Heterologous expression of the Na(+),K(+)-ATPase gamma subunit in Xenopus oocytes induces an endogenous, voltage-gated large diameter pore.

1. The gamma subunit is a specific component of the plasmalemmal Na(+),K(+)-ATPase. Like structurally related single-spanning membrane proteins such as cardiac phospholemman, Mat-8 and renal CHIF, large ion conductances are activated when gamma subunits are expressed in Xenopus oocytes. 2. Here we report critical properties of the gamma-activated conductance. The gamma-activated conductance showed non-selective cationic and anionic permeation, and extremely slow kinetics, with an activation time constant > 1 s following steps to -100 mV. 3. The gamma-activated conductance was inhibited by extracellular divalent ions including Ba(2+) (K(i) = 0.7 mM) and Ca(2+) (K(i) = 0.4 mM). 4. 2-Deoxyglucose (MW approximately 180), inulin (MW approximately 5000) and spermidine (MW approximately 148) efflux could occur through the gamma-activated conductance pathway, indicating a large pore diameter. In contrast, dextran-70 (MW approximately 70 000) did not pass through the gamma-activated channel, indicating an upper limit to the pore size of approximately 50 A (5 nm). 5. Similar conductances that are permeable to large molecules were activated by extreme hyperpolarization (> -150 mV) of uninjected oocytes. 6. We conclude that the Na(+),K(+)-ATPase gamma subunits activate Ca(2+)- and voltage-gated, non-selective, large diameter pores that are intrinsically present within the oocyte membrane.

Expression, activity and distribution of Na,K-ATPase subunits during in vitro neuronal induction.

The expression pattern of the alpha and beta isoforms and the gamma subunit of the Na,K-ATPase was investigated during in vitro induction of pluripotent murine embryonic stem (ES) cells into neuronal cells. alpha1 protein was expressed in undifferentiated ES (UES) cells and throughout all stages studied. In contrast, alpha3 protein was prominent only when neuronal cells have reached full differentiation. In this model, neuron-depleted cultures did not express the alpha3 isoform, indicating its specificity for mature neuronal cells. UES possessed Na,K-ATPase activity consistent with a single isoform (alpha1), whereas in fully mature neuronal cells a ouabain-sensitive isoform (alpha3) accounted for 27+/-4% of the activity, and a ouabain-resistant isoform (alpha1) 66+/-3%. Immunocytochemistry of mature neuronal cells for alpha1 and alpha3 proteins showed a similar distribution, including cell soma and processes, without evidence of polarization. beta1 protein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cells. While proteins of the beta2 and beta3 isoforms were not detected by immunoblots (except for beta2 in UES), their mRNAs were detected in UES and EB (beta2 and beta3), and in immature and fully differentiated neuronal cells (beta3). Message for the beta2 isoform, however, was not present in neuronal cells. gamma subunit mRNA and protein were undetectable at any stage. These results provide further characterization of neuron-like cells obtained by induction of ES cells in vitro, and establish a model for the expression of isoforms of the Na,K-ATPase during neuronal differentiation. The relation to other aspects of neuronal cell development and relevance to a specialised function for the alpha3 subunit in neurons are discussed.

The alpha4 isoform of the Na,K-ATPase is expressed in the germ cells of the testes.

In addition to the three isoforms of the catalytic subunit of the Na, K-ATPase originally identified (alpha1, alpha2, and alpha3), a fourth alpha polypeptide (alpha4) has recently been found in mammalian cells. This novel alpha-subunit of the Na,K-ATPase is selectively expressed in male gonadal tissues. In the testes, alpha4 is functionally active and comprises approximately half of the Na, K-ATPase activity of the organ. At present, the pattern of expression of the alpha4 polypeptide within the cells of the male gonad is unknown. By in situ hybridization, immunocytochemistry, and the ouabain inhibition profile of Na,K-ATPase activity, we show that the alpha4-subunit is expressed in the germ cells of rat testes. The highest amounts of the isoform are found in spermatozoa, where it constitutes two thirds of the Na,K-ATPase activity of the gametes. The other Na pump present in the cells is the ubiquitously expressed alpha1 polypeptide. The characteristic localization of alpha4 in the gonad is further supported by the drastic reduction of the polypeptide in mice that are infertile as a consequence of arrest in maturation of the germ cells. In addition, GC-1spg cells, a murine cell line derived from testis spermatogonia, also contain the Na, K-ATPase alpha4 polypeptide. However, the level of expression of the isoform in these cells is much lower than in the spermatozoa, a fact that may depend on the limited ability of the GC-1spg cells to differentiate in vitro. The particular expression of the Na,K-ATPase alpha4 isoform we encounter and the specific enzymatic properties of the polypeptide suggests its importance for ionic homeostasis of the germ cells of the testes.

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.

Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function.

The Na-K-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both its alpha- and beta-subunits. At present, as many as four different alpha-polypeptides (alpha1, alpha2, alpha3, and alpha4) and three distinct beta-isoforms (beta1, beta2, and beta3) have been identified in mammalian cells. The stringent constraints on the structure of the Na pump isozymes during evolution and their tissue-specific and developmental pattern of expression suggests that the different Na-K-ATPases have evolved distinct properties to respond to cellular requirements. This review focuses on the functional properties, regulation, and possible physiological relevance of the Na pump isozymes. The coexistence of multiple alpha- and beta-isoforms in most cells has hindered the understanding of the roles of the individual polypeptides. The use of heterologous expression systems has helped circumvent this problem. The kinetic characteristics of different Na-K-ATPase isozymes to the activating cations (Na+ and K+), the substrate ATP, and the inhibitors Ca2+ and ouabain demonstrate that each isoform has distinct properties. In addition, intracellular messengers differentially regulate the activity of the individual Na-K-ATPase isozymes. Thus the regulation of specific Na pump isozymes gives cells the ability to precisely coordinate Na-K-ATPase activity to their physiological requirements.

Differential regulation of Na,K-ATPase isozymes by protein kinases and arachidonic acid.

While several studies have investigated the regulation of the Na, K-ATPase consisting of the alpha1 and beta1 subunits, there is little evidence that intracellular messengers influence the other Na pump isozymes. We studied the effect of different protein kinases and arachidonic acid on the rat Na,K-ATPase isoforms expressed in Sf-9 insect cells. Our results indicate that PKA, PKC, and PKG are able to differentially modify the function of the Na,K-ATPase isozymes. While PKC activation leads to inhibition of all isozymes, PKA activation stimulates the activity of the Na,K-ATPase alpha3 beta1 and decreases that of the alpha1 beta1 and alpha2 beta1 isozymes. In contrast, activation of PKG diminishes the activity of the alpha1 beta1 and alpha3 beta1 isozymes, without altering that of alpha2 beta1. Treatment of cells with arachidonic acid reduced the activities of all the isozymes. The changes in the catalytic capabilities of the Na pump isozymes elicited by PKA and PKC are reflected by changes in the molecular activity of the Na,K-ATPases. One of the mechanisms by which PKA and PKC affect Na pump isozyme activity is through direct phosphorylation of the alpha subunit. In the insect cells, we found a PKA- and PKC-dependent phosphorylation of the alpha1, alpha2 and alpha3 polypeptides. In conclusion, several intracellular messengers are able to modulate the function of the Na,K-ATPase isozymes and some of them in a specific fashion. Because the Na,K-ATPase isozymes have kinetic properties that are unique, this isozyme-specific regulation may be important in adapting Na pump function to the requirements of each cell.

Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.

3beta-hydroxysteroid dehydrogenase/steroid delta5-->4-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3beta-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3beta-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3beta-HSD activity. The 3beta-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3beta-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme.

The gamma subunit of the Na,K-ATPase induces cation channel activity.

The gamma subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The gamma subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the gamma subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human gamma subunit. Confocal microscopy demonstrates that the gamma subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that gamma colocalizes with alpha1 when these proteins are coexpressed. When Sf-9 cells were coinfected with alpha1 and gamma, antibodies to the gamma subunit were able to coimmunoprecipitate the alpha1 subunit, suggesting that gamma is able to associate with alpha1. The gamma subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the gamma subunit is a functional component was supported by experiments showing gamma-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.

Translational regulation of Na,K-ATPase alpha1 and beta1 polypeptide expression in epithelial cells.

To investigate the regulation of the Na,K-ATPase, we have studied the expression of the Na,K-ATPase polypeptides in several mammalian cell lines using the vaccinia virus/T7 RNA polymerase expression system. Infection of several fibroblast-like cell lines with viral recombinants containing the Na,K-ATPase alpha and beta isoforms, the glucose transporters, GLUT 1 and GLUT 4, or the capsid protein of the Sindbis virus all result in the production of the appropriate protein products. However, all epithelial cell lines tested fail to synthesize the Na,K-ATPase viral recombinants, yet they efficiently express the other virally directed polypeptides. While Madin-Darby canine kidney (MDCK) epithelial cells infected with the Na,K-ATPase alpha1 or beta1 recombinant viruses produce both mRNAs, the messages are inefficiently translated. Furthermore, the RNA from infected MDCK cells does not direct the in vitro synthesis of the beta1 polypeptide, whereas the message from infected fibroblast-like BSC 40 cells is efficiently translated both in vivo and in vitro. Moreover, the synthesis of the H,K-ATPase alpha subunit is also limited in MDCK cells, although the H,K-ATPase beta subunit is efficiently expressed. Expression of chimeras constructed between the Na+ pump beta1 isoform and the H,K-ATPase beta subunit indicates that sequences in the 5' coding region of the beta1 message have an inhibitory effect; however, the stringent translational regulation of the beta1 isoform in MDCK cells requires the 5' and 3' regions of the coding sequence. The ability of the polarized cell lines to limit the synthesis of the Na+ pump polypeptides while expressing other vaccinia recombinants at high levels suggests that the polarized cells possess a stringent mechanism for the specific translational regulation of a select set of messages.

Substitutions of glutamate 781 in the Na,K-ATPase alpha subunit demonstrate reduced cation selectivity and an increased affinity for ATP.

The intramembrane Glu781 residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta 1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta 1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781-->Asp and Glu781-->Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781-->Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781-->Ala mutant displays a Na(+)-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781-->Ala mutant in Na+ medium produces a K(+)-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781-->Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.

Expression and characterization of the human erythrocyte anion exchanger in a baculovirus/Sf-9 cell system.

The human erythrocyte anion-exchange protein (HAE1) has been expressed in insect Sf-9 cells using a recombinant baculovirus. We subcloned the full-length cDNA encoding HAE1 into the baculovirus expression vector pVL1392 and cotransfected Sf-9 cells with the recombinant vector and wild-type AcMNPV DNA to obtain recombinant baculovirus. The expressed protein was targeted to the Sf-9 plasma membrane at an apparent density of approximately 0.5 x 10(6) copies/cell as determined by quantitative autoradiography using an HAE1-specific monoclonal antibody. Unlike native HAE1, the expressed protein was not glycosylated. Transport studies with HAE1-recombinant-infected Sf-9 cells showed saturable [Km(Cl-) = 44 mM; Vmax(Cl-) = 48 mEq/liter of cell waters min] and H2DIDS-inhibitable (K(O.5) = 34 microM) 36Cl- uptake that was not present in uninfected cells. We also found that extracellular SO4(2-) reduced 36Cl- influx [K(0.5)((SO4)2-) = 26 mM], presumably through substrate competition as in erythrocytes. Finally, we observed that H2DIDS-inhibitable 36Cl- efflux was reduced by 77% in the nominal absence of a suitable counter-anion in the external solution (HCO3(-)-free, all-glucuronate medium), thereby providing strong evidence for an obligatory exchange mechanism. We conclude that there is high-level expression of + ++HAE1 functional activity in recombinant baculovirus-infected Sf-9 cells and that this system will prove useful for kinetic and structural analyses of the HAE1 protein.

Comparison of the enzymatic properties of the Na,K-ATPase alpha 3 beta 1 and alpha 3 beta 2 isozymes.

The coexpression of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in mammalian tissues gives rise to the complex molecular heterogeneity that characterizes the Na pump. The expression of the different Na,K-ATPase isoforms in insect cells using recombinant baculoviruses represents a useful system for the analysis of Na,K-ATPase isoform function. In the present study, we use this system to direct the expression of the rat Na,K-ATPase alpha 3 beta 1 and alpha 3 beta 2 in sf-9 cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. The association of alpha 3 with either beta 1 or beta 2 results in catalytically competent Na,K-ATPase isozymes. Analysis of the kinetic characteristics of these enzymes demonstrates that the accompanying beta subunit isoform does not drastically affect the properties of the alpha 3 polypeptide. This is evidenced by the similar turnover numbers, apparent affinities for K+ and ATP, and the comparable high sensitivity to ouabain exhibited by both isozymes. The kinetic dependence on Na+, however, is different for both isozymes, with alpha 3 beta 2 displaying a 1.6-fold higher apparent affinity for the cation than alpha 3 beta 1. Comparison with other Na,K-ATPase isozymes shows that the apparent Na+ affinity of alpha 3 beta 2 is similar to that of the alpha 1 beta 1 Na pump widely expressed in every tissue; nevertheless, its reactivity toward K+, ATP, and ouabain are characteristic of the alpha 3 isoform. The most pronounced kinetic differences in Na,K-ATPase function are a result of variations in alpha isoform composition.(ABSTRACT TRUNCATED AT 250 WORDS)

A cytoplasmic region of the Na,K-ATPase alpha-subunit is necessary for specific alpha/alpha association.

While most structural studies of the Na,K-ATPase support a subunit stoichiometry of one alpha-subunit to one beta-subunit, the exact quaternary structure of the Na,K-ATPase and its relevance to enzyme function is the subject of much debate. Formation of a higher order enzyme complex is supported by our previous study demonstrating specific alpha/alpha interactions among the rat Na,K-ATPase isoforms (alpha 1, alpha 2, alpha 3), expressed in virally infected Sf-9 insect cells and among native alpha isoforms in rat brain (1). This detergent-resistant association was not observed in insect cells coexpressing the homologous gastric H,K-ATPase alpha-subunit, nor was it dependent on the coexpression of the beta-subunit. To delineate domains necessary for alpha/alpha assembly, a series of H,K-ATPase-Na, K-ATPase chimerase were constructed by combining the N-terminal, cytoplasmic midregion and C-terminal segments derived from the Na,K-ATPase (N) and the H,K-ATPase (H) alpha-polypeptides (HNN, HNH, NHH, NHN, and HHN). The alpha-subunit chimeras were coexpressed with the Na,K-ATPase alpha 1-subunit in Sf-9 cells using the baculovirus expression system. Specific and detergent-stable association is observed between the Na,K-ATPase alpha-subunit and the HNN and HNH chimeras, but not with the NHH, NHN, or HHN chimeras. Consistent with the Na,K-ATPase cytoplasmic domain as being necessary for alpha/alpha interactions, the full-length alpha-subunit stably associates with an alpha N-terminal deletion mutant (delta Gly2-Leu273), but not with an alpha cytoplasmic deletion mutant (delta Arg350-Pro785). In addition, the naturally occurring C-terminal truncated alpha 1 isoform, alpha 1T (delta Gly554 to C terminus), does not associated with the alpha 1-subunit in Sf-9 cells coexpressing both polypeptides. thus, a cytoplasmic region in the alpha-subunit (Gly554-Pro785) is necessary for specific alpha/alpha association. The same cytoplasmic region contains a strongly hydrophobic segment that, by analogy with oligomerization of water-soluble proteins, may form the interface of the extramembranous alpha/alpha contact site.