Structure/function analysis of Na+-K+-ATPase central isoform-specific region: involvement in PKC regulation

Sandrine V. Pierre, Marie-Josée Duran, Deborah L. Carr, and Thomas A. Pressley

Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Specific functions served by the various Na+-K+-ATPase alpha -isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH2 terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH2 terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat alpha 1- and alpha 2-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K+ deocclusion pattern and their response to PKC activation. Comparisons with rat full-length alpha 1- and alpha 2-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K+ deocclusion.

alpha -subunit; rat; alpha 1- and alpha 2-isoforms; chimeras; potassium deocclusion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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NA+-k+-atpase is critical for maintaining the ionic gradients across the plasma membranes of animal cells. This enzyme complex extrudes Na+ from the cell and accumulates K+, using metabolic energy derived from ATP hydrolysis. The enzyme complex consists of two dissimilar subunits, alpha  and beta , that exist in multiple forms. A fundamental question surrounding the pump is the physiological relevance of this subunit diversity. The alpha -polypeptide is the catalytic subunit and contains the binding sites for ions and substrates. Four distinct isoforms (alpha 1, alpha 2, alpha 3, and alpha 4) of the Na+-K+-ATPase alpha -subunit have been described so far, with differences in enzyme kinetics and response to second messengers. The primary structures of these isoforms are nearly identical, with the exception of the NH2 terminus and a 10-residue region near the center of the molecule (19, 24) [isoform-specific region (ISR); K489-L499; Fig. 1]. It seems likely that the specific functions served by the various Na+-K+-ATPase alpha -isoforms must originate within these regions of structural divergence.


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Fig. 1.   Structure of the alpha -subunit. Open rectangle, NH2-terminal domain; small open box, isoform-specific region (ISR) and central ISRs, with aligned amino acid sequences of alpha 1 and alpha 2 ISRs appearing as shaded rectangles and the sites of silent mutations in bold.

The NH2-terminal region has been extensively studied. Heterologous expression of NH2-terminal deletions and chimeric constructs has shown that the mutant alpha -subunits display changes in kinetics and regulation of ion transport properties. Although the NH2 terminus of alpha  is not required for Na+-K+-ATPase activity (21), a truncated enzyme lacking the first 32 amino acids has distinctive kinetics at micromolar ATP concentrations, conditions in which K+ deocclusion becomes rate limiting in the overall catalytic cycle. Thus Na+-ATPase activity of the truncated enzyme is stimulated by low concentrations of K+, whereas activity of wild-type alpha 1 is inhibited. Interestingly, the alpha 2-isoform resembles the truncated enzyme in this respect. However, unexpected characteristics of chimeras resulting from exchanges between alpha 1 and alpha 2 NH2-terminal domains suggested that this distinctive kinetic behavior of Na+-K+-ATPase alpha -isoforms is not entirely due to the NH2-terminal region but rather to its interaction with other ISRs of the alpha -protein (8).

Another isoform-specific property that clearly involves the NH2-terminal region is the regulation of Na+-K+-ATPase transport activity by PKC. Indeed, we have shown that stimulation of endogenous PKC with phorbol esters increases pump-mediated Rb+ transport in cultured opossum kidney (OK) cells expressing the exogenous rat Na+-K+-ATPase alpha 1-isoform. This increase was abolished in cells expressing a mutant missing the first 26 amino acids of the rodent alpha -subunit, consistent with a role for the NH2-terminal region in PKC regulation. Ser16 and Ser23, which are believed to be in vivo targets of PKC phosphorylation, are found within the NH2-terminal domain of the rat alpha 1-isoform. We have shown that PKC stimulation of transport is completely abolished in S16A and S23A mutants (9). However, comparisons with other species and experimental systems raise some doubts about the role of these residues. For example, Ser16 is well conserved among mammalian alpha 1-isoforms, but it is only weakly phosphorylated. Ser23, on the other hand, is missing in many species. Neither residue is contained within a PKC consensus sequence, and neither residue is conserved in alpha 2 or alpha 3. Nevertheless, there are other serines and threonines in the NH2 termini of these isoforms that may serve as phosphorylation sites. Given the variability of the response and the differences among species and isoforms, it is therefore tempting to speculate that additional regions of the alpha -subunit are contributing to the effect of PKC.

We hypothesized that the central ISR may be one of these additional regions. To test this hypothesis, chimeric molecules were constructed in which the central ISRs of the rat alpha 1- and alpha 2-isoforms were exchanged. After stable transfection into OK cells, the chimeras were characterized for their enzymatic properties. Comparisons of the chimeras with rat full-length alpha 1- and alpha 2-isoforms expressed under the same conditions suggest an involvement of the central ISR in the PKC response but not in K+ deocclusion.


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ABSTRACT
INTRODUCTION
METHODS
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Preparation of full-length alpha 1 and ouabain-resistant alpha 2 sequences. Wild-type alpha 1 and alpha 2 cDNAs, a gift from Dr. Jerry B. Lingrel and colleagues (22), were subcloned by our laboratory into pGEM-3Z (Promega, Madison, WI) as described (21). A ouabain-resistant form of the rat alpha 2-isoform (designated alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>) was constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D) (12). Use of ouabain-resistant alpha 1 and alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP> allowed us to employ the ouabain-selection strategy described below.

Preparation of chimeras. The alpha 1 and alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP> cDNAs were used to prepare chimeric molecules in which the central isoform-specific domain of alpha 1 and alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP> were exchanged. Two silent mutations were introduced in rat alpha 1 and alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP> cDNAs by conventional site-directed mutagenesis, creating unique sites for digestion by the restriction enzymes ClaI and AgeI without altering the encoded amino acids. The ClaI site was placed within the codons for Leu485, Ser486, and Ile487 (LSI), as numbered from the NH2-terminal glycine of the mature polypeptide. The AgeI site was introduced within the codons for Asp511, Arg512, and Cys513 (DRC). This was accomplished with the aid of mutagenic oligonucleotides (Table 1) and their complements, using limited amplification with Pfu polymerase, followed by restriction of the original template with DpnI. To perform the exchange of the ISRs, we proceeded in two steps. In the first, the COOH ends of each isoform were swapped, using the introduced AgeI sites (DRC). The resulting chimeras were then used as starting material for swaps of the NH2 half of the molecule, using the introduced ClaI site. The structures of the resulting mutants were then confirmed by restriction analysis and direct sequencing of the altered region.

                              
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Table 1.   Characteristics of silent mutation oligonucleotides used for the introduction of ClaI and AgeI sites into rat alpha 1 and alpha 2 sequences

Expression vectors, gene transfer, and selection. cDNAs encoding alpha 1, alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>, or the chimeras were subcloned into the HindIII and XbaI sites of the eukaryotic expression vector pRc/CMV (Invitrogen, San Diego, CA), with the aid of a SacI-XbaI adaptor. This was prepared by annealing the oligonucleotides CGCGGCCGCT and CTAGAGCGGCCGCGAGCT. The resulting expression vector contained the sequence encoding the alpha  construct downstream of the enhancer-promoter regions of the immediate early gene of human cytomegalovirus, followed by polyadenylation signals from the bovine growth hormone gene.

Heterologous expression of the alpha  constructs was achieved by transfection of OK cells (no. CRL-1840, American Type Culture Collection) and subsequent selection of recipients with ouabain. OK cells were routinely maintained at 37°C and 10% CO2 in DMEM with 10% calf serum. The expression vectors containing rat isoforms or chimeras were introduced into subconfluent cells using the cationic liposome preparation Lipofectin (GIBCO BRL, Grand Island, NY) as described (11). Transfected OK cells expressing the introduced ouabain-resistant Na+-K+-ATPase constructs were selected for their ability to grow in 3 µM ouabain, a concentration sufficient to kill control (i.e., untransfected) OK cells.

Membrane preparations. Crude plasma membranes were isolated from control and transfected OK cells. Confluent monolayers from twelve 100-mm dishes were washed twice with phosphate-buffered saline, and cells were then harvested by scraping with a rubber policeman. Crude membranes were isolated from the resulting cell suspension after hypotonic lysis and differential centrifugation, followed by treatment with sodium iodide as described (21).

Gel electrophoresis and immunoblotting. Expression of introduced alpha -subunits in transfected OK cells was confirmed by electrophoresis and subsequent immunoblotting of proteins from the isolated membranes. Electrophoresis of samples through SDS-polyacrylamide gels (7.5%), electroblotting, and probing of the blots with appropriate antibodies were performed as described (19). The presence of transferred proteins on the blots and equality of loading among the lanes were confirmed by staining with Ponceau S. For detection and characterization of rat isoform and chimera expression in OK cells, two site-directed rabbit polyclonal antibodies were used. Anti-HERED recognizes the rat alpha 2 ISR (residues 494-506). Anti-NASE recognizes the rat alpha 1 ISR (residues 494-505). However, anti-NASE also recognizes the opossum alpha 1 sequence (19); therefore, it could not be used for confirmation in constructs in which its target sequence was expected.

RNA isolation, reverse transcription, and DNA amplification. In instances where the cross-reactivity of anti-NASE precluded specific detection of the alpha 1 IRS, we confirmed the structure of the expressed mRNA. Isolation of total RNA from confluent monolayers of cells in 3.5-cm culture dishes was accomplish using a commercial preparation, Ultraspec (Biotecx Laboratories, Houston, TX), a modification of the guanidium thiocyanate-phenol-chloroform protocol of Chomczynski and Sacchi (6). The recovered RNA was dissolved in diethylpyrocarbonate-treated water, and only preparations with an A260/A280 amplification ratio >1.5 were analyzed further.

Synthesis of cDNA was achieved by reverse transcription. The RNA mixture was heated for 10 min at 65°C. Reverse transcription was then performed in a final volume of 20 µl containing 50 mM Tris · HCl (pH 8.4), 75 mM KCl, 3 mM MgCl2, 500 µM of each deoxynucleotides, 10 mM dithiothreitol, 100 pmol of a mixture of random hexamer primers, 60 units of RNAse inhibitor (Promega), and 15 units of Moloney murine leukemia virus RT. (US Biochemical). After 1 h at 37°C, the reaction was terminated by heating for 5 min at 95°C.

Various sets of oligonucleotide primers specific for the alpha 1 or alpha 2 sequence were used to confirm the expression of rat isoform and chimera RNAs in OK cells by PCR (Table 2). The design of the primers was based on the location of their target sequence (Fig. 1). Some were derived from regions on the 5'-side of the ClaI site (i.e., regions encoding amino acids to the NH2-terminal side of the ISR), such as "alpha 1 direct" or "alpha 2 direct." A second set of primers targeted regions between the ClaI and AgeI sites (i.e., the ISR itself), such as "alpha 2 ISR reverse" or "alpha 2 ISR direct b." Finally, a third set of primers had their target sequences on the 3'-side of the AgeI site (i.e., the COOH-terminal side of the ISR), such as "alpha 1-reverse." For specific amplification, 1 µl of the reverse transcribed sample was added to a PCR incubation mixture containing 10 mM Tris · HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM of each deoxynucleotide, 0.5 µM of each primer, and 2.5 units of Taq polymerase DNA (Roche Diagnostics, Mannheim, Germany) in a final volume of 100 µl. After 2 min at 94°C, samples were submitted to 30 cycles of PCR under the following conditions: 1 min at 94°C, 2 min at an annealing temperature dependent on the characteristics of the primers (Table 2), and 3 min at 72°C. After the final cycle, an additional elongation period of 4 min was performed at 72°C. The amplified products were then analyzed by electrophoresis through agarose gels (1).

                              
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Table 2.   Characteristics of primer pairs used for PCR amplifications

Enzymatic activity. The activity of Na+-K+-ATPase in isolated membranes from transfected cells was determined from the hydrolysis of radiolabeled ATP as described (17). Briefly, membranes (1 mg/ml) were treated with 0.4-0.7 mg/ml deoxycholate for 30 min at room temperature to activate latent Na+-K+-ATPase activity. Detergent-treated membranes were then diluted 1:20 and incubated at 37°C for 30 min in a total volume of 0.2 ml containing 20 mM NaCl, 25 mM histidine, 3 mM MgCl2, 0.2 mM EGTA, and 1 µM ATP (pH 7.5 at room temperature). To minimize the contribution of endogenous Na+-K+-ATPase, 3 µM ouabain was also included. The Na+-ATPase activity of the introduced enzyme was estimated by the decrement in hydrolysis in the presence of 3 mM ouabain. Various concentrations of KCl, ranging from 0.05 to 5 mM, were also added, and the resulting activity was standardized to the Na+-ATPase estimate measured in the absence of K+.

Active transport. Na+-/K+ pump-mediated transport was assessed by measuring the ouabain-sensitive uptake of the K+ congener 86Rb+ as described (20). Briefly, the difference in accumulation of the radioisotope over 5 min at 37°C was determined in cells exposed to standard culture medium plus 3 µM or 1 mM ouabain. As with the enzymatic analyses, a dose of 3 µM was used to inhibit the ouabain-sensitive endogenous opossum enzyme, whereas the 1 mM ouabain dose was necessary to inhibit the ouabain-resistant enzyme introduced. The effect of PKC activation was determined by treating confluent monolayers for 5 min with the DMSO vehicle or phorbol ester agonist PMA before the addition of radiolabeled Rb+. This was achieved by adding 5 µl of a 10 mM solution of PMA (diluted in DMSO) or 5 µl of DMSO alone (control condition) directly to the 5 ml of culture medium.


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INTRODUCTION
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Expression of full-length alpha 1- and alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>-isoforms and chimeras. Transfection into OK cells of alpha 1, alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>, alpha 1alpha 2alpha 1, and alpha 2alpha 1alpha 2 produced ouabain-resistant colonies, indicating that all four exogenous sequences were capable of producing functional Na+-K+-ATPase. To confirm the structure of the introduced subunit, membranes from transfected cells were evaluated by immunoblotting (Fig. 2). As a probe, we used anti-HERED, a polyclonal antibody directed against the alpha 2 ISR (19). A band corresponding to 116 kDa was detected in alpha 1alpha 2alpha 1, as well as rat brain membranes or membranes containing the full-length alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP> that were included as positive controls (Fig. 2A). Anti-HERED did not bind to membranes from rat kidney (known to predominantly express the Na+-K+-ATPase alpha 1-isoform), alpha 1-transfected OK membranes, or nontransfected OK membranes that were included as negative controls. No signal was detected in membranes from alpha 2alpha 1alpha 2-transfected OK cells (Fig. 2B), consistent with an absence of the alpha 2 ISR in the chimera. Taken together, these data suggest that the structure of the alpha 1alpha 2alpha 1 chimera was as intended, with substitution of the alpha 2 ISR into the alpha 1-isoform. Conversely, the alpha 2 ISR was not detected in membranes expressing alpha 2alpha 1alpha 2. The same immunoblots were probed with anti-NASE, a polyclonal antibody directed against the alpha 1 ISR. Unfortunately, given the cross-reactivity of this antibody with opossum-derived alpha 1, a band was detected in nontransfected OK cells, precluding any conclusion about exogenous expression in transfected cells (data not shown). Indeed, a previous study has shown this same cross-reactivity with opossum-derived samples (19).


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Fig. 2.   Immunodetection of HERED epitope. Immunoblot of crude membranes from representative ouabain-resistant colonies was probed with anti-HERED, a polyclonal antibody specific for the ISR of rat alpha 2-isoform. Membranes from untransfected or alpha 1-transfected opossum kidney (OK) cells and from rat kidney were included as negative controls. Membranes from ouabain-resistant rat alpha 2-isoform (alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>)-transfected OK cells and from rat brain were included as positive controls. Antibody-epitope interactions were visualized with peroxidase-conjugated goat anti-rabbit IgG and chemiluminescence.

As an alternative to immunological expression, specific oligonucleotide primers were used to confirm expression of the mRNAs encoding the introduced isoforms and chimeras. Our strategy was to design primers that recognize alpha 1 or alpha 2 nucleic acid sequences encoding regions located before, inside, or after the ISR (Fig. 2). Using reverse-transcribed DNA from OK cells and such primers, we were able to detect the expression of exogenous alpha 1- and alpha 2-isoform mRNA after amplification by PCR (Fig. 3). Indeed, bands of 747 and 1,425 bp were readily detected in alpha 1- and alpha 2-transfected cells, respectively. No signal was detected in nontransfected OK cells with any primer set. Using a direct primer specific for a region of alpha 1 before the ISR and a reverse primer specific for the alpha 2 ISR, we were able to amplify a PCR fragment of the expected mobility (556 bp) using an alpha 1alpha 2alpha 1-containing plasmid as a template (positive control) or reverse-transcribed RNA from alpha 1alpha 2alpha 1-transfected cells (Fig. 3B). As one would expect, no band of the appropriate mobility was produced when alpha 1- or alpha 2-containing plasmids or reverse- transcribed RNA from alpha 1- or alpha 2-transfected cells were used as templates. Finally, Fig. 3C shows the PCR fragment obtained using a direct primer specific for alpha 2 before the ISR and a reverse primer specific for the alpha 1 ISR when reverse-transcribed RNA from alpha 2alpha 1alpha 2-transfected cells was amplified. As a control, a band of identical mobility (743 bp) was produced from alpha 2alpha 1alpha 2-containing plasmid. PCR performed under the same conditions did not reveal any signal from reverse-transcribed RNA of nontransfected cells, alpha 1- or alpha 2-containing plasmids, or reverse-transcribed RNA from alpha 1- or alpha 2-transfected cells. Taken together, the immunological and PCR analyses strongly suggest that the structures of the various introduced isoforms were correct.


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Fig. 3.   RT-PCR amplification of rat Na+-K+-ATPase alpha 1alpha 2alpha 1- and alpha 2alpha 1alpha 2-specific sequences. PCR products were obtained with rat Na+-K+-ATPase alpha 1-, alpha 2-, alpha 1alpha 2alpha 1-, or alpha 2alpha 1alpha 2-specific primers (listed in Table 2; see METHODS for details) and plasmid or cellular cDNA as templates. A: alpha 1- and alpha 2-specific amplifications. Untransfected OK cell cDNA was used as a negative control. B and C: alpha 1alpha 2alpha 1- and alpha 2alpha 1alpha 2-specific amplifications, respectively. Untransfected, alpha 1-transfected, and alpha 2-transfected OK cell cDNAs were used as negative controls. PCR amplification products were visualized by electrophoresis on ethidium bromide (0.5 µg/ml)-stained 2% agarose gels. Negative control, cDNA was omitted in the PCR mixture.

Effect of ISR exchange on enzymatic properties. In micromolar concentrations of ATP sufficient to saturate the high-affinity phosphorylation site, the response of Na+-dependant ATP hydrolysis to varying concentrations of K+ is a convenient and sensitive indication of isoform-specific differences in the E2(K)right-arrowE1 pathway of the Na+-K+-ATPase reaction (8). This part of the reaction becomes rate limiting at low ATP concentration, and K+ inhibits Na+-ATPase activity of the alpha 1 enzyme. In contrast, alpha 2 is stimulated. Previous work using chimeric enzymes obtained by exchanges between alpha 1 and alpha 2 NH2 termini has suggested that the distinctive kinetic behavior of alpha 1 and alpha 2 was not due to the NH2-terminal domain alone but rather to its interaction with other ISRs of the protein (8). Accordingly, a series of experiments was designed to determine whether ISR sequence diversity was involved in the kinetic difference between alpha 1 and alpha 2. In Fig. 4, results are presented relative to ouabain-sensitive Na+-ATPase activity, which was 34.8 ± 6.1, 25.2 ± 2.9, 24.0 ± 7.4, and 29.4 ± 1.4 nmol Pi · mg protein-1 · h-1 for alpha 1, alpha 1alpha 2alpha 1, alpha *2, and alpha 2alpha 1alpha 2, respectively.


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Fig. 4.   Effect of alpha 1/alpha 2 ISR exchange on K+ sensitivity of Na+-ATPase. ATP hydrolysis was assayed in membranes from transfected OK cells. Hydrolysis of [gamma -32P]ATP was measured after 30 min in the presence of 1 µM ATP, 20 mM NaCl, and various concentrations of KCl. Results are presented relative to ouabain-sensitive Na+-ATPase activity for alpha 1 (), alpha 1alpha 2alpha 1 (), alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP> (triangle ), and alpha 2alpha 1alpha 2 (black-triangle), respectively. To inhibit endogenous Na+-K+-ATPase, 3 µM ouabain was present in all reactions. Values are means ± SE of data obtained from 3 different membrane preparations (assays performed in triplicate for each K+ concentration). Inset: shape of the various curves between 0 and 0.4 mM K+ shown in an expanded scale.

At 1 µM ATP, the K+ activation/inhibition profile of alpha 2alpha 1alpha 2 was indistinguishable from that of alpha 1. Na+-ATPase activity of both alpha 1 and alpha 1alpha 2alpha 1 was inhibited by 0.025-5 mM K+. In experiments with the alpha 2alpha 1alpha 2 chimera, low concentrations of K+ stimulated Na-ATPase activity and higher concentrations failed to efficiently inhibit Na+-ATPase activity, resulting in a K+ activation/inhibition profile similar to that of alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>. Therefore, switching the ISR between alpha 1 and alpha 2 did not alter differences between their K+ activation/inhibition profiles.

Effect of ISR exchange on PKC-dependent activation of cellular Na+-K+-ATPase-mediated Rb+ transport. In basal conditions, Na+-K+-ATPase-mediated Rb+ transport was in the same range for nontransfected and transfected cells, regardless of the construct introduced. Indeed, before PMA-stimulation, Na+-K+- ATPase-mediated Rb+ transport was 8.14 ± 0.6, (n = 7) in nontransfected OK cells, 6.44 ± 0.38 (n = 7) for alpha 1, 6.99 ± 0.76 (n = 7) for alpha 1alpha 2alpha 1, 6.41 ± 1.56 (n = 11) for alpha 2, and 7.99 ± 0.52 nmol · mg protein-1 · h-1 (n = 6) for alpha 2alpha 1alpha 2.This strongly suggests that Na+-K+-ATPase was not overexpressed in the transfected cells and that the overall activity was not compromised by the mutations.

We have demonstrated previously that PMA treatment of OK cells expressing the rodent alpha 1-subunit results in stimulation of Na+-K+-ATPase activity (16). The present study confirms this result for the alpha 1-isoform and extends it to alpha 2 (Fig. 5). A significant increase of 15% (P < 0.01, paired t-test) in ouabain-sensitive 86Rb+ uptake occurred after 5 min of PMA treatment in both alpha 1- and alpha 2-transfected cells. After exchange of the original alpha 1 ISR for the alpha 2 sequence (chimera alpha 1alpha 2alpha 1), the PMA-induced activation reached 30% (P < 0.001, paired t-test). This increase was significantly higher than the increase observed for full-length alpha 1 (P < 0.05, unpaired t-test comparing alpha 1 and alpha 1alpha 2alpha 1 PMA-induced activations). On the other hand, exchange of the original alpha 2 ISR for the alpha 1 sequence (chimera alpha 2alpha 1alpha 2) completely abolished the PMA-induced activation in the alpha 2 sequence. These results suggest that the ISR does indeed play a role in PKC-mediated activation of the Na+-K+-ATPase.


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Fig. 5.   Effect of alpha 1/alpha 2 ISR exchange on PMA-dependent activation of cellular Na+-K+-ATPase-mediated Rb+-transport. Na+-K+-ATPase-mediated transport was assayed in attached cells by measuring the ouabain-sensitive uptake of the K+ congener 86Rb+. PKC activation was induced by a 5-min exposure of the cells to 10 µM PMA before the addition of Rb+ and compared with paired control plates of cells exposed for 5 min to the same amount of vehicle alone (DMSO). Values are means ± SE (n = 6-11) of flux activations induced by PMA exposure, expressed as the percentage of their paired controls (same transfection group, same passage, same day), and were compared using paired 2-tailed Student's t-tests. NS, nonsignificant. **P < 0.01; ***P < 0.001.


    DISCUSSION
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ABSTRACT
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The primary structures of alpha -isoforms of the Na+-K+-ATPase are nearly identical, except for the NH2 terminus and an approximately 10-residue region near the center of the molecule, the central ISR (Ref. 19, Fig. 1). We and others have established the influence of the NH2-terminal region on isoform-specific enzyme kinetics and regulation (8, 9, 17, 26). However, this work has also raised suspicions that another site on the catalytic subunit may contribute to these differences. We thought it likely that this putative site is also a region of structural divergence between the isoforms, and we considered the ISR a prime candidate. To address this question, we produced a pair of chimeric molecules in which the ISRs of alpha 1 and alpha 2 were switched. These chimeras were then expressed in opossum renal cells in culture after DNA-mediated gene transfer.

A difficulty with heterologous expression of the Na+-K+-ATPase in mammalian cells is the nearly ubiquitous distribution of this enzyme complex. To distinguish an endogenous from introduced enzyme in the opossum cells, we took advantage of the varied sensitivity of the Na+-K+-ATPase to digitalis glycosides (10, 25). Selection of transfected cells was achieved by growth in a concentration of ouabain sufficient to kill nontransfected cells but not cells that express the more resistant, introduced form. Clearly, this strategy is dependent on the ability of the introduced subunit to sustain active transport despite the presence of digitalis glycoside. Mutations severe enough to inhibit catalytic turnover or to interfere with targeting to the plasmalemma would not produce ouabain-resistant cells and could not be studied with this procedure. For this reason, the successful production of ouabain-resistant colonies after transfection with alpha 1alpha 2alpha 1 or alpha 2alpha 1alpha 2 suggests that overall enzymatic function was not compromised, despite the switch in ISRs.

Of course, there was always the possibility that the observed ouabain resistance was not a consequence of the transfected alpha -subunit. Resistance to sublethal concentrations of ouabain is occasionally achieved, for instance, by significant overexpression of pumps rather than the introduction of a resistant enzyme (15). We ensured against this eventuality by selecting cells in ouabain at concentrations well above the K1/2 for inhibition of the endogenous enzyme. Moreover, we confirmed the expression and structure of the introduced forms by direct detection of the exogenous polypeptides and mRNAs with specific probes (Figs. 2 and 3).

Having confirmed the successful expression of the introduced isoforms and chimeras, we next evaluated the effect of ISR exchange between alpha 1 and alpha 2 on their K+ activation/inhibition profile, a kinetic parameter known to differ dramatically between these two isoforms. Indeed, the distinctive behavior of the isoforms is apparent when the reaction is carried out under micromolar ATP concentrations because the K+ deocclusion pathway of the reaction cycle becomes rate limiting (18). Under such conditions, the activity of exogenous alpha 1 expressed in HeLa and COS-1 cells is inhibited by K+, whereas alpha 2 is stimulated (8, 17). The present observations have confirmed these results in OK cells. Daly et al. (8) have argued that it is not solely the difference in amino acid sequences at the NH2 termini of alpha 1 and alpha 2 that is responsible for this kinetic difference. Rather, it is likely to be an interaction of the segment between 24 and 32 of alpha 1 with some other region of the alpha 1 protein that determines the K+-sensitive pattern displayed by this isoform. We hypothesized that the ISR could be this region. However, this seems unlikely because exchange of the alpha 1 and alpha 2 sequences did not result in a modification of the K+ activation/inhibition profile compared with the appropriate unaltered isoform (Fig. 4). Taken together, the results of Daly and co-workers and the present data suggest that it is the interaction of the NH2-terminal segment with a region of alpha 1 other than the ISR that is a determinant of the observed K+ inhibition profile.

Another possible role for the ISR region could be in the isoform-specific response to second messengers. Numerous laboratories have cataloged differences in isoform regulation after stimulation of both protein kinase A and C (reviewed in Refs. 2, 3, and 14). For the present set of experiments, we chose to focus on PKC activation of the Na+-K+-ATPase induced by the phobol ester agonist PMA. We observed a 15% stimulation of pump-mediated Rb+ transport for alpha 1 and alpha <UP><SUB>2</SUB><SUP>∗</SUP></UP>. When we replaced the ISR in alpha 1 with the alpha 2 sequence, this activation was increased twofold (Fig. 5). Conversely, substituting the alpha 2 ISR with alpha 1 in the alpha 2-isoform resulted in a nonresponding phenotype. These data clearly argue in favor of a role for the ISR in PKC regulation. Moreover, the paradoxical response to the agonist seems to imply an inhibitory effect of the alpha 1 ISR. When it was removed from alpha 1, the PMA response was increased (alpha 1alpha 2alpha 1), but when it was substituted into alpha 2 (alpha 2alpha 1alpha 2), the previously observed response was blocked.

It is tempting to speculate about what mechanism may underlie this inhibitory effect of the alpha 1 ISR. Efendiev et al. (9) have shown that PMA-induced activation of Na+-K+-ATPase in OK cells is the result of pump translocation from intracellular pools via clathrin-coated vesicles, resulting in an increased abundance in the plasma membrane (9). This process requires the phosphorylation of Ser11 and Ser18 (not present in alpha 2-isoform) of the alpha 1-isoform by the PKC beta -isoform and involves the adaptor activator protein (AP)-1 (9). Adaptors mediate the incorporation of cargo onto transport vesicles by interacting with sorting signals present in the cytosolic domain of transmembrane proteins. Four adaptors (AP-1, AP-2, AP-3, and AP-4) have been described so far. AP-1 and AP-3 mediate sorting events at the level of the trans-Golgi network and/or endosomes, whereas AP-2 functions in endocytic clathrin-coated vesicle formation (reviewed in Ref. 4). Recent evidence has shown that AP-4 participates in basolateral sorting in epithelial cells (23). Using the same transfection strategy into OK cells as was used in the present study, investigators have shown that, unlike PMA, dopamine inhibits Na+-K+-ATPase activity. Interestingly, this inhibition also involves membrane trafficking, in this case by internalization via clathrin-coated vesicles. This internalization requires activation of the atypical PKCzeta and the adaptor AP-2, as well as the binding of phosphoinositide-3 kinase to a proline-rich motif of the alpha -subunit (5, 26). Little is known about AP-3 and AP-4, but AP-1 and AP-2 are known to recognize their target by consensus signals in the cytoplasmic domain of proteins. These consensus sequences are either di-leucine motifs or tyrosine-based signals, specifically Y-X-X-Ø, where Ø is a bulky hydrophobic amino acid. Recent work by Cotta Doné et al. (7) has identified Tyr537 as an essential element for AP-2 binding and the clathrin-dependent endocytosis of Na+-K+-ATPase that mediates dopamine-induced inhibition.

In addition to this tyrosine-based signal, the alpha 1-isoform also displays a di-leucine motif, which appears to be in the ISR but is not present in alpha 2. The di-leucine motif might represent the molecular basis of the inhibitory effect that we have attributed to the alpha 1 ISR. It could act as a dynamic retention signal that favors alpha 1 internalization, even during PMA stimulation. In apparent contradiction, it has been clearly shown that mutating the second leucine of this motif (i.e., Leu500) is not sufficient to alter PMA-induced activation (7). However, this finding may not be inconsistent with an involvement of the first leucine of the motif (i.e., Leu499), which is the last amino acid of the alpha 1 ISR and is absent from the alpha 2 sequence. Moreover, dynamic retention signaling can involve an entire region, including several, sometimes redundant, consensus motifs. For instance, insulin-regulated aminopeptidase dynamic retention within the endosomal compartment requires 2 of 3 distinct motifs present in a 30-amino-acid region of its cytoplasmic tail (13). It may be that Leu499 is the more important residue of the signaling motif, but other residues might contribute to a more extensive domain that includes the alpha 1 ISR sequence but is missing in the alpha 2 sequence.

Along the same lines, it should be kept in mind that although the ISR represents one the most striking sequence variabilities among the isoforms, other regions of the intracellular domain also display various degrees of diversity, especially in the so-called large cytoplasmic loop (between TM4 and TM5; see Fig. 1 in Ref. 2). As mentioned previously, the NH2-terminal region is also clearly an ISR. By swapping ISRs, we might have disrupted an important interaction with another part of the molecule, thereby interfering with a dynamic retention signal composed of several motifs that may be far apart in the primary structure. In short, it is not clear at this point if ISR swapping is disrupting a signal contained in the sequence itself or rather an important interaction with one or several other motifs within the intracellular domains of the protein.

With previous studies, the present data, and the above-stated hypothesis taken into account, the stimulation of alpha 1 that we are observing under PMA treatment likely results from the contribution of at least two facilitating components and one inhibitory component. Accordingly, their presence in or absence from the different isoforms would determine the amplitude of the individual response to the phorbol ester. First, an isoform-specific effect is very likely to be involved in the PMA-induced increase observed with alpha 1 It may involve Ser11 and Ser18 phosphorylation (which are not present in alpha 2) and AP-1. The effect is an increase in the number of Na+-K+-ATPase complexes expressed at the membrane (9). It is also possible that tyrosine-based motifs such as Tyr469, expressed by alpha 1 but not alpha 2, could mediate the interaction. Second, a mechanism of internalization mediated by the di-leucine motif and the surrounding area in the ISR (present only in alpha 1) may contribute to the response. This effect would be a dynamic retention, taking place even in basal conditions. Third, an activator mechanism seems to be shared by alpha 1 and alpha 2. Accordingly, removing the inhibitory ISR of the alpha 1 sequence and replacing it by the "neutral" alpha 2 ISR results in an increased response to PMA as shown for alpha 1alpha 2alpha 1. On the same basis, the PMA-induced stimulation of alpha 2 could be compromised by the addition of the inhibitory sequence of the alpha 1 ISR, as shown by the absence of PMA-induced stimulation in alpha 2alpha 1alpha 2.

Clearly, the chimeras used in the present study are just the first step in a systematic evaluation of isoform-specific structure and its influence on function. Future experiments will be needed to determine the individual amino acid residues contributing to the PKC response, as well as the role played by the ISRs of other alpha -isoforms.


    ACKNOWLEDGEMENTS

This work has been supported by American Heart Association, Texas Affiliate, Grant 98G-385 and National Center for Research Resources Grant RR-19799.


    FOOTNOTES

Address for reprint requests and other correspondence: S. V. Pierre, Dept. of Physiology, Texas Tech Univ. Health Sciences Ctr., 3601 4th St., Lubbock, TX 79430 (E-mail: Sandrine.Pierre{at}ttuhsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 4, 2002;10.1152/ajprenal.00153.2002

Received 19 April 2002; accepted in final form 28 May 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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