Na,K-ATPase activity in the myocardial sarcolemma in ischemia

The total time-controlled ischemia (up to 45 min) was studied for its effect on the Na,K-ATPase activity, content of nonesterified fatty acids (NEFA) and intensity of lipid peroxidation (LP) in sarcolemmal (SL) preparations and soluble fractions (SF) from the rat and guinea-pig left ventricles. A strong correlation between Na, K-ATPase inhibition and NEFA accumulation was revealed in the SF. On the contrary, changes in the NEFA content and LP level both in SL and SF did not correlate with a decrease in the enzymic activity. Pretreatment with albumin (0.5 mg/ml) induced equally small increase both in the control and in the ischemic SL preparations. It is suggested that the Na,K-ATPase activity during a short period of myocardial ischemia (up to 45 min) may be due to the NEFA accumulation in the cytosolic and/or extracellular space, but not in SL.     

Expression of Na,K-ATPase isoforms in human heart.

The expression pattern of the multiple isoforms of Na,K-ATPase was examined in the human heart. Isoform specific oligonucleotide probes for the alpha 1, alpha 2, alpha 3 and beta 1 subunits were used to probe Northern blots. The adult human ventricle expresses mRNAs for all three alpha subunit isoforms in addition to beta 1 subunit mRNA.     

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⁺.     

Na,K-ATPase is essential for embryonic heart development in the zebrafish

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.     

Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase

There are four isoforms of the α subunit (α1–4) and three isoforms of the β subunit (β1–3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. α2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An α2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of α2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing α1β1, α2β1, and α3β1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, β-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for α2/α3 over α1 (K_D α1 > α2 = α3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for α1 over α2 (K_D α1 ≤ α3 < α2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K_D α1 = α3 = α2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K⁺ ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately α2-selective. This supports a major role of α2 in cardiac contraction and cardiotonic effects of digitalis glycosides.     

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.     

RT-PCR detection of the Na,K-ATPase beta3-isoform in human heart.

The Na,K-ATPase is a heterodimer composed of an alpha-catalytic and a beta-glycoprotein subunit. At present, three different alpha-polypeptides (alpha1, alpha2, alpha3) and two distinct beta-isoforms (beta1 and beta2) have been detected in human heart. The aim of the present study was to determine whether or not the beta3-isoform of the Na,K-ATPase can be detected in human heart. Using the highly sensitive method of RT-PCR, we here show that human heart expresses the beta3-isoform of the Na,K-ATPase. Given the differences in pharmacological properties of the nine different Na,K-ATPase isoenzymes (containing all combinations of the subunit isoforms), the study of beta3-isoform regulation in human heart may be of interest in understanding the altered response of human myocardium to digitalis therapy during heart failure.     

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.     

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.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

Active transport of Na+ by modified Na,K-ATPase

The ability of ATP, CTP, ITP, GTP and UTP to induce ouabain-sensitive accumulation of Na+ by proteoliposomes with a reconstituted Na/K-pump was studied. At low Na+/K+ ratio (20 mM/50 mM), a correlation was observed between the proton-accepting capacity of the nucleotide and its efficiency as an active transport substrate. In order to test the hypothesis on the role of the negative charge in position 1 of the purine (3-pyrimidine) base of the nucleotide in the reversible transitions from the Na- to the K-conformations of Na,K-ATPase, two ATP analogs (N1-hydroxy-ATP possessing a proton-accepting ability and N1-methoxy-ATP whose molecule carries a negative charge quenched by a methyl group) were used. The first substrate provides for active accumulation of Na+ by proteoliposomes at a rate similar to that of ATP, whereas the second substrate is fairly ineffective.     

Na,K-ATPase: radiation inactivation studies

Na,K-ATPase from duck salt gland and ox brain in the membrane-bound or solubilized form was studied by the radiation inactivation technique using ATP, CTP, GTP or p-NPP as substrates. The values of radiation inactivation size (RIS) were compared with the target size (TS) for the alpha-subunit of the enzyme obtained by an independent method as well as with analytical centrifugation data obtained for C12E8-solubilized enzyme. It was concluded that during ATP (CTP) hydrolysis the enzyme operates as an oligomeric structure; the complex formation requires the presence of K+ and adenosine triphosphate binding to the sites with a low affinity for the nucleotide. Specially designed experiments revealed that the degree of enzyme oligomerization increases with an increase in the microviscosity of the membrane lipid environment.     

Lipid-protein interactions with the Na,K-ATPase

Studies of lipid interactions with membranous Na,K-ATPase by using electron spin resonance spectroscopy in conjunction with spin-labelled lipids are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are related directly to the intramembranous structure of the Na,K-ATPase, and can be used to check the integrity of extensively trypsinised preparations.     

Na,K-ATPase from duck salt glands

The protein and lipid composition of Na,K-ATPase from duck salt glands were characterized. A kinetic analysis of hydrolysis of two substrates, one of which (ATP) provides and the other (ITP) does not provide for cation active transport was carried out. In both cases two Km values were obtained and were found equal to 10 and 330 microM for ATP and 35 and 710 microM for ITP, respectively. This suggests the existence of substrate sites with high and low affinities. The Hill coefficient for the ATP hydrolysis was equal to 1.4-1.6; the ITP hydrolysis was non-cooperative. It was assumed that positive cooperative interactions between Na,K-ATPase protomers are necessary for active translocation of Na+ and K+.     

Indanedione spin labelling of Na,K-ATPase

The indanedione series of vinyl ketone spin-labelling reagents has been extended in two ways: by increasing the length of the rigid spacer between the reactive centre and the nitroxide ring, or by introducing an electrophilic substituent (that could also hinder its rotation) at the bridge head position of the nitroxide ring. Three reagents of this new series have been used to spin label the Class II thiol groups of membranous Na,K-ATPase from Squalus acanthias. With a conjugated diene spacer, the majority of spin labels are strongly held but a minor population is relatively mobile at 37 degrees C. With a conjugated triene spacer, the nitroxide is still strongly held but a portion of the label is non-covalently bound. The 4-bromo-pyrroline derivative (with short vinyl spacer) is tightly held at the attachment site, and the conventional electron paramagnetic resonance (EPR) spectra distinguish between the two enantiomeric structures which differ in their mobility at 37 degrees C. Saturation transfer EPR (ST-EPR) spectra of this label at 4 degrees C have been used to determine the dependence of the protein rotational mobility on ionic strength. Electrostatic repulsion contributes to the lateral interactions between Na,K-ATPase molecules.     

The reaction mechanism of Na, K-ATPase

To help characterize the Na,K-ATPase active site with enzyme incorporated into phospholipid vesicles, the activities with alternative substrates were compared, 22Na/Na-transport was equivalent with ATP, CTP, carbamylphosphate and acetylphosphate, but slower with CTP, 3-O-methylfluoresceinphosphate (3-O-MFP), nitrophenylphosphate and umbelliferonephosphate. It indicates a slower rate of formation of phosphorylating enzyme complex in conformation position of E1 (E1P) when the second group of substrates is bound with enzyme active center. 22Na/K-transport was half as effective with CTP as with ATP and was far slower with the other substrates. It indicates a more stringent selectivity at the low-affinity site of enzyme in conformation E2 that accelerates the slow step of this transport mode. Although enzyme modification with fluoresceinisothiocyanate blocks the high-affinity site to ATP, the K-phosphatase reaction catalyzed by E2 is retained, even with a substrate, 3-O-MFP, that binds to the adenine pocket. Dimethylsulfoxide inhibits hydrolysis of the nucleotides and of the carboxylic phosphate substrates of the K-phosphatase reaction, but stimulates hydrolysis of the phenolic phosphate substrates (nitrophenylphosphate and umbelliferone phosphate) which normally are hydrolyzed more slowly than the other substrates. On the basis of these data the authors propose the model of Na,K-ATPase active center.