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.     

Na(+)/K(+) pump expression in the L8 rat myogenic cell line: effects of heterologous alpha subunit transfection

We have characterized the physiological and biochemical properties of the Na(+)/K(+) pump and its molecular expression in L8 rat muscle cells. Pump properties were measured by [(3)H]ouabain binding and (86)Rb uptake. Scatchard plot analysis of specific ouabain binding indicated the presence of a single family of binding sites with a B(max) of approximately 135 fmol/ mg P and a K(D) of 3.3 x 10(-8). (86)Rb uptake due to specific pump activity was found to be 20% of the total in L8 cells. The results indicated lower affinity of L8 cells for ouabain and lower activity of the pump than that reported for chick or rat skeletal muscle in primary culture. Both the alpha(1) and beta(1) protein and mRNA isoforms were expressed in myoblasts and in myotubes, while the alpha(2), alpha(3), and beta(2) isoforms were not detectable. We attempted to overcome low physiological expression of the Na(+)/K(+) pump by employing a vector expressing an avian high affinity alpha subunit. This allowed identification of the transfected subunit separate from that endogenously expressed in L8 cells. Successful transfection into L8 myoblasts and myotubes was recognized by anti-avian alpha subunit monoclonal antibodies. Fusion index, Na(+)/K(+) pump activity, and the level of the transmembrane resting potential were all significantly greater in transfected L8 (tL8) cells than in non-tL8. The total amount of alpha subunit (avian and rat) in tL8 cells was greater than that (only rat) in non-tL8 cells. This relatively high abundance of the Na(+)/K(+) pump in transfected cells may indicate that avian and rat alpha subunits hybridize to form functional pump complexes.     

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

Access of extracellular cations to their binding sites in Na,K-ATPase: role of the second extracellular loop of the alpha subunit

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na(+) and K(+) gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na(+) ions and releases them on the extracellular side of the membrane, and then binds two extracellular K(+) ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the alpha subunit of Na,K-ATPase are known to be involved in Na(+) and K(+) binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat alpha1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K(+), and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K(+) to its binding site.     

Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

Brefeldin A influences the cell surface abundance and intracellular pools of low and high ouabain affinity Na+, K(+)-ATPase alpha subunit isoforms in articular chondrocytes

The catalytic alpha isoforms of the Na+, K(+)-ATPase and stimuli controlling the plasma membrane abundance and intracellular distribution of the enzyme were studied in isolated bovine articular chondrocytes which have previously been shown to express low and high ouabain affinity alpha isoforms (alpha 1 and alpha 3 respectively; alpha 1 > alpha 3). The Na+, K(+)-ATPase density of isolated chondrocyte preparations was quantified by specific 3H-ouabain binding. Long-term elevation of extracellular medium [Na+] resulted in a significant (31%; p < 0.05) upregulation of Na+, K(+)-ATPase density and treatment with various pharmacological inhibitors (Brefeldin A, monensin and cycloheximide) significantly (p < 0.001) blocked the upregulation. The subcellular distribution of the Na+, K(+)-ATPase alpha isoforms was examined by immunofluorescence confocal laser scanning microscopy which revealed predominantly plasma membrane immunostaining of alpha subunits in control chondrocytes. In Brefeldin A treated chondrocytes exposed to high [Na+], Na+, K(+)-ATPase alpha isoforms accumulated in juxta-nuclear pools and plasma membrane Na+, K(+)-ATPase density monitored by 3H-ouabain binding was significantly down-regulated due to Brefeldin A mediated disruption of vesicular transport. There was a marked increase in intracellular alpha 1 and alpha 3 staining suggesting that these isoforms are preferentially upregulated following long-term exposure to high extracellular [Na+]. The results demonstrate that Na+, K(+)-ATPase density in chondrocytes is elevated in response to increased extracellular [Na+] through de novo protein synthesis of new pumps containing alpha 1 and alpha 3 isoforms, delivery via the endoplasmic reticulum-Golgi complex constitutive secretory pathway and insertion into the plasma membrane.     

Deletion of the Na/K-ATPase alpha1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo

Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation and are associated with increases in Na(+)-pump subunit mRNA and protein expression. We have hypothesized that the alpha1-isozyme of the Na/K-ATPase is required to mediate blastocyst formation. We have tested this hypothesis by characterizing preimplantation development in mice with a targeted disruption of the Na/K-ATPase alpha1-subunit (Atp1a1) using embryos acquired from matings between Atp1a1 heterozygous mice. Mouse embryos homozygous for a null mutation in the Na/K-ATPase alpha1-subunit gene are able to undergo compaction and cavitation. These findings demonstrate that trophectoderm transport mechanisms are maintained in the absence of the predominant isozyme of the Na(+)-pump that has previously been localized to the basolateral membranes of mammalian trophectoderm cells. The presence of multiple isoforms of Na/K-ATPase alpha- and beta-subunits at the time of cavitation suggests that there may be a degree of genetic redundancy amongst isoforms of the catalytic alpha-subunit that allows blastocyst formation to progress in the absence of the alpha1-subunit.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

Transport and pharmacological properties of nine different human Na, K-ATPase isozymes

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 alpha and beta isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K(+)-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 from digitalis inhibition at physiological K(+) levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.     

Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase.

Previous studies showed that the alpha 1, alpha 2, and alpha 3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na(+), and K(+). For the rat isoforms transfected into HeLa cells, K'(ATP) for ATP binding at its low affinity site is lower for alpha 2 and alpha 3 compared with alpha 1; relative to alpha 1 and alpha 2, alpha 3 has a higher K'(Na) and lower K'(K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925--16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668--16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that alpha 2 differs from alpha1 primarily by a shift in the E(1) E(2) equilibrium in favor of E(1) form(s) as evidenced by (i) a approximately 20-fold increase in IC(50) for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'(ATP) compared with alpha 1, the E(1) E(2) poise of alpha 3 is not shifted toward E(1). Distinct intrinsic interactions with Na(+) ions are underscored by the marked selectivity for Na(+) over Li(+) of alpha 3 compared with either alpha1 or alpha 2 and higher K'(Na) for cytoplasmic Na(+), which persists over a 100-fold range in proton concentration, independent of the presence of K(+). The kinetic analysis also suggests alpha 3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na(+) and K(+).     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C

Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).     

The A2 isoform of rat Na+,K(+)-adenosine triphosphatase is active and exhibits high ouabain affinity when expressed in transfected fibroblasts.

The alpha isoforms of the Na+,K(+)-ATPase (Na+ pump) are expressed with developmental and tissue heterogeneity in rodents and possess different sensitivity to inhibition by ouabain. We directly characterized the ouabain sensitivity of the rat A2 (alpha 2) isoform by transfecting NIH 3T3 cells with rat A2. The treated cells exhibit high affinity (40 nM) ouabain binding with a density of 2 pmol/mg protein. 86Rb+ flux studies confirm that A2 is functional in this system and that A2 is inhibited by submicromolar concentrations of ouabain. These findings are consistent with measurements of ouabain affinity in tissues which express the A2 isoform.     

Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.     

Residues within transmembrane domains 4 and 6 of the Na,K-ATPase alpha subunit are important for Na+ selectivity

The Na,K- and H,K-ATPases are plasma membrane enzymes responsible for the active exchange of extracellular K(+) for cytoplasmic Na(+) or H(+), respectively. At present, the structural determinants for the specific function of these ATPases remain poorly understood. To investigate the cation selectivity of these ATPases, we constructed a series of Na,K-ATPase mutants in which residues in the membrane spanning segments of the alpha subunit were changed to the corresponding residues common to gastric H,K-ATPases. Thus, mutants were created with substitutions in transmembrane domains TM1, TM4, TM5, TM6, TM7, and TM8 independently or together (designated TMAll). The function of each mutant was assessed after coexpression with the beta subunit in Sf-9 cells using baculoviruses. The enzymatic properties of TM1, TM7, and TM8 mutants were similar to the wild-type Na,K-ATPase, and while TM5 showed modest changes in apparent affinity for Na(+), TM4, TM6, and TMAll displayed an abnormal activity. This resulted in a Na(+)-independent hydrolysis of ATP, a 2-fold higher K(0.5) for Na(+) activation, and the ability to function at low pH. These results suggest a loss of discrimination for Na(+) over H(+) for the enzymes. In addition, TM4, TM6, and TMAll mutants exhibited a 1.5-fold lower affinity for K(+) and a 4-5-fold decreased sensitivity to vanadate. Altogether, these results provide evidence that residues in transmembrane domains 4 and 6 of the alpha subunit of the Na,K-ATPase play an important role in determining the specific cation selectivity of the enzyme and also its E1/E2 conformational equilibrium.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells

Cyclic AMP (cAMP) stimulates the transport of Na(+) and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCD(c14) collecting duct cells. db-cAMP (10(-3) M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of (86)Rb(+) uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20 degrees C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.