Reconstitution of detergent-solubilized Na,K-ATPase and formation of two-dimensional crystals

Very pure, detergent-solubilized Na,K-ATPase from dog or lamb kidneys has been successfully reconstituted at high protein-to-lipid weight ratios. Studies have been conducted to establish the orientation of the Na,K-ATPase molecules in the reconstituted membranes and to assess the functional activity and the conformational state of the reconstituted enzyme. Results indicate that reincorporation of the Na,K-ATPase molecules in the lipid bilayer is unidirectional and that the reconstituted enzyme retains its functional and structural integrity. Two-dimensional crystals have been induced in these preparations by vanadate ions. The arrays, with a dimeric structure in the unit cell, have a morphology similar to that of the crystals that had previously formed in the native membranes. Filtered images show that in projection, the molecule had an asymmetrical mass distribution, which at the resolution of 2.5 nm is identical to that of the earlier crystals. These sheets, although small, represent the first crystals of Na, K-ATPase to be formed by reconstitution. We expect that optimization of the reconstitution and crystallization parameters will lead to larger and better-ordered sheets, suitable for electron crystallography.     

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.     

Structure of two-dimensional crystals of membrane-bound Na,K-ATPase as analyzed by correlation averaging

The structure of two-dimensional crystals of membrane-bound Na,K-ATPase from rabbit kidney has been analyzed with a correlation averaging procedure. Two principally different crystal forms are observed with p1 and p21 symmetry, respectively. In the p1 form the averaged projection structure shows a triangular shaped protein domain interpreted as a protomer (alpha beta-unit) of Na,K-ATPase. In the p21-form the stain-deficient area is extended toward a twofold symmetry axis. The results are in good agreement with a previous analysis where Fourier methods were applied to well ordered crystals of pig kidney Na,K-ATPase and illustrate that the correlation averaging procedure can be used for the analysis of membrane crystals of Na,K-ATPase showing curved lattice lines.     

Three-dimensional structure of Na,K-ATPase determined from membrane crystals induced by cobalt-tetrammine-ATP.

The three-dimensional structure of Na,K-ATPase has been analyzed with electron microscopy and image processing. The enzyme, purified from pig kidney outer medulla, was arranged in a new form of tetragonal two-dimensional membrane crystals after incubation with cobalt-tetrammine-ATP, a stable MgATP complex analogue. Each continuous protein domain, as delineated by negative stain, consists of two alpha beta-protomers related by a dyad axis. The two rod-like regions are connected by a bridge displaced about 20 A away from the center of the structure toward the lipid bilayer. The domain connecting the two promoters is more constricted and closer to the center of the structure in the Co(NH3)4ATP-induced crystals than in the vanadate-induced p21 crystals. These observations suggest that the difference between previously analyzed dimers of two-dimensional p21 crystals induced with vanadate/magnesium and dimers of p4 crystals induced with Co(NH3)4ATP reflects two different conformational states of the enzyme.     

Transmembrane potential and electrogenicity of thrombocyte Na, K-ATPase in different forms of hypertension in rats

The fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide, dis-C3-(5), was used to study the membrane potential of platelets in rats with renal, DOCA-induced and spontaneous hypertension. The magnitude of the transmembrane potential in platelets combines the activity of electrogenic Na, K-ATPase and the rate of ion diffusion across the plasma membrane. The basal potential is 12 = 14 mB as lowered only in spontaneously hypertensive rats. The decrease of the basal potential is made for electrogenicity of platelet Na, K-ATPase in spontaneously hypertensive rats.     

Some properties of glial membrane Na, K-ATPase]

Na,K-ATPase activity in glial membranes is rather low that in the nerve ending membranes, but is characterized by the same kind of Na+/K+-dependence. Glial Na,K-ATPase is insensitive to acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA) while norepinephrine activates Na,K-ATPase at low concentrations and inhibits it at high concentrations. Participation of Na,K-ATPase in the regulatory mechanisms of the neuron-neuroglia relations is discussed.     

Hyperpolarization of the cell membrane during Na,K-ATPase inactivation

The relationship equation between the resting potential and potassium and sodium active currents is deduced in terms of a generally accepted model of electrogenesis. It is demonstrated that an increase of Na,K-ATPase activity to the estimated magnitude results in hyperpolarization of the cell membrane (CM), but the subsequent increase of the activity led to CM depolarisation. CM depolarisation results in an increase of the cell volume.     

Activity of erythrocyte membrane Na,K-ATPase in rats with experimental botulism

The effect of type C botulinum toxin on Na, K, Mg-ATPase activities of erythrocyte membranes of white rats was studied in experiments in vivo and in vitro. The activity of Na, K, Mg-ATPase was found to be markedly inhibited in the preclinical period of poisoning, 2 hours after intraperitoneal injection of the toxin. In this case Mg-ATPase activity noticeably increased. In the presence of the development of a grave paralytic syndrome one day after intraperitoneal injection of the toxin, the activity of Na, K-ATPase of the erythrocyte membrane remained decreased as was the case in the preclinical period of poisoning, whereas the activity of Mg-ATPase returned to normal. The experiments in vitro with preincubation of erythrocyte membranes with botulinum toxin in the concentrations corresponding to the mean calculated ones in the experiments in vivo demonstrated inhibition of Na, K-ATPase. The magnitude of Mg-ATPase activity remained virtually unchanged in all the modifications of the experiments with boiled and native botulinum toxin. The in-vivo experiments with intraperitoneal injection of glutathione and unithiol to the pretreated animals attested to normalization of Na, K-ATPase in the preclinical period of poisoning, with this normalization being brought about by unithiol. In the in-vitro experiments with addition of unithiol or glutathione into the incubation medium, each of the donators of sulphhydryl groups prevented Na, K-ATPase inhibition with botulinum toxin.     

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na⁺ for extracellular K⁺ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na⁺ into and for K⁺ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an -subunit of 112-kD and a -subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the -subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na⁺ and K⁺.     

Configuration of subunits within crystals of Na, K-ATPase maintained in the frozen-hydrated state

Two-dimensional crystalline sheets of Na, K-ATPase were studied in the vitrified, frozen-hydrated state by electron microscopy and image processing. The technique of correlation averaging was used to determine the projected structure. The projection map shows asymmetry between the pair of "alpha beta" protomers comprising a dimer of Na, K-ATPase molecules. The two protomers differ in overall density as well as in shape. One protomer has an oblong shape, whereas the other with higher density has a head and a hook region. Such an asymmetry has not been reported by other laboratories. This asymmetry may either be due to the coexistence of two different conformations of the enzyme in the dimeric form or due to the simultaneous existence of two molecular species of Na, K-ATPase.     

Role of cell membrane Na,K-ATPase for survival of human lymphocytes in vitro

Lymphocytes are primordial immune cells with variable life times. Besides genetic programming, extracellular factors interacting with cell surface receptors might alter cell survival. We investigated whether the activity of the membrane-embedded Na,K-ATPase (EC 3.6.1.37) or sodium pump (NKA) plays a role for cell survival since this ubiquitous system establishes the vital transmembrane Na and K gradients as well as the resulting high intracellular K/Na ratio required for macromolecule synthesis; furthermore, the system exposes an extracellular inhibitory receptors for cardioactive steroids and palytoxin. Isolated human lymphocytes were incubated in vitro and their viability assessed by exclusion of trypan blue. Various incubation conditions were compared; in RPMI-1640 medium cell viability was preserved for 30 h at 37 °C. Externally added ouabain, a hydrophilic cardioactive steroid, blocked the [⁸⁶Rb]potassium uptake at nanomolar concentrations. Despite pump inhibition ouabain did not alter lymphocyte survival, even at 10 mM for 30 h. By contrast, the hydrophilic toxin palytoxin, the most potent animal poison described so far, killed all cells within 2 h at 10 nM; this toxin is known to act via the sodium pump and to provoke deadly cation-leaks by unmasking a channel component. Intracellular Na increased and K decreased as measured by atomic absorption spectrometry in presence of palytoxin; cell swelling was seen by electron microscopy. Ouabain protected the cells from the toxic effect of palytoxin. The results reveal a pivotal role of NKA integrity for lymphocyte survival.Abbreviations BCA bicinchonic acid - D-PBS Dulbecco's Phosphate Buffered Saline - HBSS Hanks' Balanced Salt Solution - PYX palytoxin (used in figures only) - NKA Na,K-ATPase     

Phosphorylation of the Na,K-ATPase and the H,K-ATPase

Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na⁺,K⁺- and H⁺,K⁺-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations.     

Alteration of erythrocyte membrane Na,K-ATPase in children with borderline or essential hypertension

The aim of this study was to evaluate the substrate (ATP) kinetics of erythrocyte membrane Na, K-ATPase in children with borderline or essential hypertension. Although the activity of Na, K-ATPase in the presence of in vivo concentrations of ATP was not significantly altered, kinetic studies showed an obvious inhibition of enzyme activity in the erythrocyte membrane of children with borderline or essential hypertension. Hanes plot analysis revealed a decrease of V_max from 7·19 in erythrocytes from control subjects to 4·93 and 3·33 in those from children with borderline or essential hypertension, respectively. A mean value of the K_m decreased from 0·10 in the control to 0·08 and 0·02 in children with borderline or essential hypertension, respectively. The energy status of erythrocytes, estimated by ATP, ADP and AMP levels, ATP/ADP ratio, and adenylate energy charge (AEC) was not significantly changed in the cells from hypertensive children. The use of a free radical-generating system (FeSO₄/ascorbate) in vitro significantly reduced enzyme activity in the control erythrocytes while in those from hypertensive children it was abolished completely. The level of lipid peroxides was considerably higher (+37 per cent) in the plasma, while that of reduced glutathione was significantly lower both in the erythrocytes and the plasma of children with essential hypertension than in healthy children. These results indicate significant alterations of the antioxidant status which could be the cause of the inhibited Na,K-ATPase activity in erythrocyte membranes from hypertensive children.     

Transport and pharmacological properties of nine different human Na, K-ATPase isozymes

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 alpha and beta isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K(+)-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 from digitalis inhibition at physiological K(+) levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.     

Accessibility of tryptophan residues in Na,K-ATPase

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.     

Localization of Na,K-ATPase in lingual epithelia

Na,K-ATPase was localized in canine fungiform and circumvallfltepapillae by immunocytochemical and histochemical methods. Monoclonalantibodies raised against the -subunit of Na,K-ATPase showedspecific staining in the stratum basale and in the lower layersof the stratum spinosum. Small stained wavy lines, interpretedas trigerrunal fibers, were found in the epithelium near tastebuds. In contrast, conventional histochemical methods showedno staining in the epithelium. In both immunocytochemical and histochemical methods taste budswere densely stained. The histochemical stain in taste budswas essentially eliminated by levamisole and L-cysteine butremained in the presence of 10 mM ouabain or in the absenceof potassium. These data suggest that the majority of the histochemicalstain arises from phosphatases other than Na,K-ATPase.     

Detection of membrane protein two-dimensional crystals in living cells

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.