Na, K-ATPase a subunit

Yang PY, Menter DG (2009) Carrie Cartwright, et al. Oleandrin-mediated inhibition of human tumor cell proliferation importance of Na, K-ATPase a subunits as drag targets. Mol Cancer Ther 8 2319-2328... 

Skelly, P.J., Dougan, P.M., Maule, A.G., Day, T.A. and Shoemaker, C.B. (2001) Cloning and characterization of a muscle isoform of a Na-, K-ATPase alpha subunit (SNaK1) from Schistosoma mansoni. Parasitology 123, 277-284. 

Thompson, C. B., and McDonough, A. A. (1996). Skeletal musde Na,K-ATPase a and B subunit protein levels respond to hypokalemic challenge with isoform and muscle type specificity. / Biol. Cfiam. 271, 32653-32658. 

Young, R.M., G.E. Shull and J.B. Lingrel. Multiple iiiRNAs from rat kidney and brain encode a single Na,K-ATPase beta subunit protein. J. Biol. Chem. 262 4905-4910, 1987. 

Exposure of rat alveolar type II cells plated on polycarbonate filters in serum-free medium to 10 ng keratinocyte growth factor/ml on day 4 resulted in dose-dependent increases in short-circuit current (7sc) across alveolar epithelial cell monolayers compared with controls by day 5, with further increase occurring through day 8 (Borok et al. 1998). Relative Na -K -ATPase Qi-subunit mRNA abundance was increased by 41 % on day 6 and 8 after exposure to keratinocyte growth factor, whereas a2-subunit mRNA remained only marginally detectable in both the absence and presence of keratinocyte growth factor. Levels of mRNA for the Pi-subunit of Na" -K -ATPase did not increase, whereas cellular Oi-and Pi-subunit protein increased 70 and 31 %, respectively, on day 6. mRNA for a-, P-, and y-rat epithelial Na channel all decreased in abundance after treatment with keratinocyte growth factor. 

In the case of the Na,K ATPase, the last 161 amino adds of the a subunit are essential for effective assodation with the 6 subunit. Further, the last four or five C-terminal hydrophobic amino adds of the Na -pump 6 subunit are essential for interaction with the a subunit, whereas the last few hydrophilic amino adds are not. Expression of the Na -pump a subunit, along with the 6 subunit of either the sodium or proton pump in Xenopus oocytes, has shown that the B subunit of the gastric proton pump can act as a surrogate for the 6 subunit of the sodium pump for membrane targeting and Rb uptake. This implies homology in the associative domains of the 6 subunits of the two pumps. The H,K ATPase a subunit requires its a subunit for effident cell-surface expression. Expression of chimers of the a subunits of the Na,K and Ca ATPases showed that the C-terminal half of the a subunit assembled with the B subunit. 

Feng J, Lingrel JB. Analysis of amino acid residues in the H5-H6 transmembrane and extracellular domains of Na,K-ATPase alpha subunit identifies threonine 797 as a determinant of ouabain sensitivity. B/ocftem/stry 1994 33 4218-4224. 

Biser, P.S., Thayne, K.A., Fleming, W.W. and Taylor, D.A., Na, K+ ATPase alpha-subunit isoform distribution and abundance in guinea-pig longitudinal muscle/myenteric plexus after exposure to morphine. Brain Res., 931, 186-193 (2002). 

The expansion of our knowledge of the structure and function of Na,K-ATPase is reflected in a rapid succession of reviews on Na,K-ATPase genes and regulation of expression [17], subunit assembly and functional maturation [20], the isozymes of Na,K-ATPase [18], and the stability of a subunit isoforms during evolution [21], physiological aspects and regulation of Na,K-ATPase [22], reconstitution and cation exchange [23], chemical modification [24], and occlusion of cations [25]. Other valuable sources are the review articles [26] and recent developments [27] reported at the International Na,K-pump Conference in September 1990. 

The membrane-bound preparation from kidney is easily solubilized in non-ionic detergent and analytical ultracentrifugation shows that the preparation consists predominantly (80 85%) of soluble af units with 143000 [28]. The soluble a)S unit maintains full Na,K-ATPase activity, and can undergo the cation or nucleotide induced conformational transitions that are observed in the membrane-bound preparation. A cavity for occlusion of 2K or 3Na ions can be demonstrated within the structure of the soluble a)S unit [29], as an indication that the cation pathway is organized in a pore through the aji unit rather than in the interphase between subunits in an oligomer. 

In an ideal pure preparation of Na,K-ATPase from outer renal medulla, the al subunit forms 65 70% of the total protein and the molar ratio of a to is 1 1, corresponding to a mass ratio of about 3 1 [1,5]. Functionally the preparation should be fully active in the sense that each a/ unit binds ATP, Pj, cations and the inhibitors vanadate and ouabain. The molecular activity should be close to a maximum value of 7 000-8 000 Pj/min. The highest reported binding capacities for ATP and phosphate are in the range 5-6 nmol/mg protein and close to one ligand per otjS unit [29], when fractions with maximum specific activities of Na,K-ATPase [40 50 pmo Pj/min mg protein) are selected for assay. 

Proteolytic cleavage has proven to be an efficient tool for exploring the structure and function of the Na,K-ATPase. Exposure and protection of bonds on the surface of the cytoplasmic protrusion provides unequivocal evidence for structural changes in the a subunit accompanying E1-E2 transition in Na,K-ATPase [52]. Localization of the proteolytic splits provided a shortcut to identification of residues involved in E1-E2 transition [33,53,54] and to detection of structure-function correlations [33]. Further proteolysis identifies segments at the surface of the protein and as the cytoplasmic protrusion is shaved off all ATP-dependent reactions are abolished. 

Fig. 3. (A) Disposition of afi unit in the membrane, based on sequence information [14,15], selective proteolytic digestion of the a subunit [5,6] and hydrophobic labelling (Table 1). The model for the (S subunit is based on sequencing of surface peptides and identification of S-S bridges [64,65]. T, T2 and C3 show location of proteolytic splits. CHO are glycosylated asparagines in the P subunit. (B) Peptide fragments remaining in the membrane after extensive tryptic digestion of membrane-bound Na,K-ATPase from outer medulla of pig kidney as described by Karlish et al. [7,58].

In the family of cation pumps, only the Na,K-ATPase and H,K-ATPase possess a p subunit glycoprotein (Table II), while the Ca-ATPase and H-ATPase only consist of an a subunit with close to 1 000 amino acid residues. It is tempting to propose that the p subunit should be involved in binding and transport of potassium, but the functional domains related to catalysis in Na,K-ATPase seem to be contributed exclusively by the a subunit. The functional role of the P subunit is related to biosynthesis, intracellular transport and cell-cell contacts. The P subunit is required for assembly of the aj8 unit in the endoplasmic reticulum [20]. Association with a j8 subunit is required for maturation of the a subunit and for intracellular transport of the xP unit to the plasma membrane. In the jSl-subunit isoform, three disulphide... 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

A sequence of ten amino acids (ICS-D-KTGTLT) around the phosphorylation site of Na,K-ATPase (Asp ) is highly conserved among the Na,K-, H,K-, Ca-, and Id-pumps [6]. There is also homology with the subunit of FpATP synthetase of mitochondria and chloroplasts (see [6]) except that Asp is replaced by Thr. Accordingly a covalent phosphorylated intermediate is not formed in Fi-ATPase. Mutagenesis of the phosphorylated aspartate residue in Na,K-ATPase [82], Ca-ATPase [87], or H-ATPase [88] completely blocks activity. 

Definition of Ej and E2 eonformations of the a subunit of Na,K-ATPase involves identification of cleavage points in the protein as well as association of cleavage with different rates of inactivation of Na,K-ATPase and K-phosphatase activities [104,105]. In the Ei form of Na,K-ATPase the cleavage patterns of the two serine proteases are clearly distinct. Chymotrypsin cleaves at Leu (C3), Fig. 3A, and both Na,K-ATPase and K-phosphatase are inactivated in a monoexponential pattern [33,106]. Trypsin cleaves the E form rapidly at Lys ° (T2) and more slowly at Arg (T3) to produce the characteristie biphasic pattern of inactivation. Localization of these splits was determined by sequencing N-termini of fragments after isolation on high resolution gel filtration columns [107]. 

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