Identification of a region of the polypeptide chain of Na,K-ATPase α-subunit interacting with 67-kDa melittin-like protein

It was shown earlier that a 67-kDa protein purified from mouse kidney using polyclonal antibodies against melittin (a peptide from bee venom) interacted with Na,K-ATPase from rabbit kidney. In this study, a 43-kDa proteolytic fragment of Na,K-ATPase α-subunit interacting with the 67-kDa melittin-like protein was found. The α-subunit was hydrolyzed by trypsin in the presence of 0.5 mM ouabain (E2-conformation of Na,K-ATPase). A proteolytic fragment interacting with the 67-kDa melittin-like protein that was identified by mass-spectrometry is a region of the cytoplasmic domain of Na,K-ATPase α-subunit located between amino acid residues 591 and 775. The fragment includes a conservative DPPRA motif that occurs in many P-type ATPases. It was shown earlier that this motif of H,K-ATPase from gastric mucosa binds to melittin. We suggest that namely this motif of P-type ATPases is able to interact with proteins containing melittin-like modules.     

Immunodetection of Na,K-ATPase α3-isoform in renal and nerve tissues

At least three types of mRNA of the catalytic subunit of Na,K-ATPase namely alpha-,alpha+- and alpha 3-isoforms are identified in different tissues. Only two of them alpha and alpha+ have well known structural and catalytic properties. Here we present immunochemical data indicating that the alpha 3 protein really exists in pig and human kidney, and human brain. Crude membrane fractions and purified membrane-bound Na,K-ATPases were immunoblotted with alpha 3-specific antibodies raised against the synthetic peptide corresponding to the unique sequence of this isoform. The mature alpha 3-subunit is shown to include the sequence GDKKDDKSSPK followed by the initiating methionine residue. Nephron collecting tubules are proposed to specifically contain Na,K-ATPase alpha 3-isoform.     

G-protein-coupled receptor-mediated traffic of Na,K-ATPase to the plasma membrane requires the binding of adaptor protein 1 to a Tyr-255-based sequence in the alpha-subunit

Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase alpha1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase alpha1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase alpha1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase alpha1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA alpha1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors.     

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (∼100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (∼67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Defective activity and isoform of the Na,K-ATPase in the dilated cardiomyopathic hamster.

The Na,K-ATPase function appears impaired in human heart failure with dilation; however little is known in animal model with idiopathic dilated cardiomyopathy. We studied Na,K-ATPase isoform composition and activity from cardiomyopathic hamsters of the MS 200 strain with pure dilated cardiomyopathy and compared them with those of healthy Syrian hamsters. 150-day-old male MS 200 Syrian hamsters (n = 16) and sex- and age-matched healthy Syrian hamsters (n = 15) were used. Antibodies specific for the three alpha-isoforms and against the beta1-isoform were used to study Na,K-ATPase isoform expression in ventricular myocardium. Na,K-ATPase activity was quantified in homogenate and membrane fractions. There was no significant change in left ventricular mass. Morphological examination revealed a decreased septum thickness in the dilated cardiomyopathy compared with control hamster. Idiopathic dilated cardiomyopathy in hamsters presented significantly reduced membrane alpha1 and beta1 abundances and reduced Na,K-ATPase activity (-35% vs. healthy control, p<0.05). Chronic heart failure had no effect on the Na,K-ATPase alpha2-subunit protein. We have demonstrated for the first time that dilated cardiomyopathy induces a specific reduction of both membrane alpha1- and beta1-isoform abundance and Na,K-ATPase activity in hamsters similar to those previously reported in human dilated heart failure.     

Revealing of Proteins Interacting with Na,K-ATPase

Proteins interacting with 11-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

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.     

Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase alpha subunit. This amino acid, Asp-121, is the penultimate residue of the first extracellular domain of the alpha subunit. The border residues of this particular extracellular loop of the alpha subunit have been shown to be determinants of ouabain sensitivity (Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 is involved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant alpha subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase alpha subunit containing either Asn, Ala, Glu, or Ser in place of Asp-121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase alpha subunit participates in the binding interaction between the enzyme and ouabain.     

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.     

Ouabain sensitivity of the alpha 3 isozyme of rat Na,K-ATPase

The Na,K-ATPase of rat brainstem axolemma membranes contains two isozymes of its catalytic subunit, alpha 2 and alpha 3. To isolate the alpha 3 isozyme functionally, purified axolemma Na,K-ATPase was treated with trypsin. Immunoblot analysis of trypsin-treated Na,K-ATPase using isozyme-specific antibodies showed that alpha 3 was significantly more resistant to digestion than alpha 2. The trypsin-resistant alpha 3 isozyme fraction, devoid of alpha 2, contained 50-60% of the ATPase activity, and was inhibited by ouabain half-maximally at 0.13 microM. This indicates that the alpha 3 Na,K-ATPase isozyme has a high sensitivity to cardiac glycosides.     

Glutathionylation of Na,K-ATPase Alpha-Subunit Alters Enzyme Conformation and Sensitivity to Trypsinolysis

We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the enzyme conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different conformations with different extent of glutathionylation. Incubation of the protein in the E1 conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the enzyme in both E1 and E2 conformations. The pattern of tryptic digestion of the α-subunit in E2 conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 conformation is similar to that of the native enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN conformation than in the E1-and E2-conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects enzyme conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of enzyme glutathionylation and increase in the enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.     

Synthesis and assembly of functional mammalian Na,K-ATPase in yeast.

The yeast Saccharomyces cerevisiae was investigated as an in vivo protein expression system for mammalian Na,K-ATPase. Unlike animal cells, yeast cells lack endogenous Na,K-ATPase. Expression of high affinity ouabain binding sites, ouabain-sensitive ATPase activity, or ouabain-sensitive p-nitrophenylphosphatase activity in membrane fractions of yeast cells was observed to require the expression of both alpha subunit and beta subunit polypeptides of Na,K-ATPase in the same cell. High affinity ouabain binding sites are also expressed at the cell surface of intact yeast cells containing both the alpha subunit and the beta subunit of Na,K-ATPase. These observations demonstrate that both the alpha subunit and the beta subunit of Na,K-ATPase are required for the expression of functional Na,K-ATPase activity and that yeast cells can correctly assemble this oligomeric membrane protein and transport it to the cell surface.