Kathleen J. Sweadner

Mutations in the Na+/K+-ATPase α3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism

Rapid-onset dystonia-parkinsonism (RDP, DYT12) is a distinctive autosomal-dominant movement disorder with variable expressivity and reduced penetrance characterized by abrupt onset of dystonia, usually accompanied by signs of parkinsonism. The sudden onset of symptoms over hours to a few weeks, often associated with physical or emotional stress, suggests a trigger initiating a nervous system insult resulting in permanent neurologic disability. We report the finding of six missense mutations in the gene for the Na+/K+ -ATPase alpha3 subunit (ATP1A3) in seven unrelated families with RDP. Functional studies and structural analysis of the protein suggest that these mutations impair enzyme activity or stability. This finding implicates the Na+/K+ pump, a crucial protein responsible for the electrochemical gradient across the cell membrane, in dystonia and parkinsonism.

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, α and β, but in kidney it is also associated with a 7.4-kDa protein, the γ subunit. Stable transfection was used to determine the effect of γ on Na,K-ATPase properties. When isolated from either kidney or transfected cells, αβγ had lower affinities for both Na+ and K+ than αβ. A post-translational modification of γ selectively eliminated the effect on Na+ affinity, suggesting three configurations (αβ, αβγ, and αβγ*) 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 α and β subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect γ in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked γ, 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 γ subunit in renal physiology.

ISOFORMS OF NA,K-ATPASE ALPHA AND BETA SUBUNITS IN THE RAT CEREBELLUM AND IN GRANULE CELL CULTURES

There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the α subunit, and at least two of the β subunit. The α subunit is the catalytic subunit. The β subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of β subunit isoforms modifies enzyme kinetics, implying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase β subunit isoforms β1 and β2. These antibodies, along with antibodies against the α subunit isoforms, were used to stain sections of the rat cerebellum and cultures of cerebellar granule cells to ascertain expression and subcellular distribution in identifiable cells. Comparison of α and β isoform distribution with double-label staining demonstrated that there was no preferential association of particular α subunits with particular β subunits, nor was there an association with excitatory or inhibitory neurotransmission modes. Isoform composition differences were seen when Purkinje, basket, and granule cells were compared. Whether β1 and β2 are specific for neurons and glia, respectively, has been controversial, but expression of both β subunit types was seen here in granule cells. In rat cerebellar astrocytes, in sections and in culture, α2 expression was prominent, yet the expression of either β subunit was low in comparison. The complexity of Na,K-ATPase isoform distribution underscores the subtlety of its regulation and physiological role in excitable cells.

Phosphorylation of Na,K-ATPase by Protein Kinase C at Ser18 Occurs in Intact Cells but Does Not Result in Direct Inhibition of ATP Hydrolysis

Na,K-ATPase activity has been demonstrated to be regulated by a variety of hormones in different tissues. It is known to be directly phosphorylated on its α-subunit, but the functional effects of protein kinases remain controversial. We have developed a sensitive, antibody-based assay for detection of the level of phosphorylation of the α1-isoform of rat Na,K-ATPase at the serine residue that is most readily phosphorylated by protein kinase C (PKC)in vitro, Ser18. By stimulation of endogenous PKC and inhibition of phosphatase activity, it was possible to consistently obtain a very high stoichiometry of phosphorylation (close to 0.9) in several types of intact cells. This demonstrates the accessibility and competency of the site for endogenous phosphorylation. The cells used were derived from rat (NRK 52E, C6, L6, and primary cultures of cerebellar granule cells, representing epithelial cells, glia, muscle cells, and neurons). In the presence of the phosphatase inhibitor calyculin A, full phosphorylation was preserved during subsequent assays of enzyme activity in vitro. Assay of the hydrolysis of ATP in NRK and C6 cells, however, indicated that there was no significant effect of phosphorylation on the V max of the Na,K-ATPase or on the apparent affinity for Na+. Any regulatory effect of PKC on sodium pump activity thus must be lost upon disruption or permeabilization of the cells and is not a direct consequence of enzyme alteration by covalent phosphorylation of Ser18.

Immunologic identification of Na+,K(+)-ATPase isoforms in myocardium. Isoform change in deoxycorticosterone acetate-salt hypertension.

There are three isoforms of the catalytic (alpha) subunit of the Na+,K(+)-ATPase, each derived from a different gene, that differ in their sensitivity to inhibition by cardiac glycosides. Antibodies specific for the three isoforms were used to study Na+,K(+)-ATPase isoform expression in ventricular myocardium, where an understanding of digitalis receptor diversity is most important. In the rat heart, there is simultaneous expression of two isoforms in adult ventricle, and immunofluorescence studies demonstrated that both isoforms are expressed uniformly in cardiomyocytes. Hypertension and hypertrophy have been reported to selectively depress alpha 2 isoform mRNA levels, and we show in the present study that alpha 2 protein levels were correspondingly depressed in rats made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate and a high-salt diet. In the human heart, where mRNA for all three alpha isoforms has been reported, we detected all three isoform proteins (alpha 1, alpha 2, and alpha 3). Two isoforms (alpha 1 and alpha 3) predominated in the macaque heart; dissection of the heart showed uniformity of isoform expression in different ventricular regions but markedly less alpha 3 in the atrium. Finally, isoform-specific antibodies were used to detect which alpha isoforms were expressed in the ventricles of several commonly used experimental animals to test the correlation of isoform expression with cardiac glycoside-response heterogeneity. Two isoforms (alpha 1 and alpha 3) were found in canine myocardium, whereas only one (alpha 1) was found in sheep and guinea pig. Expression of Na+,K(+)-ATPase isoforms can thus be readily followed and related to the physiology of the digitalis receptor.

Distinct neurological disorders with ATP1A3 mutations

Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in-vitro and animal model systems, and the role of Na(+)/K(+)-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases.

Tissue-specific Expression of the Na,K-ATPase β3 Subunit THE PRESENCE OF β3 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 (α) 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 β. Recently a new isoform (β3) has been identified in mammals. Here we describe structural features and tissue distribution of the β3 protein, utilizing an antiserum specific for its N terminus. β3 was the only β detected in Na,K-ATPase purified from C6 glioma. Treatment with N-glycosidase F confirmed that β3 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 β1 and β2 subunits. Detection of β subunits has historically been difficult in certain tissues. Sensitivity was improved by deglycosylating, and expression was evaluated by obtaining estimates of β3/α ratio. The proportion of β3 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 β3 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 β subunit in membranes or in discrete cellular or subcellular structures.

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.

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ 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 °C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785–8796). The β subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the α subunit were extruded, while the rest of α 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 α 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 α subunits as dimers, trimers, and tetramers without β or γ subunits. This implies specific α-α contact. Third, the γ subunit, like the C-terminal spans of α, 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 α, β, and γ subunits in which α can associate in assemblies as large as tetramers via its cytoplasmic domain, while β and γ subunits associate with α primarily in its C-terminal portion, which has a unique structure and thermal instability.

Differential regulation of renal Na,K-ATPase by splice variants of the gamma subunit

Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the γ subunit (γ, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human γ occur in two splice variants, γa and γb, with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified duringin vitro translation with microsomes, indicating that there are four possible forms of γ. Site-directed mutagenesis revealed Thr2 and Ser5 as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of γa into NRK-52E cells resulted in reduction of apparent affinities for Na+ and K+. Individual clones differed in γ post-translational modification, however, and the effect on Na+ affinity was absent in clones with full modification. Here, transfection of γb also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na+ affinity, but modification was required for the effect on K+ affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na+ affinity was shown by the slower growth of γa, γb, and γb′ transfectants in culture. The differential influence of the four structural variants of γ on affinities of the Na,K-ATPase for Na+ and K+, together with our previous finding of different distributions of γa and γb along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.