Thermal denaturation of the Na,K-ATPase provides evidence for alpha-alpha oligomeric interaction and gamma subunit association with the C-terminal domain.

Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55 degrees C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The beta subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the alpha subunit were extruded, while the rest of alpha retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of alpha was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of alpha subunits as dimers, trimers, and tetramers without beta or gamma subunits. This implies specific alpha-alpha contact. Third, the gamma subunit, like the C-terminal spans of alpha, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of alpha, beta, and gamma subunits in which alpha can associate in assemblies as large as tetramers via its cytoplasmic domain, while beta and gamma subunits associate with alpha primarily in its C-terminal portion, which has a unique structure and thermal instability.

Genomic organization of the human FXYD2 gene encoding the gamma subunit of the Na,K-ATPase.

Although the gamma subunit of the Na,K-ATPase has only 66 or 68 amino acids, its human gene (FXYD2) was found to span 9.2 kb and have seven exons, including two alternatively spliced exons encoding different N-termini. Two candidate promoters with consensus sites for transcription factors Sp1, AP-1, and AP-2 are present, consistent with independent transcription of the splice variants. Multiple ESTs support the transcriptional competence of the identified gene elements. In the FXYD2 gene, there are two closely spaced polyadenylation signals, and both are used. A proposed third splice variant encoding a 31-residue N-terminal extension was not found in the gene, nor was the predicted larger protein found in human kidney Na,K-ATPase. Instead, evidence was found for the origin of the larger cDNA clone in homologous recombination with unrelated DNA from chromosome 2. FXYD2 is on chromosome 11q23 close to a site of tumorigenic chromosomal translocations, and has a number of repeat elements.

Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase.

The Na,K-ATPase, which catalyzes the active transport of Na(+) and K(+), has two principal subunits (alpha and beta) that have several genetically distinct isoforms. Most of these isoforms are expressed in the nervous system, but certain ones are preferentially expressed in glia and others in neurons. Of the beta isoforms, beta1 predominates in neurons and beta2 in astrocytes, although there are some exceptions. Here we demonstrate that beta3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified beta3 in oligodendrocytes of rat brain white matter in typical linear arrays of cell bodies between fascicles of axons. The intensity of stain peaked at 20 postnatal days. beta3 was identified in cortical oligodendrocytes grown in culture, where it was expressed in processes and colocalized with antibody to galactocerebroside. In the mouse and rat optic nerve, beta3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the beta1 and beta2 subunits, showing distinct patterns of labelling of axons (beta1) and astrocytes (beta2). The C6 glioma cell line was also found to express the beta3 isoform preferentially. Since beta3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage.

The gamma subunit modulates Na(+) and K(+) affinity of the renal Na,K-ATPase.

The Na(+),K(+)-ATPase catalyzes the active transport of ions. It has two necessary subunits, alpha and beta, but in kidney it is also associated with a 7.4-kDa protein, the gamma subunit. Stable transfection was used to determine the effect of gamma on Na, K-ATPase properties. When isolated from either kidney or transfected cells, alphabetagamma had lower affinities for both Na(+) and K(+) than alphabeta. A post-translational modification of gamma selectively eliminated the effect on Na(+) affinity, suggesting three configurations (alphabeta, alphabetagamma, and alphabetagamma*) conferring different stable properties to Na, K-ATPase. In the nephron, segment-specific differences in Na(+) affinity have been reported that cannot be explained by the known alpha and beta subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect gamma in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked gamma, correlating with higher Na(+) affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the gamma subunit in renal physiology.

Cellular and subcellular specification of Na,K-ATPase alpha and beta isoforms in the postnatal development of mouse retina.

The Na,K-ATPase is a dominant factor in retinal energy metabolism, and unique combinations of isoforms of its alpha and beta subunits are expressed in different cell types and determine its functional properties. We used isoform-specific antibodies and fluorescence confocal microscopy to determine the expression of Na,K-ATPase alpha and beta subunits in the mouse and rat retina. In the adult retina, alpha1 was found in Müller and horizontal cells, alpha2 in some Müller glia, and alpha3 in photoreceptors and all retinal neurons. beta1 was largely restricted to horizontal, amacrine, and ganglion cells; beta2 was largely restricted to photoreceptors, bipolar cells, and Müller glia; and beta3 was largely restricted to photoreceptors. Photoreceptor inner segments have the highest concentration of Na,K-ATPase in adult retinas. Isoform distribution exhibited marked changes during postnatal development. alpha3 and beta2 were in undifferentiated photoreceptor somas at birth but only later were targeted to inner segments and synaptic terminals. beta3, in contrast, was expressed late in photoreceptor differentiation and was immediately targeted to inner segments. A high level of beta1 expression in horizontal cells preceded migration, whereas increases in beta2 expression in bipolar cells occurred very late, coinciding with synaptogenesis in the inner plexiform layer. Most of the spatial specification of Na,K-ATPase isoform expression was completed before eye opening and the onset of electroretinographic responses on postnatal day 13 (P13), but quantitative increase continued until P22 in parallel with synaptogenesis.

Plasticity of Na,K-ATPase isoform expression in cultures of flat astrocytes: species differences in gene expression.

The Na,K-ATPase plays an active role in glial physiology, contributing to K+ uptake as well as to the Na+ gradients used by other membrane carriers. There are multiple isoforms of Na,K-ATPase alpha and beta subunits, and different combinations result in different affinities for Na+ and K+. Isoform choice should thus influence K+ and Na+ homeostasis in astrocytes. Prior studies of astrocyte Na,K-ATPase subunit composition have produced apparently conflicting results, suggesting plasticity of gene expression. Purified flat astrocytes from the cerebral cortex and cerebellum of both mouse and rat were systematically investigated here. Using antibodies specific for the alpha1, alpha2, alpha3, beta1, beta2, and beta3 subunits, isoform level was assessed with Western blots, and cellular distribution was visualized with immunofluorescence. Although alpha1 was always expressed, differences were observed in the expression of alpha2 and beta2, subunits that can be expressed in astrocytes in vivo and in coculture with neurons. In addition, abundant alpha subunit was expressed in rat astrocytes and in mouse cerebellar astrocytes without an equivalent level of any of the known beta isoforms, suggesting that an additional beta subunit important for glia is yet to be discovered. Conditions that have been shown to increase Na,K-ATPase activity in astrocyte cultures, such as dibutyryl cAMP, high extracellular K+, and glutamate, did not specifically induce missing subunits, suggesting that cellular interactions are required to alter the ion transporter phenotype.

Tissue-specific expression of the Na,K-ATPase beta3 subunit. The presence of beta3 in lung and liver addresses the problem of the missing subunit.

The Na,K-ATPase belongs to a family of P-type ion-translocating ATPases sharing homologous catalytic subunits (alpha) that traverse the membrane several times and contain the binding sites for ATP and cations. In this family, only Na,K- and H,K-ATPases have been shown to have a second subunit, a single-span glycoprotein called beta. Recently a new isoform (beta3) has been identified in mammals. Here we describe structural features and tissue distribution of the beta3 protein, utilizing an antiserum specific for its N terminus. beta3 was the only beta detected in Na,K-ATPase purified from C6 glioma. Treatment with N-glycosidase F confirmed that beta3 is a glycoprotein containing N-linked carbohydrate chains. Molecular masses of the glycosylated protein and core protein were estimated to be 42 and 35 kDa, respectively, which are different from those of the beta1 and beta2 subunits. Detection of beta subunits has historically been difficult in certain tissues. Sensitivity was improved by deglycosylating, and expression was evaluated by obtaining estimates of beta3/alpha ratio. The proportion of beta3 protein in the rat was highest in lung and testis. It was also present in liver and skeletal muscle, whereas kidney, heart, and brain contained it only as a minor component of the Na,K-ATPase. In P7 rat, we found skeletal muscle and lung Na,K-ATPase to be the most enriched in beta3 subunit, whereas expression in liver was very low, illustrating developmentally regulated changes in expression. The substantial expression in lung and adult liver very likely explains long-standing puzzles about an apparent paucity of beta subunit in membranes or in discrete cellular or subcellular structures.

Hormonal and neurogenic control of Na-K-ATPase and myosin isoforms in neonatal rat cardiac myocytes.

In the rat heart there is a postnatal switch in the expression of isoforms of both Na-K-ATPase and myosin heavy chain (MHC). Here we investigated factors controlling isoform changes in cultures of neonatal cardiomyocytes. In serum-free medium, the compositions of either Na-K-ATPase or MHC isoforms resembled the neonatal phenotype. Thyroid hormone induced the MHC isoform switch but increased expression of all Na-K-ATPase isoforms to various extents. Dexamethasone failed to induce the MHC switch and inhibited Na-K-ATPase alpha 1 isoform expression without inducing the other isoforms. With both hormones, the adult phenotype for both MHC and Na-K-ATPase was seen but with low Na-K-ATPase alpha 2. The paucity of alpha 2 protein was not predicted by studies of mRNA levels. In serum, there was a gradual decline of Na-K-ATPase alpha 3 and the appearance of alpha 2, but again at a relatively low level. Expression of Na-K-ATPase alpha 2 was significantly upregulated when cardiomyocytes were cocultured with sympathetic neurons from superior cervical ganglia, without changes in the MHC isoforms. Thus innervation is postulated to play a specific role in modulating Na-K-ATPase gene expression.

Isoform-specific monoclonal antibodies to Na,K-ATPase alpha subunits. Evidence for a tissue-specific post-translational modification of the alpha subunit.

Monoclonal antibodies to isoforms of the Na,K-ATPase have become important tools in the study of the enzyme's distribution, physiological roles, and gene regulation, and when their epitopes are defined, they are useful in the study of enzyme structure as well. Evidence is presented that the alpha3-specific antibody McBX3 recognizes an unusual epitope that is not present on alpha3 in the heart. The epitope, which is also found in kidney alpha1 from some species, was mapped to a site on the large intracellular loop near the ATP binding site. DNA sequencing of reverse transcribed-PCR products encompassing the corresponding regions from alpha3 from brain (where McBX3 recognizes alpha3) and heart demonstrated that the tissue difference in epitope is not due to alternative splicing of the mRNA. Instead, hydroxylamine sensitivity indicated that the antibody recognizes a post-translational modification. The epitope for a new antibody for alpha3, XVIF9-G10, was mapped to a site near the N terminus, a location analogous to the sites for the well-characterized antibodies McK1 (alpha1) and McB2 (alpha2). The antibody XVIF9-G10 reacted with the alpha3 of the heart as well as that of the brain; however, McBX3 and XVIF9-G10 both stained the same cellular structures in sections of the rat retina. A new alpha1-specific antibody, 6F, was characterized and mapped to another site near the N terminus; this antibody has broader species specificity than the other well-characterized alpha1 antibody, McK1.

Presence of two isoforms of Na, K-ATPase with different pharmacological and immunological properties in the rat kidney.

Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the alpha 1 isoform of the catalytic subunit, whereas the collecting duct expresses an alpha 3-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC50 approximately 5.10(-6) M) which is recognized by an anti-alpha 3 antibody and another moiety of lower affinity for ouabain (IC50 approximately 5.10(-4) M) which is recognized by an anti-alpha 1 antibody. Whether these two subpopulations correspond to different isoforms of the alpha subunit of Na,K-ATPase (alpha 1 and alpha 3-like) remains to be determined.

Topology of the Na,K-ATPase. Evidence for externalization of a labile transmembrane structure during heating.

The topological organization of the Na,K-ATPase alpha subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The beta subunit is predominantly extracellular and could mask the surface of the alpha subunit. Most of the tested proteases cleaved beta, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the alpha subunit. Reduction (at 50 degrees C) of disulfide bonds that might stabilize the beta subunit fragments, or heating alone at 55 degrees C, permitted tryptic digestion of alpha at a site close to the C terminus, while simultaneously increasing digestion of beta. A 90-kDa N-terminal fragment of alpha was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the beta subunit and the extrusion of a segment of alpha that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of alpha. We conclude that this portion of the alpha subunit contains a transmembrane structure with unique lability to heating.

Immunoelectron microscopy of epitopes on Na,K-ATPase catalytic subunit. Implications for the transmembrane organization of the C-terminal domain.

The transmembrane folding of the alpha subunit of Na,K-ATPase was studied by using immunoelectron microscopy to determine whether monoclonal antibodies with defined epitopes bind to the extracellular or cytoplasmic surface. In double labeling experiments, an antibody and a reference marker were bound to purified membrane-associated Na,K-ATPase and were visualized by employing colloidal gold particles of two different sizes. Wheat germ agglutinin and a previously characterized monoclonal antibody were used as control markers for the exoplasmic and cytoplasmic surfaces, respectively. Three antibodies, VG4, VG2, and IIC9, unambiguously bound to the extracellular surface. Previously IIC9 had been assigned to the cytoplasmic surface because, in immunofluorescence studies, it stained intact cells only when they were detergent-permeabilized. To investigate the basis for this contradiction, a third assay for sidedness was used: competition binding in solution to right-side-out renal medullary vesicles. IIC9 was found to bind to the extracellular surface of sealed vesicles, but only in certain experimental conditions. It was concluded that IIC9 has an epitope that is not always accessible and that in this instance, studies of binding to intact and detergent-treated cells had given misleading results. An extracellular disposition for all three antibodies is not compatible with existing folding models, and new models are presented.

Are there several isoforms of Na,K-ATPase alpha subunit in the rabbit kidney?

Previous pharmacologic and kinetic studies have demonstrated the axial heterogeneity of the rabbit kidney tubule with regard to Na,K-ATPase. To evaluate whether this heterogeneity might reflect the presence of distinct isoforms of the alpha subunit of Na,K-ATPase, we used two monoclonal antibodies, IIC9 and IIE2 (G8), specific for the alpha 1 and alpha 3 isoforms, respectively, as probes for changes in the specific activity of Na,K-ATPase at the level of successive segments of the rabbit nephron. Single, well defined nephron segments were obtained by microdissection of collagenase-treated kidney. Results indicate that IIC9 antibody inhibited Na,K-ATPase activity by > 90% in all the segments of the nephron except the collecting duct. Conversely, IIE2 (G8) antibody abolished Na,K-ATPase activity in the collecting duct, whereas it had no effect in other nephron segments. These findings suggest that the rabbit collecting duct preferentially expresses a distinct isoform of Na,K-ATPase catalytic subunit, which is presumably alpha 3-like, in agreement with previous pharmacologic and kinetic observations, whereas other nephron segments would express the alpha 1 isoform.

Constraints on models for the folding of the Na,K-ATPase.

We have attempted to bring together in graphic fashion the available evidence on the structure of the Na,K-ATPase and the H,K-ATPase. There appears to be much room for modification of the existing models for transmembrane folding. More sites on each side of the membrane need to be identified. Whether these will be antibody epitopes, sites of covalent modification, or tags inserted by mutagenesis is less important than that there be many of them and that each be verified by alternative approaches. If any single principle has emerged from the study of the topography of membrane proteins, it is that it is easy to reach conclusions too soon.

Na,K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding.

The Na,K-stimulated ATPase is inhibited by extracellular cardiac glycosides, which bind to the enzyme's alpha subunit. We used a monoclonal antibody, VG4, as a probe of the extracellular surface. The antibody was specific for Na,K-ATPase and bound to intact cells. The epitope was mapped to the first extracellular loop (H1-H2) of alpha, using a combination of techniques including trypsinolysis, N-terminal sequence of a fragment containing the determinant, and analysis of the effects of species-specific sequence differences. The antibody inhibited Na,K-ATPase activity under certain circumstances, indicating that the H1-H2 loop participates in conformational changes that are transmitted to the active site. Mutations in the H1-H2 loop have been shown by others to affect ouabain affinity. Ouabain and the antibody acted synergistically to inhibit the enzyme, which seemingly supported the hypothesis that the H1-H2 loop is an essential part of the cardiac glycoside binding site. Direct measurements of the binding of [3H]ouabain, however, indicated that VG4 enhanced rather than inhibited binding, presumably by promoting favorable conformation changes. The data suggest the possibility that the cardiac glycoside binding site may be intramembrane rather than extracellular.

Immunoaffinity isolation of Na,K-ATPase alpha 3 isoform from pig kidney.

The Na,K-ATPase alpha 3 isoform of the catalytic subunit has been isolated from pig kidney microsomes. The procedure employs immunoaffinity chromatography on Sepharose 4B covalently coupled with monospecific antibodies a-II against the synthetic peptide including the putative alpha 3 N-terminus. The structural analysis provides unambiguous proof that the isolated protein corresponds to the third transcript for the alpha 3 isoform. The N-terminal amino acid sequence determined. Met-Gly-Asp-Lys-Lys-Asp-Asp, shows that unlike the alpha 1 and alpha 2 proteins, the mature Na,K-ATPase isoform lacks post-translational proteolytic processing.

Immunodetection of Na,K-ATPase alpha 3-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.

Topology of Na+,K+-ATPase. Identification of the extra- and intracellular hydrophilic loops of the catalytic subunit by specific antibodies.

To study the topology of Na+,K+-ATPase monoclonal antibodies (MAbs) specific for membrane-bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra- or intracellular portions of the alpha-subunit. The extracellular location of peptide loop 804-841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween-20), was shown to bind to the hydrophilic loop 868-945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887-904 and 810-825, respectively. The C-terminus of the alpha-subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity-purified antibodies to the synthetic peptide corresponding to fragment 999-1008.

Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy.

Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.