Isoforms of Na+,K+-ATPase catalytic subunit in animal tissues

The tissue and species distribution of Na+,K(+)-ATPase isoenzymes is considered. Criteria for identification of the above isoforms are presented as attributed to their structural and functional peculiarities. Specificity inherent in functional properties of isoenzymes of Na+,K(+)-ATPase and physiological significance of the latter are discussed.     

Phosphorylation of lens membrane: identification of the catalytic subunit of Na+, K+-ATPase

Receptor mediated endocytosis appears to depend on the action of a transglutaminase (TGase). Endocytosis can be induced in intact human RBC by the action of several classes of drugs. We tested the hypothesis that drugs acted by stimulating TGase activity. Of the endocytosis inducing drugs tested, neither primaquine nor vinblastine nor chlorpromazine enhanced TGase activity. We next tested the hypothesis that TGase activity was required for drug endocytosis in RBC by adding known TGase inhibitors. Paradoxically, m-Dansyl cadaverine, the most potent TGase inhibitor, produces endocytosis in human RBC. Therefore despite apparent striking morphologic similarities, drug induced endocytosis in RBC appears to proceed via different mechanisms from those involved in receptor mediated endocytosis in other cells.In the receptor-mediated endocytosis of some hormones and growth factors, it appears that the receptor-ligand complex forms clusters over clathrin coated pits which are then internalized as endocytic vacuoles. Both the clustering and internalization of ligands are inhibited by a variety of agents shown to inhibit transglutaminase (TGase) and it is therefore proposed that TGase participates in receptor-mediated endocytosis (1–3). Human erythrocytes undergo endocytosis when exposed to drugs like primaquine, chlorpromazine, and vinblastine (4), all of which are amphipathic cations (4). However, the mechanism of drug action is not known nor is it clear that this is a form of receptor-mediated endocytosis (4). Furthermore, clustering of receptors can occur in neonatal but not adult human RBC (5). TGase has been measured in human red cells (6) although its physiologic role is unknown. Like all TGases, it is calcium dependent (6,7), and primaquine induced red cell endocytosis is enhanced by Ca⁺⁺ addition (8). Therefore, we tested the hypothesis that TGase participates in drug induced endocytosis in intact human red cells.     

Phosphorylation of the catalytic subunit of rat renal Na+, K+-ATPase by classical PKC isoforms

In this study we have evaluated the specificity of different PKC isozymes for the phosphorylation of the catalytic alpha1 subunit of rat renal Na+,K+-ATPase (alpha1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, betaI, and gamma efficiently phosphorylate alpha1 Na+,K+-ATPase. However, alpha1 Na+,K+-ATPase was a poor substrate for the novel PKCs (nPKCs) delta and epsilon. Two-dimensional phosphopeptide mapping revealed a similar pattern of phosphorylation by all cPKCs. The functional significance of this finding was evaluated by measuring Na+,K+-ATPase activity (assessed by 86Rb+ uptake) in COS-7 cells expressing the rat alpha1 Na+,K+-ATPase. 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a nonselective PKC activator, inhibited Na+,K+-ATPase activity in this system. On the other hand, 12-deoxyphorbol-13-phenylacetate (DPP), which preferentially activates nPKCepsilon, did not affect 86Rb+ uptake. These results indicate a differential pattern of phosphorylation and regulation of rat renal Na+,K+-ATPase activity by PKC isoforms and suggest an important role for cPKCs in the physiological regulation of the pump.     

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.     

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.     

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 (+).     

Study of expression of Na+,K+-ATPase subunit genes in the human brain using cDNA libraries

Human brain cDNA libraries were screened with cDNA inserts corresponding to the mRNA for the Na+,K(+)-ATPase alpha-subunit from pig kidney. The results obtained demonstrate the existence of two highly homologous mRNAs encoding the alpha- and alpha III-isoforms of the Na+,K(+)-ATPase catalytic subunit.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

Study of ADP effect on brain Na+, K+-ATPase]

A mechanism of K-insensitive, ouabain-dependent liberation of Na+ from the cell during an increase in ADP intracellular concentration is studied. It is shown that the increase in the ADP/ATP ratio does not change the Na+, K+-ATPase affinity to K+ ions and does not result in the Na-activated, K-independent ATPase reaction. ADP protects ATPase from the inhibition by ouabain which is accounted for by a decrease in the concentration of a glycoside-sensitive form of the enzyme E2-P due to a turnover of the phosphokinase step of the reaction, but not due to the binding of free Mg2+ ions. The results obtained suggest that the increase in ADP concentration within the cell activates Na-Nan exchange along Na-transporting channels of the ionic pump.     

Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.     

Up-regulation of Na(+),K(+)-ATPase alpha 3-isoform and down-regulation of the alpha1-isoform in human colorectal cancer

We investigated expression levels of Na(+),K(+)-ATPase alpha-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of the alpha1-isoform protein was observed in all sampled cancer tissues compared with the normal mucosae. The level of ouabain (5 microM)-sensitive Na(+),K(+)-ATPase activity in carcinomas was 81+/-5% that of in the normal mucosae. The mRNA expression of alpha2- and alpha 4-isoforms was decreased in almost all the carcinoma samples. Interestingly, the expression level of the alpha 3-isoform protein in the cancer tissue was higher than that of the normal mucosa. These results indicate that a decrease in the alpha1-isoform expression and an increase in the alpha 3-isoform expression may be associated with colorectal cancer.     

Expression of ouabain-sensitive H+,K+-ATPase catalytic subunit gene in the rat 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 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 alpha-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. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.