Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry

Na,K-ATPase (EC, 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis, is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes take up Na,K-ATPase-vesicles in a dose-and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37°C together with up to about 200,000 renal Na,K-ATPase molecules.     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K⁺ so that the membrane voltage approaches the Nernst potential for K⁺. With constant external K⁺ concentration, the time course of internal K⁺ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K⁺ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.     

Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the receptor-Ni interaction, i.e. carbachol stimulation of the activities of Ni and the GTP gamma S effect on carbachol binding, were no longer observed, when this IAP-treated Ni, instead of the nontreated Ni, was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated Ni in their basal activities observable without carbachol. No, the protein with a character very similar to Ni in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.     

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.     

Ultrastructure of the purified and reconstituted Na/K-ATPase of the avian salt gland

Na/K-ATPase of salt-stressed salt glands of the domestic duck (Anas platyrhynchos) was purified in membrane-bound form by incubation of the microsomal fraction with sodium dodecylsulphate and ATP followed by discontinuous sucrose gradient centrifugation. Gel electrophoresis of the purified plasma membrane preparation substantially showed the two polypeptide subunits of the Na/K-ATPase both of which stained with the periodic acid-Schiff reagent. About 99% of the total ATPase activity was ouabain-inhibitable amounting to 1300 mumol Pi/(mg protein X h) of specific activity. The anion-stimulated, ouabain-insensitive ATPase increased parallel to the Na/K-ATPase up to the microsomal fraction until it totally vanished during SDS incubation. Electron microscopy of thin sections revealed that the purified fraction consisted of flat and cup-shaped triple-layered membrane fragments. Particles arranged into clusters and strands were visible as 3 to 5 nm surface particles in negatively stained suspensions and as 8 to 10 nm intramembraneous particles in freeze fracture replicas. The differential distribution of the intramembraneous particles on the fracture faces reflected the structural membrane asymmetry. Solubilization of Na/K-ATPase led to the disappearance of intramembraneous particles. Incorporation of the solubilized enzyme into phosphatidylcholine vesicles again showed 8 to 10 nm particles apparently orientated at random in the artificial membrane. Control liposomes prepared in the absence of solubilized enzyme were devoid of intramembraneous particles. These results clearly demonstrate that the avian salt gland Na/K-ATPase exists as 8 to 10 nm particles in both the purified plasma membrane and the artificial phospholipid membrane.     

Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals

The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.     

Uncoupled Na+-efflux on reconstituted shark Na,K-ATPase is electrogenic

In liposomes with reconstituted shark Na,K-ATPase produced to contain sucrose addition of external Na+ and ATP induce an uncoupled Na+-efflux on inside-out oriented pumps which can be inhibited by digitoxigenin. This flux mode is found to be electrogenic and accompanied by hydrolysis of ATP. The coupling ratio of Nacyt transported per ATP split is 3:1 measured as the initial rate of rise in transmembrane potential and initial rate of liberated Pi.     

Covalent binding of negatively charged phospholipid to Na,K-ATPase

Phosphatidylglycolaldehyde and its lyso derivative were applied as probes in order to study lipid-protein interactions with purified, membrane-bound Na,K-ATPase. Reduction with [3H]NaCNBH3 led to formation of a stable chemical derivative between added lipid and protein. The extent of modification of the two subunits of Na,K-ATPase was similar. Extensive tryptic digestion of derivatized ATPase resulted in cleavage of the alpha-subunit without hydrolysis of the beta-subunit.     

Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles

Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase.     

Variable stoichiometry in reconstituted shark Na,K-ATPase engaged in uncoupled efflux

In liposomes with reconstituted shark Na,K-ATPase produced to contain no internal K+ or Na+ addition of external Na+ and ATP induce an uncoupled Na+ efflux on inside-out oriented pumps which is electrogenic and accompanied by hydrolysis of ATP (Cornelius, F. (1989) Biochem. Biophys. Res. Commun. 160, 801-807). At saturating cytoplasmic Na+ the net-charge translocated per ATP molecule split is compatible with a coupling ratio of Nacyt transported per ATP split of 3:1 at pH greater than or equal to 7.0. However, this ratio decreases to 1.5:1 below pH 7.0. At non-saturating cytoplasmic Na+ the 3:1 stoichiometry is attained at pH 7.0-7.5, whereas outside this range of pH the net-charge translocated per ATP molecule split decreases.     

Sodium transport defect of ouabain-resistant renal Na,K-ATPase

The murine renal Na,K-ATPase is resistant to cardiac glycosides. It is not yet known however whether altered active transport is associated with the drug-resistance. To investigate this problem Na,K-ATPases were purified from the outer medulla of both rat and rabbit kidneys and reconstituted identically into liposomes. The Na-stimulation of the Na,K-ATPase activity before reconstitution and of the Na-transport after reconstitution was measured. A Na-defect inherent in the ouabain-resistant rat Na,K-ATPase was discovered indicating a link between the cardiac glycoside sensitivity and the Na-transport.     

Fusion of phospholipid vesicles reconstituted with cytochromec oxidase and mitochondrial hydrophobic protein

Summary Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes and Ca⁺⁺ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca⁺⁺ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 Å; fusion yielded vesicles with diameters in excess of 1000 Å.     

The three-dimensional structure of the Na,K-ATPase from electron microscopy

The structure of Na,K-ATPase has been studied by electron microscopy and image reconstruction. A three-dimensional structure of this enzyme has been obtained to an overall resolution of 2.5 nm using data from specimens of negatively stained dimer sheets tilted through a range of angles +/- 60 degrees. The reconstruction shows a complex mass distribution consisting of ribbons of paired molecules extending approximately 6.0 nm from the cytoplasmic side of the membrane. The molecular envelope consists of a massive "body" with "lobe" and "arm" structures projecting from it. The body has a columnar shape and is tilted with respect to the plane of the membrane. The region of interaction responsible for dimer formation is located between two bodies and is clearly visible in the reconstruction. It has been identified as a segment in the amino-terminal portion of the alpha subunit. The arms that interconnect the ribbons are located close to the membrane and are most probably formed by the beta subunits.     

Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes

Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.     

Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode

Na,K-ATPase from rabbit kidney outer medulla was reconstituted in large unilamellar lipid vesicles by detergent dialysis. Vesicles prepared in the presence or absence of potassium allowed to study two different transport modes: the (physiological) Na,K-mode in buffers containing Na+ and K+ and the Na-only mode in buffers containing Na+ but no K+. The ATP hydrolysis activity was obtained by determination of the liberated inorganic phosphate, Pi, and the inward directed Na+ flux was measured by 22Na-tracer flux. Electrogenic transport properties were studied using the membrane potential sensitive fluorescence-dye oxonol VI. The ratio upsilon(Na,K)/upsilon(Na) of the turnover rates in the Na,K-mode and in the Na-only mode is 6.6 +/- 2.0 under otherwise identical conditions and nonlimiting Na+ concentrations. Strong evidence is found that the Na-only mode exhibits a stoichiometry of 3Na+cyt/2Na+ext/1ATP, i.e. the extracellular (= intravesicular) Na+ has a potassium-like effect. In the Na-only mode one high-affinity binding side for ATP (KM congruent to 50 nM) was found, in the Na,K-mode a high- and low-affinity binding side with equilibrium dissociation constants, KM, of 60 nM and 13 microM, respectively. The sensitivity against the noncompetitively inhibiting ADP (KI = 6 microM) is higher by a factor of 20 in the Na-only mode compared to the Na,K-mode. From the temperature dependence of the pumping activity in both transport modes, activation energies of 160 kJ/mol for the Na,K-mode and 110 kJ/mol for the Na-only mode were determined.     

Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles

Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides.     

Electron microscopy and hydrodynamic properties of blood clotting factor V and activation fragments of factor V with phospholipid vesicles

The electron microscopic and hydrodynamic properties of factor V and factor Va-vesicle complexes were determined. Images of negatively stained factor V bound to vesicles showed the protein as a relatively large globular domain (9.5 nm diameter) connected to the membrane through a narrow protein region 0.5-3 nm in length. This connecting region was not always visible and was measured as the distance between the globular region and the apparent vesicle edge. Factor V protein alone usually appeared as two connected globular regions of 10.2 and 6.5 nm diameter. The two-domain protein structure appeared consistent with both the image of factor V alone and bound to the membrane. Factor V had no biological activity in a phospholipid-free prothrombinase assay system used. The proteolytically activated form of factor V generated by digestion with thrombin (factor Va) was at least 30,000 times more active. The electron microscopic images of factor Va-vesicle complexes showed a smaller protein that was more closely associated with the vesicle surface than was factor V. The light chain (Mr about 80,000) component of factor Va also bound to the surface of the vesicles and appeared to be largely external to the membrane. Protein-induced hydrodynamic radius changes for the factor V-vesicle and factor Va-vesicle complexes were 12.8 and 6.3 nm, respectively. The images observed in the electron microscope were used to calculate protein-induced radius changes. Comparison of these values with the experimentally determined hydrodynamic radius changes showed approximate agreement for factor Va-membrane complexes. However, the images of factor V-vesicle complexes suggested smaller hydrodynamic radius changes than were actually observed.