Involvement of protons in the ion transport cycle of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase

The effect of pH on electrogenic sodium transport by the Na⁺,K⁺-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na⁺,K⁺-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na⁺, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na⁺,K⁺-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.     

Electrogenic transport of sodium ions in cytoplasmic and extracellular ion access channels of Na+,K+-ATPase probed by admittance measurement technique

Electrogenic movements of sodium ions in cytoplasmic and extracellular access channel of the Na⁺,K⁺-ATPase have been studied by the admittance measurement technique which allows the detection of small changes of the membrane capacitance and conductance induced by phosphorylation of the ion pump. The measurements were carried out on a model system consisting of a bilayer lipid membrane, to which membrane fragments with ion pumps were adsorbed that contain the ion pumps in high density. Small changes of the membrane capacitance and conductance were induced by a fast release of ATP from caged ATP. The effect was measured at various frequencies and in solutions with different Na⁺ concentrations. The experimentally observed frequency dependences were explained using a theoretical model assuming that Na⁺ movement through the cytoplasmic access channel occurs in one step and through the extracellular access channel, in two steps. The phosphorylation of the protein by ATP leads to a block of the cytoplasmic access channel and an opening the extracellular access channel. The disappearance of electrogenic Na⁺ movements on the cytoplasmic side produces a negative change of capacitance and conductance, while the emergence of extracellular Na⁺ movements generates a positive change. Fitting the experimental dependences of capacitance and conductance by theoretical curves allowed the determination equilibrium and kinetic parameters of sodium transport in the access channels. The text was submitted by the authors in English.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Confining the sodium pump in a phosphoenzyme form: the effect of lead(II) ions

The effect of Pb²⁺ ions on the Na⁺,K⁺-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Experiments were performed by using the electrochromic styryl dye RH421. It is shown that Pb²⁺ ions can bind reversibly to the protein and do not affect the Na⁺ and K⁺ binding affinities in the E₁ and P-E₂ conformations of the enzyme. The pH titrations indicate that lead(II) favors binding of one H⁺ to the P-E₂ conformation in the absence of K⁺. A model scheme is proposed that accounts for the experimental results obtained for backdoor phosphorylation of the enzyme in the presence of Pb²⁺ ions. Taken together, our results clearly indicate that Pb²⁺ bound to the enzyme stabilizes an E₂-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb²⁺ ions are able to confine the Na⁺,K⁺-ATPase into a phosphorylated E₂ state.     

Effects of tricyclohexylhydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents

Tricyclohexylhydroxytin, commonly known as Plictran® inhibited Na⁺, K⁺ -ATPase activity of rat brain synaptosomes in a concentration-dependent manner with median inhibitory concentration (IC-50) of 2 μM. Both K⁺ -stimulated para-nitrophenylphosphatase and [3-H]-ouabain binding to synaptosomes were also inhibited by Plictran with IC-50 values of 11 and 30 μM, respectively. Altered pH and Na⁺, K⁺ -ATPase activity curves demonstrated comparable inhibition in buffered neutral and alkaline pH ranges, and no inhibition was observed in acidic pH. The inhibition of Na⁺, K⁺ -ATPase was independent of temperature. Kinetic studies of substrate (ATP) activation of Na⁺, K⁺ -ATPase indicated uncompetitive inhibition. Results also showed noncompetitive inhibition for p-nitrophenylphosphate and uncompetitive inhibition for K⁺ activations of p-nitrophenylphosphatase. Preincubation of synaptosomes with dithiothreitol, a sulfhydryl (SH) agent, resulted in the complete protection of Plictran inhibition of Na⁺, K⁺ -ATPase, K⁺ -para-nitrophenylphosphatase, and [3-H]-ouabain binding. The protection was specific and concentration dependent since cysteine and glutathione did not afford protection. These results indicate that Plictran inhibited Na⁺, K⁺ -ATPase by interacting with dephosphorylation of the enzyme-phosphoryl complex and exerted a similar effect to that of SH-blocking agents.     

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.     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

Identification of Electric-Field-Dependent Steps in the Na,K-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Identification of Electric-Field-Dependent Steps in the Na+,K+-Pump Cycle

The charge-transporting activity of the Na⁺,K⁺-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement, we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with BTEA (benzyltriethylammonium), which binds from the extracellular medium to the Na⁺,K⁺-ATPase’s transport sites in competition with Na⁺ and K⁺, but is not occluded within the protein. We find that only the occludable ions Na⁺, K⁺, Rb⁺, and Cs⁺ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na⁺ or K⁺ binding in a high field access channel is a major electrogenic reaction of the Na⁺,K⁺-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation.     

Electrogenic Properties of the Na+,K+-ATPase Probed by Presteady State and Relaxation Studies

Electrical measurements on planar lipid bilayers, patch/voltage clamp experiments, and spectroscopic investigations involving a potential sensitive dye are reviewed. These experiments were performed to analyze the kinetics of charge translocation of the Na⁺,K⁺-ATPase. High time resolution was achieved by applying caged ATP, voltage-jump, and stopped-flow techniques, respectively. Kinetic parameters and the electrogenicity of the relevant transitions in the Na⁺,K⁺-ATPase reaction cycle are discussed.     

Expression of the Catalytic Subunit of Ouabain-Sensitive H+,K+-ATPase in Rat Skin Epidermis

A comparative localization of Na⁺,K⁺-ATPase and ouabain-sensitive H⁺,K⁺-ATPase in rat skin was performed using in situ RNA hybridization and immunohistochemistry. Na⁺,K⁺-ATPase was predominantly detected in the basal layer of the epithelium, whereas the ouabain-sensitive H⁺,K⁺-ATPase, in the granular and prickle cell layers. The genes of these ATPases are thus expressed in epithelial cells at different stages of their development. The hypothesis was advanced that the ouabain-sensitive H⁺,K⁺-ATPase is involved in maintaining the skin pH value. The probes specific to the mRNAs of the full-size -subunit of the ouabain-sensitive H⁺,K⁺-ATPase and its truncated form were used to establish a similar distribution of both mRNA variants in skin.     

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.     

Short-Term Regulation of the Proximal Tubule Na+,K+-ATPase: Increased/Decreased Na+,K+-ATPase Activity Mediated by Protein Kinase C Isoforms

In different species and tissues, a great variety of hormones modulate Na⁺,K⁺-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na⁺,K⁺-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na⁺,K⁺-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na⁺,K⁺-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.     

Overexpression of Na+/K+-ATPase Parallels the Increase in Sodium Transport and Potassium Recycling in an In Vitro Model of Proximal Tubule Cellular Ageing

Na⁺/K⁺-ATPase plays a key role in the transport of Na⁺ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na⁺ and K⁺ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na⁺/K⁺-ATPase activity and K⁺ conductance were evaluated using electrophysiological methods. Na⁺K⁺-ATPase α₁– and β₁-subunit expression was quantified by Western blot techniques. Na⁺/H⁺ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na⁺ transport, which results from an increase in the number of Na⁺/K⁺-ATPase α₁- and β₁-subunits, in the membrane. Increases in Na⁺/K⁺-ATPase activity were accompanied by increases in K⁺ conductance as a result of functional coupling between Na⁺/K⁺-ATPase and basolateral K⁺ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na⁺/H⁺ exchanger activity were observed. H₂O₂ production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H₂O₂ had no effect on Na⁺/K⁺-ATPase expression. Ouabain (100 nM) increased α₁-subunit, but not β₁-subunit, Na⁺/K⁺-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.     

Sodium ATPase and Sodium/proton Antiporter Are Not Obligatory for Sodium Homeostasis of Enterococcus hirae at Acid pH

Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na⁺-ATPase and Na⁺/H⁺ antiporter, does not grow in high Na⁺ medium at pH above 7.5. We found that 7683 grew normally in high Na⁺ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na⁺ and K⁺ were normal in this mutant. The Na⁺ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na⁺ elimination of this bacterium at acid pH is achieved by a decrease in Na⁺ entry and a normal K⁺ uptake.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Purification,characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol

A protein isolated from goat testis cytosol is found to inhibit Na⁺,K⁺-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I₅₀) occurs is in the nanomolar range. The inhibitor seems to bind Na⁺,K⁺-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na⁺,K⁺-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na⁺,K⁺-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.     

Ligand-Dependent Effects on the Conformational Equilibrium of the Na,K-ATPase As Monitored by Voltage Clamp Fluorometry

Voltage clamp fluorometry was used to monitor conformational changes associated with electrogenic partial reactions of the Na⁺,K⁺-ATPase after changes in the concentration of internal sodium (Na⁺_i) or external potassium (K⁺_o). To probe the effects of the Na⁺_i concentration on the Na⁺ branch of the Na⁺,K⁺-ATPase, oocytes were depleted of Na⁺_i and then loaded with external sodium (Na⁺_o) using the amiloride-sensitive epithelial sodium channel. The K⁺ branch of the Na⁺,K⁺-ATPase was studied by exposing the oocytes to different K⁺_o concentrations in the presence and absence of Na⁺_o to obtain additional information on the apparent affinity for K⁺_o. Our results demonstrate that lowering the concentration of Na⁺_i or increasing the amount of K⁺_o in the external solution shifts the equilibrium toward E₁/E₁P. Furthermore, the K⁺_o-induced relocation toward E₁ occurs at a much lower K⁺_o concentration when Na⁺_o is absent, indicating a higher apparent affinity. Finally, voltage-dependent steps associated with the K⁺ branch or the Na⁺ branch of the Na⁺,K⁺-ATPase are affected by the K⁺_o concentration or the Na⁺_i concentration, respectively.     

Modulation of H,K-ATPase activity by the molecular chaperone ERp57 highly expressed in gastric parietal cells

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H⁺,K⁺-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H⁺,K⁺-ATPase significantly increased the ATPase activity without changing the expression level of H⁺,K⁺-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H⁺,K⁺-ATPase activity. In contrast, knockdown of endogenous ERp57 in H⁺,K⁺-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H⁺,K⁺-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na⁺,K⁺-ATPase. These results suggest that ERp57 positively regulates H⁺,K⁺-ATPase activity apart from its chaperoning function.     

Demonstration of an endogenous activator for the Na+, K+-ATPase system

A heat-labile, non-dialysable and protease-sensitive endogenous activator (NaAF) capable of stimulating the Na⁺, K⁺-ATPase system has been demonstrated. The activator (NaAF) activity was partially enriched (about 10 fold) by dialysis (30 kDa cutoff) under negative pressure and pH 4.8 precipitation. The NaAF has been found to occur in the cytosolic fractions of tissues such as the kidney and brain from two different species (rabbit and pig) tested so far. Also, the factor from one tissue stimulates with equal efficacy the Na⁺, K⁺-ATPase systems of other tissues regardless of the species; thus demonstrating universal nature of the activator. Some degree of cross-reactivity was noted between the activating effects of this activator (for the Na⁺,K⁺-ATPase) and that for the H⁺,K⁺-ATPase recently described (J. Biol. Chem. 262:5664–5670, 1987). The purified NaAF obtained from sephacryl S-300 column chromatography activates the pure renal medullary Na⁺,K⁺-ATPase in a dose dependent manner.A preliminary account of this work was published in Fed. Proc. 46(4): 4466, 1987