Peter Leth Jorgensen

Expression in High Yield of Pig α1β1 Na,K-ATPase and Inactive Mutants D369N and D807N in Saccharomyces cerevisiae

Studies of structure-function relationships in Na,K-ATPase require high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The α1- and β1-subunit cDNAs were inserted into a single 2-μm-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 ± 3,000 [3H]ouabain sites/cell. In one bioreactor run 150-200 g of yeast were produced with 54 ± 5 μg of Na,K-pump protein/g of cells. Through purification in membrane bound form the activity of the recombinant Na,K-ATPase was increased to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min) were close to those of native pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same α-subunit concentration and [3H]ouabain binding capacity as the wild type Na,K-ATPase. The high yield and absence of endogenous activity allowed assay of [3H]ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).

STRUCTURE-FUNCTION RELATIONSHIPS OF E1-E2 TRANSITIONS AND CATION BINDING IN NA, K-PUMP PROTEIN

Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for equilibrium ATP binding, occlusion of T1+, K+ displacement of ATP, and Na(+)-dependent phosphorylation with determinations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic substitution of the phosphorylated residue (Asp369Ala) reveals an intrinsic high affinity for ATP binding (Kd 2.8 vs. 100 nM for wild type) and causes a shift of conformational equilibrium towards the E2 form. Substitution of Glu327, Glu779, Asp804 or Asp808 in transmembrane segments 4, 5, and 6 shows that each of these residues are essential for high-affinity occlusion of K+ and for binding of Na+. Substitution of other residues in segment 5 shows that the carboxamide group of Asn776 is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr774 as specific determinant of Na+ binding in the E1P[3Na] form, whereas Ser775 is a specific participant of high-affinity binding of the E2[2K] form, suggesting that these residues engage in formation of a molecular Na+/K+ switch. The position of the switch may be controlled by rotating or tilting the helix during the E1-E2 transition.

Trafficking of Na,K-ATPase Fused to Enhanced Green Fluorescent Protein Is Mediated by Protein Kinase A or C

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3–4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.

Transmission of E1-E2 structural changes in response to Na+ or K+ binding in Na,K-ATPase.

Abstract: The extensive E1‐E2 conformational changes in response to Na+ or K+ binding in the absence of other ligands must be driven by motion of the side chains contributing to cation coordination, but the differences in structure of Na+ and K+ sites have not been resolved. The recent high resolution structure model of the E2 conformation of Ca‐ATPase offers the first opportunity to examine and model the changes accompanying the adjustment of the cation sites from an E1 form with specificity for Na+ to an E2 form with specificity for K+. The model of the E2 form provides a remarkable fit to the data of direct Tl+ or K+ binding after site‐directed mutagenesis of residues Asp804 and Asp808 in M6, Glu 779, Gln776, and Ser775 in M5, and Glu327 in M4. Cytoplasmic domain movements during E1↔ E2 conformational transition can be monitored by proteolytic cleavage. Protection of the chymotrypsin‐sensitive bond at Leu266 in L2/3 and rotation of the A domain is more complete in the E2Mg‐vanadate‐ouabain complex than in the E2[2K] form.

Homology Modeling of Na,K‐ATPase

Identification of the third Na+ binding site would be crucial in interpretation of the electrophysiological behavior of Na,K‐ATPase. To address this question a three‐dimensional homology model of Na,K‐ATPase was built from the known crystallographic structure of Ca‐ATPase (1EUL). Phe760, which is conserved in virtually all Ca‐ATPases, is replaced by Ser768 in Na,K‐ATPase, resulting in a small cavity between M4, M5, and M6. A partially hydrated Na+ ion can be bound at this third site on the cytoplasmic side of cation binding sites 1 and 2. This leads to the proposal that the conductance of the ‘third Na+’ ion across ∼70% of the membrane dielectric may be achieved by adding up the passage of one Na+ ion from the described cytoplasmic cavity to cation site 1 and the further conductance of the previously bound Na+ ion from cation site 1 to the extracellular phase. This relay mechanism may therefore be compatible with the electrogenic profile of Na+ translocation.

Regulation and Function of Lysine-Substituted Na,K Pumps in Salt Adaptation of Artemia franciscana

The brine shrimp Artemia thrives at extreme conditions of up to 300 g/l salt in hypersaline lakes, but the molecular aspects of this salt adaptation are not clarified. To examine the influence of salt on the expression of two isoforms of Na,K-ATPase, adult Artemia franciscana were cultured for 39 days with the microalga Dunaliella salina as fodder at increasing salt from 30 to 280 g/l. Quantitative reverse-transcriptase polymerase chain reaction showed that the abundance of mRNA of the lysine-substituted α2(KK)-subunit was very low at 30 g/l salt but rose steeply in the range of 70–200 g/l to a level at 200–280 g/l salt, similar to the abundance of the mRNA of the α1(NN)-subunit, which was insignificantly affected by increasing salt. Site-directed mutagenesis showed that Asn324Lys and Asn776Lys in the α1-subunit of pig kidney Na,K-ATPase reduced the stoichiometry of 204Tl binding from 2 to about 1 Tl+(K+) per α-subunit and Na+-dependent phosphorylation from ATP to 25–30%. In structure models, the ε-amino group of Lys776 is located at cation site 1 in the E1P form and near cation site 2 in the E2 conformation, while the side chain of Lys324 points away from the cation sites. Salt-induced expression of the α2(KK)-subunit Na,K-ATPase in A. franciscana may reduce the Na+/ATP ratio and enable the Na,K pump to extrude Na+ against steeper gradients and, thus, contribute to salt adaptation.

Chapter 1 Na, K-ATPase, structure and transport mechanism

Publisher Summary The Na,K-pump is ubiquitous and located at the surface membrane of most animal cells. Primary active Na,K-pumping is a key process for the active uptake of nutrients, salts, and water and for the regulation of fluid and electrolyte homeostasis in mammals. The application of recombinant DNA techniques led to the primary structure of the α subunit and β subunit of the Na,K-pump in the mammalian kidney and a number of tissues and species. This chapter is focuses on the structural organization of this renal Na,K-pump and the molecular mechanisms behind the transformation of chemical energy to the movement of Na + and K + across the membrane. The chapter discusses the organization of the proteins in the membrane, their interaction with cytoskeletal components, and the identification of protein segments that are engaged in the binding of nucleotides or cations and in conformational changes in the protein that bring about a reorientation of cation binding sites.

Expression of Na,K-ATPase in Saccharomyces cerevisiae.

The lack of an efficient expression system that allows direct analysis of ligand binding and enzymatic intermediates of the Na+,K+ pump constitutes a bottleneck for studying the mechanisms for Na+,K+-transport across the membranes.

Increase in Affinity for ATP and Change in E1-E2 Conformational Equilibrium after Mutations to the Phosphorylation Site (Asp369) of the σ Subunit of Na,K-ATPase

The side chain of phosphorylated residue D369 is essential for ATPase activity of all P-type ATPases and mutations to residue D369 have been described to prevent assembly of a p units at the cell surface of mammalian cells( and Xenopus oocytes2 We analyzed whether the recently developed yeast expression system3 was able to target the enzymatically inactive mutations a(D369N)P and a(D369A)P to the plasma membrane. We furthermore investigated the effects of these mutations on equilibrium [3H]ATP binding, ADP binding, and [3H]ouabain binding and on the conformational equilibrium between the El and E2 forms. Equilibrium [3H]ouabain binding to intact yeast cells expressing a(wt)P, a(D369N)P, and a(D369A)P demonstrated the expression of all these enzymes at the cell surface at comparable densities (FIG. 1). The discrepancy between these results and data obtained in mammalian cells’ and oocytes2 might be due to competition between the endogenous a subunit and the a(D369N) or a(D369A) subunits for a limiting number of p subunits in those cells. The absence of endogenous a subunits in yeast cells circumvents this problem. For the interpretation of site-directed mutagenesis experiments it is essential to have a homogeneous population of heterologously expressed protein. The yeast expression system was shown to fulfill this criterion as the density of expressed a-subunit protein in yeast membranes, as quantified by Western blotting, equaled the density of [3H]ouabain sites and [3H]ATP sites. Additionally, size exclusion chromatography on a TSK 3000 SW column demonstrated that the hydrodynamic properties of recombinant a(wt)P, a(D369N)P and a(D369A)P enzymes were identical to those of purified pig kidney enzyme. Removal of the negative charge on D369 and introduction of a more hydrophobic amino acid had a dramatic effect on [3H]ATP binding at equilibrium (TABLE 1). The

Cloning and Characterization of a New Member of the Family of Na+/K+-ATPase Genes

The Na+/K+-ATPase consists of one α and one β subunit. In mammals, three isoforms of the α subunit, and two isoforms of the β subunit have been identified by molecular genetic and immunological techniques. Two additional genes (αC and αD) with high similarity to the α genes have also been cloned and partly sequenced. The functional status of these two genes remains to be determined. Here we report the data on cloning and characterization of a new Na+/K+-ATPase α subunit related gene from rabbit.