Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.     

Structural interactions between FXYD proteins and Na+,K+-ATPase: alpha/beta/FXYD subunit stoichiometry and cross-linking

Interactions of rat FXYD4 (corticosteroid hormone-induced factor (CHIF)), FXYD2 (gamma), or FXYD1 (phospholemman (PLM)) proteins with rat alpha1 subunits of Na(+),K(+)-ATPase have been analyzed by co-immunoprecipitation and covalent cross-linking. In detergent-solubilized membranes from HeLa cells expressing both gamma and CHIF or CHIF and hemagglutinin A-tagged CHIF, mixed complexes of CHIF and gamma or CHIF and hemagglutinin A-tagged CHIF with alpha/beta subunits are undetectable. This implies that the alpha/beta/FXYD protomer is the major species in detergent solution. A lipid-soluble cysteine-cysteine bifunctional reagent, dibromobimane, cross-links CHIF to alpha in colonic membranes but not gamma or PLM to alpha in kidney or heart membranes, respectively. Sequence comparisons of the FXYD proteins suggested that Cys-49 in the trans-membrane segment of CHIF could be involved. In detergent-solubilized HeLa cell membranes, dibromobimane cross-links wild-type CHIF to alpha but not the C49F mutant, and also the corresponding F36C mutant but not wild-type gammab, and F48C but not wild-type PLM. C140S, C338A, C804A, and C966S mutants of the alpha subunit have been expressed. Only the C140S mutant prevents cross-linking with CHIF. The data demonstrated the proximity of trans-membrane segments of CHIF, gamma, and PLM to M2 of alpha. Molecular modeling is consistent with location of the trans-membrane segment of all FXYD proteins between M2, M6, and M9 and the proximity of Cys-49 of CHIF or Phe-36 of gamma with Cys-140 of M2. Cross-linking also demonstrated CHIF-alpha and CHIF-beta proximities in extra-membrane regions, similar to the evidence for gamma-alpha and gamma-beta cross-links.     

Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.     

Dynamic lipid interactions in the plasma membrane Na+,K+-ATPase

The function of ion-transporting Na⁺,K⁺-ATPases depends on the surrounding lipid environment in biological membranes. Two established lipid-interaction sites A and B within the transmembrane domain have been observed to induce protein activation and stabilization, respectively. In addition, lipid-mediated inhibition has been assigned to a site C, but with the exact location not experimentally confirmed. Also, possible effects on lipid interactions by disease mutants dwelling in the membrane-protein interface remain relatively uncharacterized. We simulated human Na⁺,K⁺-ATPase α₁β₁FXYD homology models in E1 and E2 states in an asymmetric, multicomponent plasma membrane to determine both wild-type and disease mutant lipid-protein interactions. The simulated wild-type lipid interactions at the established sites A and B were in agreement with experimental results thereby confirming the membrane-protein model system. The less well-characterized, proposed inhibitory site C was dominated by lipids lacking inhibitory properties. Instead, two sites hosting inhibitory lipids were identified at the extracellular side and also a cytoplasmic CHL-binding site that provide putative alternative locations of Na⁺,K⁺-ATPase inhibition. Three disease mutations, Leu302Arg, Glu840Arg and Met859Arg resided in the lipid-protein interface and caused drastic changes in the lipid interactions. The simulation results show that lipid interactions to the human Na⁺,K⁺-ATPase α₁β₁FXYD protein in the plasma membrane are highly state-dependent and can be disturbed by disease mutations located in the lipid interface, which can open up for new venues to understand genetic disorders.     

Localization of Na+, K+-ATPase in brain.

Enzyme activity, representing the sites of K+-stimulated p-nitrophenylphosphatase, a component of the sodium, potassium-stimulated-adenosinetriphosphatase system, has been localized in the somatosensory cortex of the rat brain. The reaction product is most obviously associated with fibers that are thought to be axons and dendrites. Large dendrite-like fibers appear to arise in layer 5 of the cortex and arborize in layers 1 through 4. Smaller, reactive fibers are found throughout the cortical layers. Neuron cell bodies did not exhibit substantial enzymatic activity. It did not appear that glia contributed significantly to the activity in cerebral cortex.     

Stabilization of Na(+),K(+)-ATPase purified from Pichia pastoris membranes by specific interactions with lipids

Na+,K+-ATPase (porcine alpha1/His10*beta1 or human alpha1/porcine His10*beta1) has been expressed in Pichia pastoris and purified by Co2+-chelate affinity resin chromatography, yielding about 80% pure, functional, and stable protein in a single step. The protein was eluted in nonionic detergents together with a phosphatidylserine. Size exclusion chromatography showed that the protein eluted in n-dodecyl beta-d-maltoside is an alpha1/beta1 protomer, whereas that in octaethylene glycol dodecyl monoether contains a mixture of alpha1/beta1 protomer and higher order oligomers. The Na+,K+-ATPase activity (8-16 (mumol/min)/mg of protein) is similar in both detergents. Thus, the minimal functional unit is the alpha1/beta1 protomer, and activity is unaffected by the presence of oligomeric forms. Screening of phospholipids for stabilization of the Na+,K+-ATPase activity shows that (a) acid phospholipids are required and phosphatidylserine is somewhat better than phosphatidylinositol and (b) optimal stabilization is achieved with asymmetric phosphatidylserines having saturated (18:0 >or= 16:0) and unsaturated (18:1 > 18:2) side chains at sn-1 an sn-2 positions, respectively. In the presence of phosphatidylserine, cholesterol stabilizes the protein at 37 degrees C, but not at 0 degrees C. Cholesterol also increases the "apparent affinity" of the phosphatidylserine and stabilizes optimally in the presence of phosphatidylserines with a saturated fatty acyl chain at the sn-1 position. Ergosterol is a poor stabilizer. We propose that phosphatidylserine and cholesterol interact specifically with each other near the alpha1/beta1 subunit interface, thus stabilizing the protein. These interactions do not seem to affect Na+,K+-ATPase activity.     

Tissue distribution of mRNAs encoding the alpha isoforms and beta subunit of rat Na+,K+-ATPase

The tissue distribution of the multiple forms of rat Na+,K+-ATPase was examined at the molecular level with cDNA probes specific for the alpha, alpha (+), alpha III and beta subunit mRNAs. Northern and slot blot analyses demonstrate that these mRNAs are produced in a tissue-specific manner. RNAs encoding the alpha (+) isoform are detected in kidney, brain, heart, adipose, muscle, stomach and lung, whereas alpha III RNA is detected in brain, stomach and lung. Both alpha and beta mRNAs are present in all the tissues studied, although at very different levels. Examination of heart tissue in greater detail demonstrates that the levels of mRNA encoding the alpha subunit are greater in the atria than in the ventricles, while the converse is true for alpha (+).     

Thermolability of alpha+-isoform of the catalytic subunit of brain Na+,K+-ATPase

Thermal stabilities of Na+,K(+)-ATPase isozymes from the rat brain and kidney tissues are compared. It is established that heat treatment of Na+,K(+)-ATPase preparations from the brain decreases the high affinity component of the ouabain inhibition of the enzyme activity due to selective inactivation of alpha-isoform. Its higher thermal lability in comparison with alpha-isoform is confirmed.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Location of signal sequences for membrane insertion of the Na+,K+-ATPase alpha subunit.

To study the membrane insertion of the Na+,K+-ATPase (EC 3.6.1.37) alpha subunit with six to eight transmembrane segments, mRNAs encoding the entire alpha subunit and its four different domains were prepared and translated in rabbit reticulocyte lysate with rough microsomal membranes. On the basis of the resistance of the membrane-inserted products to alkali extraction and the failure to insert the translation products into N-ethylmaleimide-treated membranes, it is suggested that at least two signal sequences are contained within the four transmembrane segments from the amino terminus of the alpha subunit.     

Purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris

Human beta2-adrenergic receptor is a G-protein-coupled receptor with seven transmembrane helices, and is important in pharmaceutical targeting on pulmonary and cardiovascular diseases. N-terminal histidine-tagged gene constructs with optimized codon usage were designed so as to obtain Pichia pastoris transformants with a high expression level. The constructs were inserted into the pPIC9 vector, and then electroporated into the SMD1168 strains. The highest expression level obtained was about 4 mg/liter-culture broth. The dissociation constant of the receptor in the membrane fraction was 1.2 nM toward CGP-12177 antagonist. The receptor was solubilized with sucrose monolaurate and purified with a series of chromatography steps including anion-exchange, Ni-Sepharose, alprenolol-Agarose, and hydroxyapatite columns. The receptor was heterogeneously glycosylated, showing broad SDS-PAGE bands around 70-90 kDa. After endoglycosidase treatment, the receptor appeared as a single band around 45 kDa, and was further purified with hydroxyapatite and gel-filtration columns. The receptor was eluted as a sharp peak at the gel-filtration elution volume corresponding to a molecular mass of 117 kDa. The saccharide-trimmed receptor thus purified is homogeneous as analyzed with SDS-PAGE, shows the dissociation constant of 4.7 nM toward CGP-12177 antagonist, and is suitable for crystallization experiments.     

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.     

Dissociation of Na+,K+-ATPase inhibition from digitalis inotropy.

Recent findings from our laboratory as well as those of other laboratories do not support the postulation that the mechanism of the positive inotropic action of digitalis is due to inhibition of NA,K-ATPase. Using short-acting digitalis steroids and drug washout experiments, in isolated myocardial preparations, it has been demonstrated that Na,K-ATPase isolated from such preparations is still significantly inhibited, whereas the positive inotropic effect is no longer present. Also, based on kinetic measurements the two exponential rate constants observed for drug half-life, a rapid and slow phase, were found to be associated, respectively, with the very short inotropic half-life and the very long enzyme inhibition half-life. In addition, a dissociation of the transient inotropic effects of digitalis was observed from the long lasting cardiotoxic effects of digitalis during drug washout. Moreover, a temporal correlation was noted between the persistent inhibitory effects of digitalis on Na,K-ATPase and the persistent cardiotoxic effects of digitalis. Therefore, it is concluded that inhibition of Na,K-ATPase is not responsible for the positive inotropic action of digitalis, but may be the mechanism, at least in part, for certain cardiotoxic effects of digitalis.     

Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction

The Kd for ouabain-sensitive K+ or Rb+ binding to Na+,K(+)-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the Kd from 15-17 microM to 5 or 0.8 microM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0 degrees C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperativity of the alpha beta-protomer structure in Na+,K+-ATPase functioning. A scanning microcalorimetry study

Heat denaturation of the free and ligand-bound forms of purified Na+,K+-ATPase from pig kidney is studied with the scanning microcalorimetry technique. A single two-state transition is observed during denaturation of the free enzyme, the molar concentration of the cooperatively melting units being equal to the concentration of alpha beta-protomers (Mr approximately equal to 140 000). Upon interaction of the enzyme with phosphate, Mg2+, and strophanthidin, but not with Na+, the cooperativity of the protomer unfolding is lost, and the protein stabilization enthalpy becomes approximately equal to 230 kJ/mol higher. The data suggest that in a functionally active enzyme form, the alpha beta-protomers possess a rigid structure with tight association of their subunits and domains, this structural rigidity is essential for the Na+,K+-ATPase functioning and there is a unique non-active conformation of the enzyme which may play an important role in its in vivo regulation.     

Distribution of immunoreactive Na+,K+-ATPase in gerbil cochlea

The distribution of Na+,K+-ATPase was mapped in cochleas of mature gerbils with normal hearing, using a specific and sensitive immunocytochemical method. Na+,K+-ATPase was abundant in the basolateral plasma membrane of marginal cells in the stria vascularis. Considerable levels of enzyme were also associated with the surfaces of spiral ganglion neurons and their central and peripheral processes. An unexpected finding was the detection of high levels of immunoreactive Na+,K+-ATPase in three different populations of cells lying in the inferior portion of the spiral ligament and at the medial and lateral border of the scala vestibuli just superior to the attachment of Reissner's membrane. Cells in these areas shared the morphological characteristics of cells specialized for active transport but appeared to be nonpolarized, suggesting a uniform distribution of Na+,K+-ATPase over their entire plasmalemma. The presence of these three distinct cell populations in the cochlea of several mammalian species suggests that they play an important role in cochlear function, perhaps that of regulating the cation content of perilymph. The absence of discrete concentrations of Na+,K+-ATPase-rich cells in the perilymphatic connective tissue of the bird cochlea and the mammalian vestibular system suggests further that these cells may be involved with generating and maintaining the high endolymphatic potential unique to the mammalian cochlea.