-shaped curve with zero initial slope (Equation 4). The existence at non-saturating [K+] of pathways not requiring occlusion means that the rate of occlusion will be lower than the ATPase activity, a difference that will be cancelled at saturating [K+] when all the reaction flow passes through the E2(K2)→E1 + 2K+ step (cf. Equations 1 and 4). We have shown (5Kaufman S.B. González-Lebrero R.M. Schwarzbaum P.J. Nørby J.G. Garrahan P.J. Rossi R.C. J. Biol. Chem. 1999; 274: 20779-20790Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) that if we assume that k41 = k40, then the pathways not involving occlusion are too slow to explain why deocclusion rate is slower than ATPase activity at nonsaturating [K+]. We also showed that the most economical way to solve this discrepancy is to abandon the restriction that k41 = k40 and to posit that k41 > k40; that is, that the binding of a single K+ to E2P increases the rate of dephosphorylation. Moreover, we proposed that dephosphorylation of E2PK does not involve occlusion of the ion. The experiments in this paper are an attempt to obtain direct evidence for this hypothesis. For this, we looked at the effects of Rb+ as congener of K+ on the initial rates of occlusion and of dephosphorylation and on the ATPase activity in enzymes incubated at the same temperature and in media of identical composition. The experiments were designed to test the following predictions. (i) If occlusion were strictly dependent on the binding of two Rb+ per E2P, then the function relating the rate of occlusion to Rb+ concentration should be a sigmoid curve of zero initial slope (see Equation 4). (ii) If acceleration of dephosphorylation took place after the binding of a single Rb+ to E2P, then dephosphorylation rate should increase with [Rb+] along a function of positive initial slope. (iii) If (i) and (ii) were fulfilled, then at sufficiently low [Rb+] the velocity of dephosphorylation should be higher than that of occlusion. (iv) At micromolar [ATP], ATPase activity versus [Rb+] should first increase above the value of the Na+-ATPase as a consequence of activation by a single Rb+ and, after passing through a maximum, tend to a lower value as [Rb+] reaches saturation. This is so because it is known that under these conditions the E2(K2) → E1+ 2K+ step is slower than any other. We further analyzed the consequences of our hypothesis by simulating the behavior of the model in Fig. 1 to which we added the pathway enclosed within the shaded area. Ex was included in the reaction scheme to evaluate whether the dephosphorylation of E2PK leads to occlusion. To perform this we fitted analytical solutions of the model to the experimental data of the effects of [Rb+] on the steady-state levels of ATPase activity, EP, and occluded Rb+, measured in media of identical composition and temperature as those used to measure the rates of dephosphorylation and occlusion. The values of the kinetic parameters thus obtained were used to predict the effects of Rb+ on the above mentioned rates. Results reported here constitute the first detailed study of the time course of Rb+ occlusion through the physiological route. This allowed us also for the first time to correlate the kinetics of occlusion with that of dephosphorylation. A preliminary report of some of the results presented in this work has been published (6Kaufman S.B. González-Lebrero R.M. Garrahan P.J. Rossi R.C. Ann. New York Acad. Sci. 2003; 986: 155-158Crossref PubMed Scopus (2) Google Scholar). Na+/K+-ATPase was partially purified from pig kidney outer medulla according to Jensen et al. (7Jensen J. Nørby J.G. Ottolenghi P. J. Physiol. (Lond.). 1984; 346: 219-241Crossref Scopus (66) Google Scholar) and kindly provided by the Department of Biophysics, University of Ärhus, Denmark. All the results shown in this paper were obtained from experiments performed at 25 °C with enzyme suspended in reaction media containing 150 mm NaCl, 10 μm ATP, 0.7 mm MgCl2, 0.2 mm EDTA, and 25 mm imidazole-HCl (pH 7.4 at 25 °C) and the concentrations of Rb+ indicated in the figures. The time courses of dephosphorylation and of occlusion were measured in experiments carried out using a rapid-mixing apparatus, by The experiments were the concentrations of Rb+ to the incubation medium in which the enzyme concentration performed steady-state Na+-ATPase activity. The time course of dephosphorylation was measured by the transition of the phosphoenzyme to a state after the addition of enough of as to the concentrations indicated in the to the figures. were by with concentration 10 as by Schwarzbaum et al. P.J. S.B. Rossi R.C. Garrahan P.J. PubMed Scopus Google Scholar). The time course of occlusion was measured using by occluded Rb+, occlusion was measured after the reaction by rapid following the in our R.C. S.B. González-Lebrero R.M. Nørby J.G. Garrahan P.J. Biochem. 1999; PubMed Scopus Google Scholar). This which the structural of the is to deocclusion that with rate constants of up to 25 without of Rb+ R.C. S.B. González-Lebrero R.M. Nørby J.G. Garrahan P.J. Biochem. 1999; PubMed Scopus Google Scholar). The initial rates of dephosphorylation and of Rb+ occlusion were from (i) the at = of the first of the fitted to the time courses (Fig. 2), (ii) the slope of the fitted to data of occluded Rb+ and of phosphoenzyme concentrations measured at the time of addition of Rb+ = and after a This which was to increase the of the the initial rates of occlusion and dephosphorylation, allowed a higher of data at the lower Rb+ concentrations (Fig. 3, when the rates were enough to initial rate conditions after initial rates of dephosphorylation and of Rb+ occlusion as a function of were after the shown in the to Fig. and or obtained from (see are of the data in A for Rb+ concentrations up to μm Rb+. are by for dephosphorylation and for are solutions of used to or data of dephosphorylation and occlusion A and an function of the form = + was fitted to the the are of the scheme in Fig. 1 to conditions and (iii) and the values of the rate constants in The steady-state concentration of occluded Rb+ was measured by in the rapid-mixing apparatus equal of a of Na+/K+-ATPase of in reaction media with the same media containing ATP and different concentrations of were incubated for 3.5 into the experiments using times from to 10 showed no in the of occluded that 3.5 was enough to The steady-state concentration of phosphoenzyme was measured by the same as that used for occlusion that was the reaction was by of the reaction with of an of 25 and mm ATPase activity was according to Schwarzbaum et al. P.J. S.B. Rossi R.C. Garrahan P.J. PubMed Scopus Google the amount of from The reaction medium was identical as that used for the occlusion and dephosphorylation concentration was 10 of times and to the hydrolysis of more than of the ATP, thus initial rate were measured in the media where all Na+ was replaced by K+. were to the results by on the using and for were as the of the of experimental was as or using the of Glynn and I.M. Biochem. J. PubMed Scopus Google that no was and were from ATP, and for the of were from All were of analytical the of Dephosphorylation and measured the time courses of dephosphorylation and of Rb+ occlusion in experiments using the same enzyme preparation in media of the same composition and All time courses were after the addition of Rb+ to enzymes that perform steady-state Na+-ATPase activity. Results are shown in Fig. 2, A and We the initial of and Rb+ from the values of the initial of time are as a function of Rb+ concentration in Fig. can be that with [Rb+] along This strongly that are the initial of sigmoid that not saturation at μm A of the results in Fig. is that for Rb+ concentrations than dephosphorylation is than This difference is cancelled at concentrations higher than from becomes At μm is as This is to be if at sufficiently [Rb+] dephosphorylation the → + the initial as a function of [Rb+] have been in to values and μm Rb+. is increases with [Rb+] at the concentrations This that binding of a single Rb+ to E2P is to promote dephosphorylation. these results are compatible with our hypothesis that k41 > The of the at low [Rb+] can be analyzed that (Eq. and (Eq. where is a whose value will be 1 or on whether or not leads to the occlusion of a single Rb+. that, under the conditions of our the initial rate of dephosphorylation will be the difference the rates of and of of the addition of Rb+ the enzyme Na+-ATPase activity in the rate of dephosphorylation will be by that of For this the in Equation If each state of E2P is as in Equations 2, 3, then at low Equations and be by (Eq. and (Eq. which that include a and a in the the initial slope will be positive in the results in Fig. if k41 > k40; that is, if is activation by a single Rb+. the a positive initial slope > If one Rb+ were occluded in be equal to and the initial slope be than that (cf. in Equations and This is in with our results in which the initial slope is that the absence of occlusion. of Rb+ on ATPase and Rb+ in the whose results are shown in Fig. ATPase activity was measured in media containing 10 μm ATP and from zero to μm Rb+. can be that the to [Rb+] is with an initial activation that no of and a at μm Rb+ (see the by a decrease to an value that is lower than that observed at zero We have shown R.C. Garrahan P.J. PubMed Scopus Google Scholar) that by Rb+ as that in the in Fig. 4 is not by of Na+ from the from which it the The results in Fig. 4 are to those obtained by J. Biol. Chem. Full Text PDF PubMed Google J. Biol. Chem. Full Text PDF PubMed Google Scholar). also with the by 1999; PubMed Scopus Google Scholar) an to K+ using mm ATP in a of kidney Na+/K+-ATPase with low for The most of the activation by Rb+ is that k41 > The of in the activation the idea that activation the binding of one Rb+ to the that if k41 = k40, ATPase activity continuously decrease with This is so because the of Rb+ be to the step from that by k40 to that by the E2 to E1 whose value at 10 μm ATP is (5Kaufman S.B. González-Lebrero R.M. Schwarzbaum P.J. Nørby J.G. Garrahan P.J. Rossi R.C. J. Biol. Chem. 1999; 274: 20779-20790Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). This mechanism for the by Rb+ shown in Fig. 4 and was first proposed by et al. J. Biol. Chem. Full Text PDF PubMed Google Scholar) in work on occlusion. The steady-state levels of and of and are sigmoid of Rb+ of these results with those in Fig. that the for Rb+ is higher for the steady-state results μm) than for the effects of Rb+ on the initial The of this discrepancy is that initial velocity the rate of Rb+ whereas the steady-state include the of the and of occluded Rb+. the rate of deocclusion is low at 10 μm ATP, the enzyme in its occluded which as a of occluded Rb+ as a function of The is a of Equation for the values of the parameters The and of the scheme in Fig. 1 for the conditions and (iii) and the values of the rate constants in The is the of the up to μm Fig. we the sum of the concentrations of and of occluded enzyme as a function of can be that the sum reaches a at μm Rb+. This is with the idea that at intermediate Rb+ concentrations is an of enzyme states that are states to that in the activation of dephosphorylation by a single Rb+. The experimental data of the steady-state values of ATPase activity (Fig. phosphorylation (Fig. and occlusion (Fig. were fitted to the following (Eq. where and are the values of the steady-state measured in the absence and presence of saturating concentrations of Rb+, is the of at intermediate Rb+ and and are constants for the dissociation of Rb+ from the enzyme. can be by the that the experimental Equation an of the three steady-state of the values of the parameters in 1 the (i) For Rb+ occlusion, and tended to values that were not different from This is with the of states with one Rb+ occluded per enzyme. (ii) > > for ATPase activity the existence of the maximum, and that the activity at Rb+ concentration is than the Na+-ATPase activity (Fig. 4). (iii) the of > > which with the continuously curve of versus (iv) for Rb+ occlusion is the value of for EP, as it is to be if all the enzyme from to as [Rb+] from zero to The for is equal to the value of at saturating this times the rate for the → E1+ step at 10 μm ATP the value of for the ATPase activity. This the idea that at saturating [Rb+] all the reaction flow takes place through this The values of and for each steady-state are sufficiently as to it that the three the same if the value of and for the three are used for and the the experimental data not values of the parameters of Equation after of the results in in a The of with k40 or is no experimental that k41 from k40 or (cf. Equation with Equations and 4). view of this we used an to k41 on binding of Rb+ to E2P, in which the initial rate of dephosphorylation will be (Eq. [Rb+] Equation 10 will (Eq. where (Eq. Equations and that, the values of E2P0 and of k40, the values of k41, and can be by Equation to the experimental results this is using the in Fig. 3, the value of k41 is or on the value of E2P0 used (see values the usually accepted values for k40 and for values of the parameters of Equation from the model in Fig. 1 at [Rb+] = using the rate constants in measured in the absence of E2P from the model in Fig. 1 at [Rb+] = using the rate constants in measured in the absence of Rb+. in a of the kinetic parameters used for of the scheme in Fig. 1 for different = k40 = k41 = k41 = et al. and Schwarzbaum et al. et al. and Schwarzbaum et al. et al. and Schwarzbaum et al. et al. and Schwarzbaum et al. et al. and Schwarzbaum et al. that a value higher than and that et al. a value higher than et al. and González-Lebrero et al. et al. and González-Lebrero et al. was as (see González-Lebrero et al. et al. and González-Lebrero et al. et al. (5Kaufman S.B. González-Lebrero R.M. Schwarzbaum P.J. Nørby J.G. Garrahan P.J. Rossi R.C. J. Biol. Chem. 1999; 274: 20779-20790Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and Schwarzbaum et al. P.J. S.B. Rossi R.C. Garrahan P.J. PubMed Scopus Google that J. Biol. Chem. Full Text PDF Google Scholar) a value higher than and that et al. J. Physiol. PubMed Scopus Google Scholar) a value higher than et al. (5Kaufman S.B. González-Lebrero R.M. Schwarzbaum P.J. Nørby J.G. Garrahan P.J. Rossi R.C. J. Biol. Chem. 1999; 274: 20779-20790Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and González-Lebrero et al. R.M. S.B. Nørby J.G. Garrahan P.J. Rossi R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was as (see González-Lebrero et al. R.M. S.B. Nørby J.G. Garrahan P.J. Rossi R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in a the Results and the of the in Fig. experimental results presented are with our hypothesis that the binding of a single Rb+ to E2P dephosphorylation without to the occlusion of Rb+. To if the of our hypothesis into the model (Fig. 1) is to predict the experimental results in this we fitted the analytical of the scheme in Fig. 1 to the data in three (i) k41 = k40 and no occlusion of Rb+ in Ex takes place = (ii) k41 of k40, and Ex one Rb+ = and (iii) k41 of k40, and Ex not Rb+ = all the was performed using the of steady-state experimental values in To the of parameters to be fitted we some of as indicated in This is because is experimental information on the values of these parameters from our work and the work of (see the of 3). The of parameters obtained by this were used to values of the initial rates of dephosphorylation and occlusion and to with the experimental results (Fig. 3, can be that (i) is to the A is obtained k41 of k40 but that Ex one Rb+ and the is no occlusion in Ex and k41 of k40 (iii) also gives the of the steady-state values in The of (i) to the results of ATPase activity is (Fig. 4). be that (iii) does not for the experimental This is so for the initial rates of occlusion and dephosphorylation in it is the one that a difference (iii) the slope of the rate of occlusion at low be that, for our experimental the slope when [Rb+] → is not zero because one Rb+ is occluded in Ex = 1) but that a of the occluded Rb+ is out its If this were the rate of deocclusion be the rate of the Ex → E1 reaction The value of this 3) the values of deocclusion rate that can be by our without of Rb+ (see and Rossi et al. R.C. S.B. González-Lebrero R.M. Nørby J.G. Garrahan P.J. Biochem. 1999; PubMed Scopus Google an to the of occluded Rb+ its does not to be the of our A more be that Rb+ occlusion does place but in one of the states of of these states be of E2P that one Rb+ and one Na+, as has been by 1999; PubMed Scopus Google Scholar). is not known if the ATPase by a single Rb+ is to drive active This be in view of the by et al. L.A. Glynn I.M. J. Physiol. (Lond.). 1979; Scholar) showed that a as that observed in Fig. 4 is by extracellular K+. The data presented here were in a that does not if transport if a were to measure the of K+ with 10 μm ATP with a low is a For this all the of of transport in the Na+ have been performed with concentrations of ATP and Na+ and K+ P.J. Glynn I.M. J. Physiol. Scopus Google Scholar). results in this paper the presence of an of behavior of the Na+/K+-ATPase, whose further study to the mechanism of active transport of Na+ and K+.