Quantitative immunogold localization of Na+,K(+)-ATPase alpha-subunit in the tympanic wall of rat cochlear duct

Ultrastructural localization of Na+,K(+)-ATPase was quantitatively investigated in the tympanic wall of rat cochlear duct by use of the protein A-gold method, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. A moderate number of gold particles were found on the basolateral membrane of the interdental cells of the spiral limbus. A small number of gold particles were found on the basolateral surfaces of the border cells and Hensen's cells. On the inner and outer sensory hair cells, however, the plasma membranes were rarely labeled by gold particles. The general pattern of labeling densities in cochlear structures determined here and in a previous communication from our laboratory shows good correlation with the distribution of Na+,K(+)-ATPase activity as previously estimated biochemically, cytochemically, and autoradiographically.     

Na+,K(+)-ATPase activity in human colorectal carcinoma

Na+,K(+)-ATPase activities in macroscopically unchanged mucosa (conditionally normal tissue) and human colorectal carcinoma (mainly low-grade and moderately differentiated adenocarcinomas) have been investigated. Microsomal fractions are similar by dimensions of the membrane fragments detected by photon correlation spectroscopy analysis. The activation optima under digitonin pretreatment of the membrane fractions differ significantly for Na+,K(+)-ATPase and concomitant Mg(2+)-ATPase activity, but are the same in conditionally normal and cancerous tissues. This allows to detect correctly total levels of the Na+,K(+)-ATPase activity in the detergent-pretreated preparations. The moderate decrease of the Na+,K(+)-ATPase activity is revealed in carcinomas. It is concluded that a decrease of activity of the ouabain-sensitive human Na+,K(+)-ATPase is characteristic of colorectal carcinoma.     

Kidney Na+,K(+)-ATPase is associated with moesin

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).     

Polarized distribution of Na+,K+-ATPase in giant cells elicited in vivo and in vitro

Giant cell formation was analyzed to determine whether it results in the high level of Na+,K+-ATPase expression that characterizes multinucleated cells such as osteoclasts. Giant cells and fusing alveolar macrophages were subjected to morphological, immunological, and biochemical studies. Both subunits of the Na+,K+-ATPase were found to be present on the plasma membrane of giant cells. Their localization was restricted to the non-adherent domain of the cell surface. Dynamic studies of giant cell differentiation demonstrated that on culture and/or multinucleation, an increase in sodium pump alpha-subunit synthesis occurred and led to a high level of expression of Na pumps. Conversely, the adherent plasma membrane of giant cells was enriched in a lysosomal membrane antigen. This study demonstrates that culture and/or multinucleation induces a significant increase in the expression of sodium pumps. The polarized distribution of these pumps and of a lysosomal component suggests that fusing macrophages undergo biochemical and morphological alterations which prepare them for a new and specialized function in chronic inflammatory reactions. Giant cells may offer a suitable model system to study the differentiation of other related multinucleated cells, such as osteoclasts.     

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides

Three-subunit isoforms of the sodium pump, which is the receptor forcardiac glycosides, are expressed in human heart. The aim of this studywas to determine whether these isoforms have distinct affinities forthe cardiac glycoside ouabain. Equilibrium ouabain binding to membranesfrom a panel of different human tissues and cell lines derived fromhuman tissues was compared by an F statistic to determinewhether a single population of binding sites or two populations ofsites with different affinities would better fit the data. For alltissues, the single-site model fit the data as well as the two-sitemodel. The mean equilibrium dissociation constant(K_d) for all samples calculated using thesingle-site model was 18 ± 6 nM (mean ± SD). No differencein K_d was found between nonfailing and failinghuman heart samples, although the maximum number of binding sites infailing heart was only ~50% of the number of sites in nonfailingheart. Measurement of association rate constants and dissociation rateconstants confirmed that the binding affinities of the different human-isoforms are similar to each other, although calculatedK_d values were lower than those determined byequilibrium binding. These results indicate both that the affinity ofall human -subunit isoforms for ouabain is similar and that theincreased sensitivity of failing human heart to cardiac glycosides isprobably due to a reduction in the number of pumps in the heart ratherthan to a selective inhibition of a subset of pumps with differentaffinities for the drugs.

     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Ouabain sensitivity of Na+,K+-ATPase in neuronal membranes]

Na+, K(+)-ATPase preparations of the rat and bovine brain and kidney were studied for ouabain sensitivity. Differences in apparent affinities to inhibitor of alpha(+)- and alpha-isozymes of Na+, K(+)-ATPase catalytic subunit were detected only in rat tissues but not in bovine ones. It is concluded that glycoside-sensitive and glycoside-resistant enzymic forms are not fully identical to alpha(+)- and alpha-subunit forms of Na+, K(+)-ATPase.     

Involvement of the alpha-subunit N-terminus in the mechanism of the Na+,K+-ATPase

Previous studies have shown that cytoplasmic K⁺ release and the associated E2 → E1 conformational change of the Na⁺,K⁺-ATPase is a major rate-determining step of the enzyme's ion pumping cycle and hence a prime site of acute regulatory intervention. From the ionic strength dependence of the enzyme's distribution between the E2 and E1 states, it has also been found that E2 is stabilized by an electrostatic attraction. Any disruption of this electrostatic attraction would, thus, have profound effects on the rate of ion pumping. The aim of this paper is to identify the location of this interaction. Using enhanced-sampling molecular dynamics simulations with a predicted N-terminal structure added to the X-ray crystal structure of the Na⁺,K⁺-ATPase, a previously postulated salt bridge between Lys32 and Glu233 (rat sequence numbering) of the enzyme's α-subunit can be excluded. The residues never approach closely enough to form a salt bridge. In contrast, strong interactions with anionic lipid head groups were seen. To investigate the possibility of a protein-lipid interaction experimentally, the surface charge density of Na⁺,K⁺-ATPase-containing membrane fragments was estimated from zeta potential measurements to be 0.019 (± 0.001) C m⁻². This is in good agreement with the charge density previously determined to be responsible for stabilization of the E2 state of 0.023 (± 0.009) C m⁻² and the membrane charge density estimated here from published electron-microscopic images of 0.018C m⁻². The results are, therefore, consistent with an interaction of the Na⁺,K⁺-ATPase α-subunit N-terminus with negatively-charged lipid head groups of the neighbouring cytoplasmic membrane surface as the origin of the electrostatic interaction stabilising the E2 state.     

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.     

The heterogeneity of the Na+, K(+)-ATPase ouabain sensitivity in microsomal membranes of rat colon smooth muscles

The dose dependence of the Na+, K(+)-ATPase ouabain inhibition in the rat colon smooth muscle permeabilized microsomes has been analyzed according to the model of two independent binding sites of inhibitor to determine the activity of separate molecular forms of the enzyme that differ by affinity for cardiac glycosides. The two-phase inhibition curve with moderate content of the high-affinity activity component was revealed. The apparent inhibition constant of the low-affinity component corresponds to the value for the rat kidney microsomal Na+, K(+)-ATPase (alpha1-isoform). The specific role of the alpha2- and alpha1- Na+, K(+)-ATPase catalytic subunit isoforms in colonic smooth muscle electromechanical coupling is considered.     

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 (+).     

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)     

Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.     

Time-dependent increases in Na+,K(+)-ATPase content of low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation of rabbit fast-twitch muscle induced time-dependent increases in the concentration of the sarcolemmal Na+,K(+)-ATPase and in mitochondrial citrate synthase activity. The almost twofold increase in Na+,K(+)-ATPase preceded the rise in citrate synthase and was complete after 10 days of stimulation. We suggest that the increase in Na+,K(+)-ATPase enhances resistance to fatigue of low-frequency-stimulated muscle prior to elevations in aerobic-oxidative capacity.     

Effect of lead on Na+, K(+)-ATPase activity in Penaeus indicus postlarvae

In vivo effect of lead on Na, K(+)-ATPase was studied in plasma membrane/mitochondrial fraction of P. indicus post-larvae (PL), exposed to 30 days to a sublethal concentration (1.44 ppm) of lead. A significant (P < 0.05) decrease in the enzyme activity was observed for exposed PL with respect to their controls at different intervals except 24hr. Further the substrate (ATP) and ion (Na+ and K+)-dependent kinetics of Na+, K(+)-ATPase was studied with the plasma membrane/mitochondrial fractions of control and 30 days exposed PL. The apparent KM and V(max). values were calculated to determine the nature of inhibition. Both the control and exposed PL showed almost the same apparent KM values in the presence of different substrate or ion concentrations indicating that lead interacts with the enzyme at a different binding site.