Functional analysis of human Na~+/K~+-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes

AIM: Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine(FHM2, SHM). METHODS: cRNA of human Na+/K+-ATPase α2- and β1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/β interaction sites or in the α2-subunit's C-terminal region were investigated. Mutants were analyzed by the twoelectrode voltage-clamp(TEVC) technique on Xenopus oocytes. Stationary K+-induced Na+/K+ pump currents were measured, and the voltage dependence of apparent K+ affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na+ buffers to analyze kinetics and voltage-dependent presteady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS: Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant dif-ferences in the voltage dependence of K+-induced currents, and analysis of the transient currents indicated that the extracellular Na+ affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K+ interaction, D999H affected the voltage dependence of K+-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na+ was reduced. CONCLUSION: The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.     

Membrane (Na+ + K+)-ATPase of canine brain,heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures

Effects of commonly used purification procedures on the yield and specific activity of (Na⁺ + K⁺)-ATPase (Mg²⁺-dependent, Na⁺ + K⁺-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na⁺ + K⁺)-ATPase and the ratio between (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na⁺ + K⁺)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na⁺ + K⁺)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter.     

Extracellular Allosteric Na Binding to the Na,K-ATPase in Cardiac Myocytes

Whole-cell patch-clamp measurements of the current, I_p, produced by the Na⁺,K⁺-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I_p over the extracellular Na⁺ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na⁺,K⁺-ATPase mechanism, where extracellular Na⁺ should act as a competitive inhibitor of extracellular K⁺ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K⁺ ions into the cytoplasm. The increase in I_p is consistent with Na⁺ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K⁺ to the cytoplasm, E2(K⁺)₂ → E1 + 2K⁺. At normal physiological concentrations of extracellular Na⁺ of 140 mM, it is to be expected that binding of Na⁺ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme.     

Dual Mechanisms of Allosteric Acceleration of the Na,K-ATPase by ATP

Investigations of the E2 → E1 conformational change of Na⁺,K⁺-ATPase from shark rectal gland and pig kidney via the stopped-flow technique have revealed major differences in the kinetics and mechanisms of the two enzymes. Mammalian kidney Na⁺,K⁺-ATPase appears to exist in a diprotomeric (αβ)₂ state in the absence of ATP, with protein-protein interactions between the α-subunits causing an inhibition of the transition, which occurs as a two-step process: E2:E2 → E2:E1 → E1:E1. This is evidenced by a biphasicity in the observed kinetics. Binding of ATP to the E1 or E2 states causes the kinetics to become monophasic and accelerate, which can be explained by an ATP-induced dissociation of the diprotomer into separate αβ protomers and relief of the preexisting inhibition. In the case of enzyme from shark rectal gland, the observed kinetics are monophasic at all ATP concentrations, indicating a monoprotomeric enzyme; however, an acceleration of the E2 → E1 transition by ATP still occurs, to a maximum rate constant of 182 (± 6) s⁻¹. This indicates that ATP has two separate mechanisms whereby it accelerates the E2 → E1 transition of Na⁺,K⁺-ATPase αβ protomers and (αβ)₂ diprotomers.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

Structural Changes in the Catalytic Cycle of the Na,K-ATPase Studied by Infrared Spectroscopy

Pig kidney Na⁺,K⁺-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na₃⁺ to an E2P state was initiated by photolysis of P³-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg²⁺ and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na⁺,K⁺-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15°C. Several controls ensured that the observed difference signals were due to structural changes of the Na⁺,K⁺-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca²⁺-ATPase and H⁺,K⁺-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm⁻¹, respectively, for αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na₃⁺ to an E2P state. A negative band at 1730 cm⁻¹ is in line with the presence of a protonated Asp or Glu residue that coordinates Na⁺ in E1Na₃⁺. Infrared signals were also detected in the absorption regions of ionized carboxyl groups.     

Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

The Na⁺/K⁺-ATPase mediates electrogenic transport by exporting three Na⁺ ions in exchange for two K⁺ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb⁺ ions in the crystal structures of the Na⁺/K⁺-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na⁺ or K⁺ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na⁺ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na⁺/K⁺-ATPase α₂ subunit decreases the affinity for extracellular and intracellular Na⁺, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na⁺ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K⁺, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na⁺ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na⁺/Na⁺ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na⁺ release/binding mechanism. In analogy to the mechanism proposed for the H⁺ leak currents of the wild-type Na⁺/K⁺-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na⁺ occlusion reaction, thus destabilizing the Na⁺-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na⁺/K⁺-ATPase α subunit represents a structural and functional relay between Na⁺ binding site III and the intracellular cation occlusion gate.     

Species-specific peculiarities of functional reactions of the sodium pump to phosphorylation by protein kinase A

The Na⁺K⁺-ATPase isolated from shark rectal gland or pig kidney was inserted into liposomes and phosphorylated with cAMP-dependent protein kinase without detergent. Stoichiometry of phosphorylation of a-subunit of the enzyme was 0.9 and 0.2 mol P_i/mol α-subunit of the pig kidney and shark gland, respectively. The phosphorylation of the shark Na⁺, K⁺-ATPase led to an increase in maximum of hydrolytic activity dependent on the cytoplasmic sodium concentration and the extracellular activation with potassium ions. On the contrary, the phosphorylation of the sodium pump of the pig kidney did not produce any significant functional effect.     

Species-specific peculiarities of functional reactions of the sodium pump to phosphorylation by protein kinase A

The Na⁺,K⁺-ATPase isolated from shark rectal gland or pig kidney was inserted into liposomes and phosphorylated with cAMP-dependent protein kinase without detergent. Stoichiometry of phosphorylation of α-subunit of the enzyme was 0.9 and 0.2 mol P_i/mol α-subunit of the pig kidney and shark gland, respectively. The phosphorylation of the shark Na⁺,K⁺-ATPase led to an increase in maximum of hydrolytic activity dependent on the cytoplasmic sodium concentration and the extracellular activation with potassium ions. On the contrary, the phosphorylation of the sodium pump of the pig kidney did not produce any significant functional effect.     

Two gears of pumping by the sodium pump

The kinetics of the phosphorylation and subsequent conformational change of Na⁺,K⁺-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na⁺)₃ state. On mixing with ATP in the presence of Mg²⁺, a fluorescence increase occurred, due to enzyme conversion into the E2P state. The fluorescence change accelerated with increasing ATP concentration until a saturating limit in the hundreds of micromolar range. The amplitude of the fluorescence change (ΔF/F₀) increased to 0.98 at 50 μM ATP. ΔF/F₀ then decreased to 0.82 at 500 μM. The decrease was attributed to an ATP-induced allosteric acceleration of the dephosphorylation reaction. The ATP concentration dependence of the time course and the amplitude of the fluorescence change could not be explained by either a one-site monomeric enzyme model or by a two-pool model. All of the data could be explained by an (αβ)₂ dimeric model, in which the enzyme cycles at a low rate with ATP hydrolysis by one α-subunit or at a high rate with ATP hydrolysis by both α-subunits. Thus, we propose a two-gear bicyclic model to replace the classical monomeric Albers-Post model for kidney Na⁺,K⁺-ATPase.     

Sensitivity of the (Na+ + K+)-ATPase to state-dependent inhibitors: Effects of digitonin and triton X-100

Treatment of a purified (Na⁺ + K⁺)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na⁺ + K⁺)-ATPase reaction inhibited more than the K⁺-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na⁺ + K⁺)-ATPase reaction but not the K⁺-phosphatase reaction; however, treatment with digitonin made the K⁺-phosphatase reaction almost as sensitive to oligomycin as the (Na⁺ + K⁺)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K⁺-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K⁺-phosphatase reaction more than the (Na⁺ + K⁺)-ATPase reaction). Both digitonin and Triton markedly increased the K_0.5 for K⁺ as activator of the K⁺-phosphatase reaction, with little effect on the K_0.5 for K⁺ as activator of the (Na⁺ + K⁺)-ATPase reaction. In contrast, increasing the K_0.5 for K⁺ in the K⁺-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K⁺-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E₁ conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E₂ state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K_0.5 for K⁺ in the K⁺-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na⁺ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na⁺ plus nucleotide.     

Different domain organization of two main conformational states of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase

The Na⁺/K⁺-ATPase generates an electrochemical gradient of Na⁺ and K⁺, which is necessary for the functioning of animal cells. During the catalytic act, the enzyme passes through two principal conformational states, E₁ and E₂. To assess the domain organization of the protein in these conformations, thermal denaturation of Na⁺/K⁺-ATPases from duck salt gland and from rabbit kidney has been studied in the absence and in the presence of Na⁺ or K⁺, which induce the transition to E₁ or E₂. The melting curves for the ion-free forms of the two ATPases have different shapes: the rabbit protein shows one transition at 56.1°C, whereas the duck protein shows two transitions, at 49.8 and 56.9°C. Addition of Na⁺ or K⁺ ions abolishes the difference in thermal behavior between these enzymes, but through opposite effects. The melting curves for the E₂ conformation (K⁺ bound) in both cases exhibit a single peak of heat absorption at ∼63°C. For the E₁ conformation (Na⁺ bound), each melting curve has three peaks, indicating denaturation of three domains. The difference in the domain organization of Na⁺/K⁺-ATPase in the E1 and E2 states may account for the different sensitivity to temperature, proteolysis, and oxidative stress observed for the two enzyme conformations.     

Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes ofXenopus laevis

Summary Electrophysiological experiments were performed to analyze the Na⁺/K⁺-ATPase in full-grown prophase-arrested oocytes ofXenopus laevis. If the Na⁺/K⁺-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na⁺-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na⁺/K⁺-ATPase and varies with intra-cellular Na⁺ activity (a _Na ⁱ ), and extracellular K⁺ (K ₀ ⁺ ) It is concluded that variations ofa _Na ⁱ among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes ofX. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence ona _Na ⁱ that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na⁺/2K⁺ pump. The voltage dependence of the pump current was investigated at saturating values ofa _Na ⁱ (33 mmol/liter) and of K ₀ ⁺ (3 mmol/liter) in the range from –200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

Capsazepine (CPZ) inhibits Na⁺,K⁺-ATPase-mediated K⁺-dependent ATP hydrolysis with no effect on Na⁺-ATPase activity. In this study we have investigated the functional effects of CPZ on Na⁺,K⁺-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na⁺,K⁺-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na⁺,K⁺-ATPase current in the presence of extracellular K⁺. In contrast, CPZ stimulated pump current in the absence of extracellular K⁺. Similar conclusions were attained using HEK293 cells loaded with the Na⁺ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na⁺,K⁺-ATPase indicated that CPZ stabilizes ion interaction with the K⁺ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na⁺ congener at the Na⁺ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na⁺,K⁺-ATPase. CPZ increased oxonol VI fluorescence in the absence of K⁺, reflecting increased Na⁺ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K⁺, likely indicating opening of an intracellular pathway selective for K⁺. As revealed by the recent crystal structure of the E₁.AlF₄ ^-.ADP.3Na⁺ form of the pig kidney Na⁺,K⁺-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K⁺ sites in Na⁺,K⁺-ATPase.     

Na+−K+-ATPase activity of glial,neuronal, and synaptosomal enriched fractions from normal and freezing-injured rabbit cerebral cortex

This paper investigates the kinetic parameters of Na⁺–K⁺-ATPase in glial, neuronal, and synaptosomal enriched fractions isolated from rabbit cerebral cortex. Under normal conditions, kinetic parameters-V_max and K _0.5 ^K+ -of Na⁺–K⁺-ATPase are the same in the three fractions, suggesting that this enzyme behaves as the same molecular entity. Following a cryogenic lesion, the alterations of these parameters appear to be different in the different fractions. These data suggest that the same enzyme exhibits various responses when exposed to the same pathological event. The dissimilar lipid composition of the Na⁺–K⁺-ATPase environment, and/or different adaptative responses to abnormal ion concentrations in glial, neuronal, and synaptosomal fractions could account for these different responses.     

Mechanism of Mg Binding in the Na,K-ATPase

The Mg²⁺ dependence of the kinetics of the phosphorylation and conformational changes of Na⁺,K⁺-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na⁺)₃ state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg²⁺ to saturating values of 195 (± 6) s⁻¹ and 54 (± 8) s⁻¹ for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (K_d) of Mg²⁺ interaction with the E1ATP(Na⁺)₃ state was estimated as 0.069 (± 0.010) mM. This is virtually identical to the estimated value of the K_d of Mg²⁺-ATP interaction in solution. Within the enzyme-ATP-Mg²⁺ complex, the actual K_d for Mg²⁺ binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation.     

Effects of prostaglandin A2 on Na+, K+-ATPase activity in basolateral plasma membrane of rat intestine in vitro

Prostagladin A₂, which prevents intestinal ulcers produced by administration of nonsteroidal antiinflammatory compounds such as indomethacin, inhibited the Na⁺,K⁺-ATPase activity in basolateral plasma membrane of rat intestine significantly. Prostaglandin A₂ inhibited mainly the Na⁺-dependent phosphorylation step in the overall reaction of Na⁺,K⁺-ATPase. This decrease of the Na⁺,K⁺-ATPase activity by prostaglandin A₂ was due to the decrease of Vmax of the enzyme and of the affinity of the enzyme for Na⁺. It was also suggested that the presence of both Δ^5,6 and Δ^10,11 structure of prostaglandin A₂ may be necessary for the inhibition of the Na⁺,K⁺-ATPase activity.     

Mechanisms of activation of renal (Na+ + K+)-ATPase in the rat Effects of acute and chronic administration of dexamethasone

The mechanisms of activation of renal (Na⁺ + K⁺)-ATPase by administration of the synthetic glucocorticoid hormone, dexamethasone, have been investigated in adrenalectomized rats. Chronic treatment with dexamethasone (1–5 mg/100 g body wt. daily for 5 days) stimulated (Na⁺ + K⁺)-ATPase specific activity in crude homogenated and microsomal fractions of renal cortex (by approx. 100–150%) and renal medulla (by approx. 100%). Acute treatment with dexamethasone (0.5–10 mg/100 g body wt.) also stimulated enzyme activity in crude homogenates and microsomal fractions of renal cortex and medulla (by approx. 40–50%). Stimulation was dose dependent and occurred within 2h after hormone treatment. In vitro addition of dexamethasone (10^?4–10^?8 M) to microsomal fractions did not modify the specific activity of (Na⁺ + K⁺)-ATPase. Stimulation of (Na⁺ + K⁺)-ATPase activity by acute and chronic administration of the hormone was demonstrated whether specific activities were expressed as a function of cellular protein or cellular DNA. Dexamethasone treatment increased the ratios protein:DNA and, to a lesser extent, the ratios RNA:DNA. However, these effects were mainly due to a reduction in the renal contents of DNA, which suggests that the observed enzyme activation is not due to an action of the hormone on renal hypertrophy. Dexamethasone also reduced cellular DNA contents in the liver. The characteristics of the activation process were essentially similar after treatment with single or multiple doses of the hormone. There were increases in the value for Na⁺ (approx. 100%), K⁺ (approx. 40%) and ATP (approx. 160%). The K_m values for Na⁺ (approx. 17 mM) and K⁺ (approx. 1.8 mM) were unchanged and there was a small increase in the K_m value for ATP (0.7 mM as against 1.7 mM). There was no difference in the Hill coefficients for the three substrates. The levels of the high-energy P_i intermediate of the (Na⁺ + K⁺)-ATPase reaction were augmented by dexamethasone treatment and the increased levels were quantitatively correlated with the observed stimulation of (Na⁺ + K⁺)-ATPase specific activity. The apparent turnover numbers of the reaction remained unchanged. The specific activity of the ouabain-sensitive p-nitrophenylphosphatase increased proportionally to the increase in (Na⁺ + K⁺)-ATPase specific activity. Enzyme activation by acute dexamethasone treatment occurred in the absence of changes in glomerular filtration rate and tubular Na⁺ excretion.These results indicate that (Na⁺ + K⁺)-ATPase activation by acute and chronic dexamethasone treatment represents an increase in the number of enzyme units with little or no change in the kinetic properties (affinity, cooperativity) of the enzyme. In addition, the information presented suggests a direct regulatory effect of glucocorticoid hormones on the activity of renal (Na⁺ + K⁺)-ATPase and is inconsistent with the concept that changes in Na⁺ loads mediate the effects of these hormones on enzyme activity. Instead, the results suggests a primary role for glucocorticoid hormones in the renal regulation of Na⁺ homeostasis.