Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

Kinetics of K(+) occlusion by the phosphoenzyme of the Na(+),K(+)-ATPase

Investigations of K⁺-occlusion by the phosphoenzyme of Na⁺,K⁺-ATPase from shark rectal gland and pig kidney by stopped-flow fluorimetry reveal major differences in the kinetics of the two enzymes. In the case of the pig enzyme, a single K⁺-occlusion step could be resolved with a rate constant of 342 (±26) s⁻¹. However, in the case of the shark enzyme, two consecutive K⁺-occlusions were detected with rate constants of 391 (±19) s⁻¹ and 48 (±2) s⁻¹ at 24°C and pH 7.4. A conformational change of the phosphoenzyme associated with K⁺-occlusion is, thus, the major rate-determining step of the shark enzyme under saturating concentrations of all substrates, whereas for the pig enzyme the major rate-determining step under the same conditions is the E2 → E1 transition and its associated K⁺ deocclusion and release to the cytoplasm. The differences in rate constants of the K⁺ occlusion reactions of the two enzymes are paralleled by compensating changes to the rate constant for the E2 → E1 transition, which explains why the differences in the enzymes' kinetic behaviors have not previously been identified.     

Rate limitation of the Na(+),K(+)-ATPase pump cycle

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.     

Simplified digitalis-like compounds acting on Na(+), K(+)-ATPase

Digitalis compounds are used in the treatment of congestive heart failure as positive inotropic agents; their action is mainly due to the inhibition of Na(+),K(+)-ATPase. A well-known drawback is their arrhythmogenic potential. Attempts to find safer digitalis-like compounds by means of molecular simplifications of the typical 5beta,14beta-steroidal skeleton, which appeared in the medicinal chemistry literature from 1990 until 2002, are briefly reviewed. Several novel achievements were obtained in order to better understand the requisites of the digitalis binding site on Na(+), K(+)-ATPase. Only minor simplification, such as cleavage of the D ring of the digitalis skeleton, could preserve the desired inotropic activity, while highly simplified digitalis-like compounds failed to give sufficiently high inotropic potency, even in the presence of a powerful pharmacophore, such as the O-aminoalkyloxime group.     

Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway

We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.     

Mechanism of the rate-determining step of the Na(+),K(+)-ATPase pump cycle

The kinetics of the E(2) --> E(1) conformational change of unphosphorylated Na(+),K(+)-ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E(2) conformation. When rabbit kidney enzyme was mixed with NaCl alone, tris ATP alone or NaCl, and tris ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/tau, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris ATP of 54 +/- 3 s(-1) in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na(+) to the enzyme in the E(2) state with a dissociation constant of 31 +/- 7 mM, which induces a subsequent rate-limiting conformational change to the E(1) state. Similar behavior, but with a decreased saturating value of 1/tau, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly lower amplitude of the fluorescence change and a higher saturating value of 1/tau for both the NaCl and choline chloride titrations. The results suggest that Na(+) ions or salt in general play a regulatory role, similar to that of ATP, in enhancing the rate of the rate-limiting E(2) --> E(1) conformational transition by interaction with the E(2) state.     

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Extracellular signal-regulated protein kinases mediate H(+),K(+)-ATPase alpha-subunit gene expression.

Extracellular signal-regulated protein kinases (ERKs) are important in many cellular functions. We and others have previously reported that prolonged exposure of gastric parietal cells to epidermal growth factor (EGF) enhanced gastric acid secretion stimulated by secretagogues via ERKs. In this study, we examined whether ERKs regulated H(+),K(+)-ATPase alpha-subunit gene expression using a gastric cancer cell line, AGS. EGF induced ERK activity time- and dose-dependently with a maximal effect observed at 10 min and 10 nM, respectively. The MEK inhibitors, U0126 and PD-98059, dose-dependently inhibited the ERK activity stimulated by EGF. To test H(+),K(+)-ATPase alpha-subunit gene expression, we transfected AGS cells with a plasmid containing a canine H(+),K(+)-ATPase alpha-subunit gene promoter fused to a luciferase reporter gene. EGF induced luciferase activity in transfected cells; this effect was inhibited by the MEK inhibitors, suggesting that EGF-induced gene expression involved the ERK pathway. When AGS cells were transfected with the reporter plasmids in conjunction with an expression vector encoding constitutively active MEK1, luciferase activity was strongly enhanced; this effect was attenuated by the MEK inhibitors or by an additional cotransfection of dominant negative MEK1. Taken together, our results led us to conclude that the ERK pathway may mediate H(+),K(+)-ATPase alpha-subunit gene expression, contributing to gastric acid secretion in parietal cells.     

Mutation of aspartate 804 of Na(+),K(+)-ATPase modifies the cation binding pocket and thereby generates a high Na(+)-ATPase activity

A series of six different mutants (D804A, D804E, D804G, D804N, D804Q, and D804S) of aspartate 804 present in transmembrane segment 6 of the rat Na(+),K(+)-ATPase alpha(1)-subunit were prepared and expressed in Sf9 cells by use of the baculovirus expression system. In contrast to the wild-type enzyme all mutants except D804Q showed a very high Na(+)-ATPase activity, which was hardly further stimulated by the addition of K(+). The ATPase activity of the mutants was already nearly maximal at 10 microM ATP and most of them could be phosphorylated in the absence of Na(+) at pH 6.0 and 21 degrees C, suggesting that they strongly prefer the E(1) over the E(2) conformation. However, Na(+) dose-dependently lowered the steady-state phosphorylation level, as a consequence of the increased affinity for Na(+) in the dephosphorylation reaction of the mutants compared to the wild-type enzyme. Conversely, the affinity for K(+) in the dephosphorylation reaction was decreased for the mutants as compared to that for the wild-type enzyme. When the pH was increased or the temperature was decreased, the phosphorylation level of the mutants decreased and the Na(+) activation in the phosphorylation reaction became apparent. It is concluded that upon mutation of aspartate 804 the affinity of the cation-binding pocket is changed relatively in favor of Na(+) instead of K(+), as a consequence of which the enzyme has obtained a preference for the E(1) conformation.     

Nitric oxide -- superoxide cooperation in the regulation of renal Na(+),K(+)-ATPase

The aim of this study was to investigate whether endogenous superoxide anion is involved in the regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities. The study was performed in male Wistar rats. Compounds modulating superoxide anion concentration were infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. We found that infusion of a superoxide anion-generating mixture, xanthine oxidase (1 mU/min per kg) + hypoxanthine (0.2 mumol/min per kg), increased the medullary Na(+),K(+)-ATPase activity by 49.5% but had no effect on cortical Na(+),K(+)-ATPase and either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase. This effect was reproduced by elevating endogenous superoxide anion with a superoxide dismutase inhibitor, diethylthiocarbamate. In contrast, a superoxide dismutase mimetic, TEMPOL, decreased the medullary Na(+),K(+)-ATPase activity. The inhibitory effect of TEMPOL was abolished by inhibitors of nitric oxide synthase (L-NAME), soluble guanylate cyclase (ODQ) and protein kinase G (KT5823). The stimulatory effect of diethylthiocarbamate was not observed in animals pretreated with a synthetic cGMP analogue, 8-bromo-cGMP. An inhibitor of NAD(P)H oxidase, apocynin (1 mumol/min per kg), decreased the Na(+),K(+)-ATPase activity in the renal medulla and its effect was prevented by L-NAME, ODQ or KT5823. In contrast, a xanthine oxidase inhibitor, oxypurinol, administered at the same dose was without effect. These data suggest that NAD(P)H oxidase-derived superoxide anion increases Na(+),K(+)-ATPase activity in the renal medulla by reducing the availability of NO. Excessive intrarenal generation of superoxide anion may upregulate medullary Na(+),K(+)-ATPase leading to sodium retention and blood pressure elevation.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

Sodium and ATP affinities of the cardiac Na(+),K(+)-ATPase in spontaneously hypertensive rats

The Na(+),K(+)-ATPase is postulated to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembrane Na(+)-efflux from cardiac cells in spontaneously hypertensive rats (SHR). In the investigated group of SHR the systolic blood pressure and the heart weight were increased by 48% and by 60%, respectively. Upon activating the cardiac Na(+),K(+)-ATPase with substrate, its activity was lower in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (Vmax) by 28% which was accompanied with lowered affinity of the ATP-binding site as indicated by the increased value of Michaelis-Menten constant (Km) by 354% in SHR. During activation with Na(+), we observed an inhibition of the enzyme in hearts from SHR at all tested Na(+) concentrations. The value of Vmax decreased by 37%, and the concentration of Na(+) that gives half maximal reaction velocity (KNa) increased by 98%. This impairment in the affinity of the Na(+)-binding site together with decreased affinity to ATP in the molecule of the Na(+),K(+)-ATPase are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of SHR.     

Insect Na(+)/K(+)-ATPase

Na(+)/K(+)-ATPase (sodium/potassium pump) is a P-type ion-motive ATPase found in the plasma membranes of animal cels. In vertebrates, the functions of this enzyme in nerves, heart and kidney are well characterized and characteristics a defined by different isoforms. In contrast, despite different tissue distributions, insects possess a single isoform of the alpha-subunit. A comparison of insect and vertebrate Na(+)/K(+)-ATPases reveals that although the mode of action and structure are very highly conserved, the specific roles of the enzyme in most tissues varies. However, the enzyme is essential for the function of nerve cells, and in this respect Na(+)/K(+)-ATPase appears to be fundamental in metazoan evolution.     

Reduction of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to chemically induced hyperhomocysteinemia

Hyperhomocysteinemia occurs in homocystinuria, an inherited metabolic disease clinically characterized by thromboembolic episodes and a variable degree of neurological dysfunction whose pathophysiology is poorly known. In this study, we induced elevated levels of homocysteine (Hcy) in blood (500 M), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 mol/g of body weight) twice a day at 8-hr intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the hippocampus of treated Hcy- and saline-treated rats. Chronic administration of Hcy significantly decreased (40%) Na⁺,K⁺-ATPase activity but did not alter Mg²⁺-ATPase activity. Considering that Na⁺,K⁺-ATPase plays a crucial role in the central nervous system, our results suggest that the brain dysfunction found in homocystinuria may be related to the reduction of brain Na⁺,K⁺-ATPase activity.     

The Occlusion of Rb(+) in the Na(+)/K(+)-ATPase. II. The effects of Rb(+), Na(+), Mg2(+), or ATP on the equilibrium between free and occluded Rb(+).

We used the direct route of occlusion to study the equilibrium between free and occluded Rb(+) in the Na(+)/K(+)-ATPase, in media with different concentrations of ATP, Mg(2+), or Na(+). An empirical equation, with the restrictions imposed by the stoichiometry of ligand binding was fitted to the data. This allowed us to identify which states of the enzyme were present in each condition and to work out the schemes and equations that describe the equilibria between the ATPase, Rb(+), and ATP, Mg(2+), or Na(+). These equations were fitted to the corresponding experimental data to find out the values of the equilibrium constants of the reactions connecting the different enzyme states. The three ligands decreased the apparent affinity for Rb(+) occlusion without affecting the occlusion capacity. With [ATP] tending to infinity, enzyme species with one or two occluded Rb(+) seem to be present and full occlusion seems to occur in enzymes saturated with the nucleotide. In contrast, when either [Mg(2+)] or [Na(+)] tended to infinity no occlusion was detectable. Both Mg(2+) and Na(+) are displaced by Rb(+) through a process that seems to need the binding and occlusion of two Rb(+), which suggests that in these conditions Rb(+) occlusion regains the stoichiometry of the physiological operation of the Na(+) pump.     

Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).