Elena Arystarkhova

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.

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.

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.

FXYD Proteins as Regulators of the Na,K‐ATPase in the Kidney

Abstract: The FXYD gene family has seven members in mammals and others in fish. Five of these (FXYD1, FXYD2, FXYD4, FXYD7, and PLMS from shark) have been shown to alter the activity of the Na,K‐ATPase, as described by other papers in this volume. The gene structure of FXYD family members suggests assembly from protein domain modules and gene duplication. The γ subunit is unique in the family for having alternative splice variants in the coding region and can be posttranslationally modified with different final consequences for enzyme properties. The nonoverlapping distribution of γ and CHIF (FXYD4) in kidney helps to explain physiological differences in Na+ affinity among nephron segments. We also detected phospholemman (FXYD1) in kidney. By immunofluorescence, it was found in extraglomerular mesangial cells (EM cells) of the juxtaglomerular apparatus and in the afferent arteriole. Contrary to many reports that only α1 and β1 are expressed in the kidney, we found that α2 and β2 are present, although not in any nephron segment. Both were detected in arterioles, and β2 was found in the EM cells. In contrast, α1, β1, and γ were found in adjacent macula densa. Phospholemman, α2, and β2 are proposed to have distinct roles in regulating the sodium pump in structures involved in tubuloglomerular feedback.

Oligodendrocytes in brain and optic nerve express the β3 subunit isoform of Na,K-ATPase

The Na,K‐ATPase, which catalyzes the active transport of Na+ and K+, has two principal subunits (α and β) 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 β isoforms, β1 predominates in neurons and β2 in astrocytes, although there are some exceptions. Here we demonstrate that β3 is expressed in rat and mouse white matter oligodendrocytes. Immunofluorescence microscopy identified β3 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. β3 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, β3 stain was seen in oligodendrocytes, where it colocalized with carbonic anhydrase II. For comparison, optic nerve was stained for the β1 and β2 subunits, showing distinct patterns of labelling of axons (β1) and astrocytes (β2). The C6 glioma cell line was also found to express the β3 isoform preferentially. Since β3 was not found at detectable levels in astrocytes, this suggests that C6 is closer to oligodendrocytes than astrocytes in the glial cell lineage. GLIA 31:206–218, 2000.

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.

Hyperplasia of Pancreatic Beta Cells and Improved Glucose Tolerance in Mice Deficient in the FXYD2 Subunit of Na,K-ATPase

Background: Reduction in functional beta cells in pancreas is the major obstacle in diabetes. Results: Mice deficient in FXYD2 subunit of Na,K-ATPase possess a metabolic phenotype of low blood glucose along with hyperplastic pancreatic islets and hyperinsulinemia. Conclusion: The phenotype observed in Fxyd2−/− mice results from an increase in beta cell mass. Significance: FXYD2 may be a novel target for development of cell-based interventions in diabetes. Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2−/− mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2−/− mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2−/− mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.

Post-transcriptional Control of Na,K-ATPase Activity and Cell Growth by a Splice Variant of FXYD2 Protein with Modified mRNA

In kidney, FXYD proteins regulate Na,K-ATPase in a nephron segment-specific way. FXYD2 is the most abundant renal FXYD but is not expressed in most renal cell lines unless induced by hypertonicity. Expression by transfection of FXYD2a or FXYD2b splice variants in NRK-52E cells reduces the apparent Na+ affinity of the Na,K-ATPase and slows the cell proliferation rate. Based on RT-PCR, mRNAs for both splice variants were expressed in wild type NRK-52E cells as low abundance species. DNA sequencing of the PCR products revealed a base alteration from C to T in FXYD2b but not FXYD2a from both untreated and hypertonicity-treated NRK-52E cells. The 172C→T sequence change exposed a cryptic KKXX endoplasmic reticulum retrieval signal via a premature stop codon. The truncation affected trafficking of FXYD2b and its association with Na,K-ATPase and blocked its effect on enzyme kinetics and cell growth. The data may be explained by altered splicing or selective RNA editing of FXYD2b, a supplementary process that would ensure that it was inactive even if transcribed and translated, in these cells that normally express only FXYD2a. 172C→T mutation was also identified after mutagenesis of FXYD2b by error-prone PCR coupled with a selection for cell proliferation. Furthermore, the error-prone PCR alone introduced the mutation with high frequency, implying a structural peculiarity. The data confirm truncation of FXYD2b as a potential mechanism to regulate the amount of FXYD2 at the cell surface to control activity of Na,K-ATPase and cell growth.

A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1

A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures. DOI: http://dx.doi.org/10.7554/eLife.11102.001