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)     

Site-directed mutagenesis of a conserved, extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase

Site-specific mutagenesis was used to study the function of a conserved, extracellular aspartic acid residue from the sheep Na,K-ATPase alpha subunit. This amino acid, Asp-121, is the penultimate residue of the first extracellular domain of the alpha subunit. The border residues of this particular extracellular loop of the alpha subunit have been shown to be determinants of ouabain sensitivity (Price, E. M., and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to determine if Asp-121 is involved in ouabain binding, five different amino acid substitutions at this position were generated. Four of the five mutant alpha subunits, containing either Asn, Ala, Glu, or Ser in place of Asp-121, conferred ouabain resistance to HeLa cells when expressed in those cells. Cloned sublines of cells selected in ouabain were characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. The cells expressing the mutant Na,K-ATPase alpha subunit containing either Asn, Ala, Glu, or Ser in place of Asp-121 contained a component of Na,K-ATPase activity that was nearly 100-times more resistant to ouabain than the endogenous HeLa (human) or sheep enzyme. Apparently, conservative (Glu for Asp), isosteric (Asn for Asp), and nonconservative (Ala or Ser for Asp) substitutions all significantly decreased ouabain sensitivity. These data suggest that Asp-121 of the sheep Na,K-ATPase alpha subunit participates in the binding interaction between the enzyme and ouabain.     

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.     

The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha subunit, in determining the voltage and extracellular K+ (K+(o)) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+(o) dependence similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+(o) concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+(o) affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage-dependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K+(o) binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+(o) binding to the Na,K-ATPase.     

Amino acid substitutions of Na,K-ATPase conferring decreased sensitivity to cardenolides in insects compared to mammals

Mutagenesis analyses and a recent crystal structure of the mammalian Na,K-ATPase have identified amino acids which are responsible for high affinity binding of cardenolides (such as ouabain) which at higher doses block the enzyme in the phosphorylated state. Genetic analysis of the Na,K-ATPase of insects adapted to cardenolides in their food plants revealed that some species possess substitutions which confer strongly increased resistance to ouabain in the mammalian enzyme such as the substitution T797A or combined substitutions at positions 111 and 122. To test for the effect of these mutations against the background of insect Na,K-ATPase, we here expressed the ouabain sensitive Na,K-ATPase α-subunit of Drosophila melanogaster together with the β-subunit Nrv3 in baculovirus-infected Sf9 cells and introduced the substitutions N122H, T797A, Q111T-N122H, Q111V-N122H, all of which have been observed in cardenolide-adapted insects. While all constructs showed similar expression levels, ouabain affinity of mutated Na,K-ATPases was reduced compared to the wild-type fly enzyme. Ouabain sensitivity of the ATPase activity in inhibition assays was significantly decreased by all mutations, yet whereas the IC₅₀ for the single mutations of N122H (61.0 μM) or T797A (63.3 μM) was increased roughly 250-fold relative to the wild-type (0.24 μM), the double mutations of Q111V-N122H (IC₅₀ 550 μM) and Q111T-N122H (IC₅₀ 583 μM) proved to be still more effective yielding a 2.250-fold increased resistance to ouabain. The double mutations identified in cardenolide-adapted insects are more effective in reducing ouabain sensitivity of the enzyme than those found naturally in the rat Na,K-ATPase (Q111R-N122D) or in mutagenesis screens of the mammalian enzyme. Obviously, the intense selection pressure on cardenolide exposed insects has resulted in very efficient substitutions that decrease cardenolide sensitivity extremely.     

Alanine scanning mutagenesis of oxygen-containing amino acids in the transmembrane region of the Na,K-ATPase.

Oxygen-containing amino acids in the transmembrane region of the Na, K-ATPase alpha subunit were studied to identify residues involved in Na+ and/or K+ coordination by the enzyme. Conserved residues located in the polar face of transmembrane helices were selected using helical wheel and topological models of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha1 isoform and expressed in HeLa cells. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of heterologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, Ser140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu818, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those carrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were introduced into the wild-type alpha1 sheep isoform and expressed in mouse NIH3T3 cells where [3H]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. Expression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at the plasma membrane was significantly lower than that of the wild-type isoform. Thus, these substitutions appear to impair the maturation process or targeting of the enzyme to the plasma membrane, but not cation-enzyme interactions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-containing residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme function. Consequently, other chemical groups, aromatic rings or backbone carbonyls, should be considered in models of cation-binding sites.     

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.     

Progesterone binding to the alpha1-subunit of the Na/K-ATPase on the cell surface: insights from computational modeling

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [(3)H]ouabain and [(3)H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112kDa alpha1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding alpha1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar beta and hydrophobic alpha surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg(118), Asp(129) and possibly Glu(122-124) in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys(111) near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding.     

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.     

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.     

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.     

Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits

We have examined the relationship between Na,K-ATPase and FGF-2 secretion in transfected primate cells. FGF-2 lacks a classic hydrophobic export signal, and the mechanisms mediating its secretion are unknown. To monitor secretion, a FLAG epitope tag was inserted into the carboxyl terminus of the 18 kDa form of human FGF-2, and the construct was transfected into either human HEK 293 or monkey CV-1 cells. Exported FGF-2 was detected in the culture medium using the FLAG-specific monoclonal antibody M2. FGF-2 secretion from HEK 293 or CV-1 cells was linear over time and sensitive to inhibition by the cardiac glycoside ouabain, a specific inhibitor of the Na,K-ATPase. In contrast, the secretion of FGF-8 (an FGF family member that contains a hydrophobic secretory signal) was not inhibited by treatment of HEK 293 or CV-1 cells with ouabain. FGF-2 secretion was also assayed in CV-1 cells expressing the naturally ouabain-resistant rodent Na,K-ATPase alpha1 subunit. In cells expressing the rodent alpha1 subunit, FGF-2 secretion was unaffected by high levels of ouabain, indicating that the rodent alpha1 subunit was capable of rescuing ouabain-inhibitable FGF-2 export. Expression of ouabain-resistant mutants of the rodent alpha2 and alpha3 subunits, or the naturally ouabain-resistant rodent alpha4 subunit, also supported FGF-2 secretion in ouabain-treated cells. Taken together, our studies are consistent with the idea that the Na,K-ATPase plays a prominent role in regulating FGF-2 secretion, although none of the alpha subunit isoforms exhibited specificity with regard to FGF-2 export.     

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.     

Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids

P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven amino acids in Na,K-ATPase that conferred high affinity ouabain binding to gastric H,K-ATPase (Qiu, L. Y., Krieger, E., Schaftenaar, G., Swarts, H. G. P., Willems, P. H. G. M., De Pont, J. J. H. H. M., and Koenderink, J. B. (2005) J. Biol. Chem. 280, 32349-32355). Because important, but identical, amino acids were not recognized in that study, here we used the non-gastric H,K-ATPase, which is rather ouabain-insensitive, as template. The catalytic subunit of this enzyme, in which several amino acids from Na,K-ATPase were incorporated, was expressed with the Na,K-ATPase beta1 subunit in Xenopus laevis oocytes. A chimera containing 14 amino acids, located in M4, M5, and M6, which are unique to Na,K-ATPase, displayed high affinity ouabain binding. Four of these residues, all located in M5, appeared dispensable for high affinity binding. Individual mutation of the remaining 10 residues to their non-gastric H,K-ATPase counterparts yielded five amino acids (Glu312,Gly319, Pro778, Leu795, and Cys802) whose mutation resulted in a loss of ouabain binding. In a final gain-of-function experiment, we introduced these five amino acids in different combinations in non-gastric H,K-ATPase and demonstrated that all five were essential for high affinity ouabain binding. The non-gastric H,K-ATPase with these five mutations had a similar apparent affinity for ouabain as the wild type Na,K-ATPase and showed a 2000 times increased affinity for ouabain in the NH4+-stimulated ATPase activity in membranes of transfected Sf9 cells.     

Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance.

We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase.

A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.     

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.     

Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.     

Ouabain-stimulated trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate whether this mechanism is species-specific, ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular (22)Na(+) transport were compared in three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat α1). Ouabain-induced inhibition of transcellular (22)Na(+) transport is due to an ouabain-induced redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.