Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.     

cDNA cloning of the beta-subunit of the rat gastric H,K-ATPase

A cDNA encoding the beta-subunit of the rat gastric H,K-ATPase has been identified using oligonucleotide probes based on the amino acid sequences of two peptides from the pig H,K-ATPase beta-subunit (Hall, K., Perez, G., Anderson, D., Gutierrez, C., Munson, K., Hersey, S. J., Kaplan, J. H., and Sachs, G. (1990) Biochemistry 29, 701-706). The nucleotide sequence of the 1.3-kilobase cDNA has been determined and the primary structure of the protein deduced. The protein consists of 294 amino acids and has an Mr of 33,625. The amino acid sequence of the H,K-ATPase beta-subunit is similar to those of the beta 1 (29% identity) and beta 2 (37% identity) subunits of the Na,K-ATPase. Based on the hydropathy profile it seems to have the same transmembrane organization as the Na,K-ATPase beta-subunit, with a single membrane-spanning domain near the amino terminus. Seven potential N-linked glycosylation sites are located in the putative extracellular regions of the protein. Northern blot analyses of poly(A)+ RNAs from 13 tissues demonstrate that the H,K-ATPase beta-subunit mRNA is expressed at high level in stomach and is not expressed in any of the other tissues.     

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.     

cDNA cloning of the beta-subunit of the human gastric H,K-ATPase.

A full-length cDNA clone encoding the human gastric H,K-ATPase (EC 3.6.1.36)beta-subunit was isolated from a human gastric mucosal lambda gt10 library using oligonucleotide probes which were based on the cDNA sequence from rat and rabbit H,K-ATPase beta-subunits. The insert was 1407 bp in length and encoded a polypeptide of 291 amino acids with a MW = 33,367 Da. It exhibited 84.2%, 85.6% and 81.3% identity to the H,K-ATPase beta-subunits of rabbit, pig and rat, respectively.     

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080- inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit

An isozyme-specific domain of the catalytic subunit of the Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints of the Na,K-ATPase. The antibody recognized the alpha I isozyme of the rat Na,K-ATPase, but not the alpha II or alpha III isozymes. It recognized native and sodium dodecyl sulfate-denatured Na,K-ATPase and specifically stained basolateral membranes of the renal tubule. It bound to rat alpha I with highest affinity, but also cross-reacted with mouse, monkey, and human alpha I. It did not cross-react with sheep, pig, chicken, Torpedo, or dog alpha I. Fine specificity mapping was used to deduce the most likely antibody binding sites, based on comparison of eight amino acid sequences from cDNA clones. Two potential binding sites were found at widely separated locations. Limited tryptic digestion of the native enzyme was then used to demonstrate that the binding site was close to the N-terminal end of the Na,K-ATPase. The binding site is predicted to include the following essential amino acid sequence: Asp-Lys-Lys-Ser-Lys-Lys in rat alpha I or Asp-Lys-Lys-Gly-Lys-Lys in human alpha I. The antibody was found to bind to opened, but not to sealed right-side-out vesicles isolated from the rat renal medulla, demonstrating that the N-terminal end of the Na,K-ATPase is exposed at the interior of the cell.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

Use of the H,K-ATPase beta subunit to identify multiple sorting pathways for plasma membrane delivery in polarized cells

A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase beta subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the beta subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase beta subunits associate with the endogenous Na,K-ATPase alpha(1) subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase beta subunit with the Na,K-ATPase alpha(1) subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the beta subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase beta subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the beta subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric beta subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase beta subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis.     

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.     

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K- ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.     

Epitope and mimotope for an antibody to the Na, K-ATPase.

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.     

The role of the beta1 subunit of the Na,K-ATPase and its glycosylation in cell-cell adhesion

Based on recent data showing that overexpression of the Na,K-ATPase beta(1) subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the beta(1) subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase alpha(1) and beta(1) subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, beta-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca(2+) depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase beta(1) subunit and the endogenous alpha(1) subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous beta(1) subunits in the lateral membrane were substituted by unglycosylated beta(1) subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase beta(1) subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase beta(1) subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase beta(1) subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation.     

Na,K-ATPase isoform expression in sheep red blood cell precursors

Isoform expression of mammalian red cell Na,K-ATPase was analyzed using messenger RNA isolated from red cell precursor-enriched bone marrow of anemic sheep. Expression of the catalytic alpha subunit was analyzed using rat isoform-specific cDNA probes and expression of the beta 1 subunit, using a sheep beta 1-specific cDNA probe. RNA isolated from sheep kidney and brain were analyzed concurrently. In the red cell, as in the kidney, messenger RNA encoding only one isoform (alpha 1) of the catalytic subunit is detected; neither of the other isoforms (alpha 2 or alpha 3) could be detected. This holds true for bone marrow of sheep of either the low potassium or high potassium phenotype. Relative to the expression of alpha 1, beta subunit-specific message (beta 1) was extremely low in the red cell compared to either kidney (less than 5%) or brain (less than 3%). Using a rat cDNA probe specific for a beta 1-like subunit, beta 2, message was detected in brain but not in either kidney or bone marrow.