Distribution and oligomeric association of splice forms of Na+-K+-ATPase regulatory gamma -subunit in rat kidney

Elena Arystarkhova, Randall K. Wetzel, and Kathleen J. Sweadner

Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Renal Na+-K+-ATPase is associated with the gamma -subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma a and gamma b. Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha -subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na+-K+-ATPase conformation. In the cortex, the proportion of the gamma b-subunit was markedly lower, and the gamma a-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma b-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na+-K+-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma b-subunit. Because different mechanisms maintain and regulate Na+ homeostasis in different nephron segments, the splice forms of the gamma -subunit may have evolved to control the renal Na+ pump through pump properties, gene expression, or both.

sodium pump; nephron; immunofluorescence; confocal microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE KEY ENZYME that maintains a low intracellular concentration of Na+ inside the cell by generation of an electrochemical gradient across the cell membrane is Na+-K+-ATPase. The enzyme has two necessary subunits, alpha  and beta , but in the kidney it is tightly associated with a small, 7.4-kDa protein, the gamma -subunit (25, 42). The gamma -subunit is a type I, single-span membrane protein with the amino terminus exposed outside the cell (11, 59). Expression of the gamma -subunit at the mRNA or protein level is almost exclusively in the kidney (42, 59), although our recent analysis of the expressed sequence tag database revealed other tissues, such as placenta and mammary gland, as potential sources (56). On the basis of sequence homology, the gamma -subunit belongs to a family of small (7.5- to 19-kDa) single-span membrane proteins, the FXYD family. The family also includes phospholemman (46), mammary tumor antigen-8 (MAT-8) (44), channel-inducing factor (CHIF) (7), RIC (i.e., related to ion channel) (26), and two new gene products (FXYD6 and FXYD7) (56). Most of the homology is within the transmembrane region and the extracellular PFXYD motif, which is characteristic of the family. Outside the core motif, the gamma -subunit sequence is unique.

In the kidney, the gamma -subunit occurs as two splice variants, gamma a and gamma b (34, 35, 55, 56). The mRNA splice affects protein sequence only at the amino terminus. These splice forms were found in independent cDNA libraries derived from multiple organ types, predominantly from fluid- and solute-transporting tissues, and the sequences of the homologs from human, mouse, and rat were obtained (55). We also cloned and sequenced gamma b-subunit cDNA from the rat (GenBank AF233060). Recently, the genomic organization of the human FXYD2 gene encoding the gamma -subunit of Na+-K+-ATPase was described (41, 57). Two different promoter regions were identified, thus providing a potential structural basis for differential regulation of expression of the splice variants of the gamma -subunit.

It is established that the gamma -subunit is not required for catalytic activity (27, 51), but it has been implicated to play a modulatory role in the function of the Na+ pump. Experiments with different expression systems revealed that the gamma -subunit could alter Na+-K+- ATPase affinity for extracellular K+ in a voltage-dependent manner when expressed in oocytes (10, 11), increase affinity for ATP (48, 59, 60), or reduce apparent affinity for Na+ and/or K+ (5, 10, 48). The modulation of apparent Na+ affinity by the gamma -subunit appeared to be abolished by a posttranslational modification of the protein that occurred in cell culture (5). Interestingly, the mechanism of the effect on apparent Na+ affinity was ascribed to an increase in K+ antagonism of cytoplasmic Na+ activation (48), similar to tissue-specific kinetic differences observed earlier (45, 61).

With antibodies to the gamma -subunit, we recently showed that the gamma -subunit is not expressed everywhere in the kidney but instead has a very specific distribution to certain nephron segments: the gamma -subunit colocalized with the alpha 1-subunit in all nephron segments except the cortical thick ascending limb (CTAL) and cortical collecting ducts (CCD) (5, 62). Here, evidence is presented that splice variants of the gamma -subunit are differentially distributed in renal segments. Where coexpressed, the alternative splice forms appeared to be able to associate with the same macromolecular Na+-K+-ATPase complex.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies. Splice-specific antibodies RNGA (rat amino terminus of the gamma a-subunit) and RNGB (rat amino terminus of the gamma b-subunit) were raised at Quality Control Biochemicals (Hopkinton, MA) against the peptides Ac-TELSANHGGC and MDRWYLGGC, corresponding to the amino-terminal sequences of gamma a- and gamma b-subunits, respectively. The peptides were conjugated to tetanus toxoid as a carrier through the specially added cysteine residues and injected into rabbits (two for each peptide). The conjugate was chosen specifically to boost the immune response, since neither peptide was predicted to be highly immunogenic. The specificity of the sera was tested by ELISA with BSA-coupled peptides on solid phase. The titers of crude sera against the peptides were >20,000 (RNGA) and >60,000 (RNGB) for the rabbits with better titer. The RNGA antiserum was affinity purified against the immunizing peptide on a SulfoLink column (Pierce, Rockford, IL). For some immunofluorescence experiments, we employed an anti-TELSANHC affinity-purified antibody kindly provided by S. J. D. Karlish (Weizmann Institute). Another antipeptide antibody (RCT-G1), developed previously (5), was employed to detect the COOH terminus of the gamma -subunit, which is identical in both splice variants. Monoclonal anti-gamma -subunit antibody McG-11H was isolated from a BALB/c mouse injected with the synthetic peptide CGGSKKHRQVNEDEL (corresponding to the COOH-terminal 15 amino acids of the rat gamma -subunit) bound to keyhole limpet hemocyanin. Monoclonal antibody McK1 (19) was used to detect the alpha 1-subunit of Na+-K+-ATPase. Antipeptide antibody KETYY (gift of J. Kyte, University of California, San Diego), specific for the COOH terminus of the alpha -subunit of Na+-K+-ATPase (9), was used in ELISA experiments. Monoclonal antibody VG4 (gift of N. Modyanov, Medical College of Ohio) against Na+-K+-ATPase alpha -subunit was used for immunoprecipitation (4).

Membrane preparation and characterization. Isolation of membranes (microsomal fraction) containing Na+-K+- ATPase from rat kidney outer medulla or cortex by differential centrifugation was performed as described by Jørgensen (31). Purified enzyme used in Fig. 1 was prepared by SDS extraction (31). Cortical tubule cells were prepared as described previously (52). Briefly, two rats were euthanized, and the kidneys were perfused with 300 ml of PBS. Freshly isolated renal cortex was minced on ice to a pastelike consistency in 15 ml of DMEM in an atmosphere of 95% O2-5% CO2 and incubated with 6 mg/ml collagenase (Worthington; 269 U/mg) for 60 min at 37°C and then chilled on ice. A cell suspension was obtained by pouring the paste through graded sieves (180, 75, 63, and 53 µm). The cells were washed four times by centrifugation at 100 g for 4 min at 4°C. The last pellet was resuspended in DMEM. Electrophoresis was carried out in SDS-N-[2-(hydroxymethyl)ethyl]glycine (Tricine) gels (50) with 12.5% acrylamide. Proteins were transferred to nitrocellulose, incubated with antibodies, and detected with chemiluminescence using luminol reagents (Pierce). Blots were scanned with a laser densitometer (Molecular Dynamics).


View larger version (76K):
[in this window]
[in a new window]
 
Fig. 1.   Different electrophoretic mobilities of gamma a- and gamma b-subunits on SDS gels. A: rat kidney outer medulla microsomes (5 µg) were dissolved in electrophoresis sample buffer, loaded in a wide lane, run on an SDS-N-[2-(hydroxymethyl)ethyl]glycine (Tricine) gel, and transferred to nitrocellulose. The blot was cut vertically in the middle of the lane, and lane 1 was stained with RCT-G1 antibody (the shared COOH terminus of the gamma -subunit splice forms) at 1:5,000 and lane 2 with the RNGB antibody (gamma b-subunit specific) at 1:5,000. The faster-migrating band was identified as the gamma b-subunit. B: purified rat kidney outer medulla enzyme (25 µg) was similarly resolved in a wide lane of a gel. The blot was cut into 3 pieces and stained as follows: RCT-G1 at 1:10,000 (lane 3), Karlish gamma a-subunit antibody at 1:75 (lane 4), and RNGB at 1:10,000 (lane 5). The slower-migrating band was identified as the gamma a-subunit.

Immunoassay. Antibody binding was measured using an ELISA. Titers were measured on preparations of crude rat renal membranes from medulla or cortex adsorbed to Nunc MaxiSorp Immunoplates. PBS containing 0.5% BSA and 0.1% Tween 20 was used for washing and dilution. The secondary antibody was biotinylated goat anti-rabbit IgG, and color was developed with streptavidin-conjugated horseradish peroxidase and o-phenylenediamine (Bethesda Research Laboratories).

Immunoprecipitation. Membranes from rat kidney outer medulla (1 mg/ml) were solubilized with n-dodecyl octaethylene glycol monoether detergent (C12E8, 3 mg/ml; Calbiochem) for 10 min at room temperature in a buffer containing 25 mM imidazole, pH 7.3, and 1 mM EDTA. The extract was diluted with two volumes of detergent-free buffer, insoluble material was precipitated by centrifugation for 30 min at 20,000 g (4°C), and the pellet was checked for residual Na+-K+-ATPase. The supernatant was incubated overnight with primary antibodies (RNGA, RNGB, or VG4) at 4°C. The immune complexes were collected after 2 h of incubation with secondary goat anti-rabbit or goat anti-mouse IgG antibodies covalently bound to magnetic beads (Bio-Mag, PerSeptive Diagnostics, Cambridge, MA). The samples were washed four or five times in solubilization buffer containing 0.05% C12E8. Final precipitates were resuspended in electrophoresis sample buffer containing 62.5 mM Tris-Cl, pH 6.8, 2% SDS, 1% beta -mercaptoethanol, and 10% glycerol and heated for 15 min at 65°C before they were loaded on SDS-Tricine gels. Alternatively, rat kidney membranes were solubilized with 1 or 0.3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in a buffer containing 20 mM HEPES, pH 7.3, and 100 mM NaCl. The rest of the procedure was the same as that described above. To test the effect of Na+ pump ligands on the immunoprecipitation of the gamma -subunit splice variants, membranes from rat kidney outer medulla were first resuspended in a buffer containing 20 mM HEPES, pH 7.3, supplemented with 1) 100 mM NaCl, 2) 20 mM KCl, 3) 100 mM NaCl, 5 mM MgCl2, and 5 mM ATP, or 4) 5 mM MgCl2 and 5 mM Pi, with or without 1 mM ouabain. Solubilization with C12E8 was carried out as described above, and the buffer composition was the same for all subsequent steps, except detergent concentration was reduced from 0.1 to 0.05% during the washes.

Immunofluorescence. Immunocytochemistry was performed as described elsewhere in more detail (5, 62). Rats were given food and water ad libitum. Cryostat sections of periodate-lysine-paraformaldehyde-fixed rat kidney, sometimes treated with SDS for antigen retrieval, were concurrently probed with a mixture of mouse monoclonal anti-gamma -subunit COOH terminus antibody (McG-11H, 1:50) and either rabbit polyclonal anti-gamma b-subunit (RNGB, 1:500) or Karlish anti-gamma a-subunit (1:100) antibody. The sections were subsequently incubated with a mixture of Cy3-conjugated goat anti-mouse IgG (1:300; Accurate, Westbury, NY) and FITC-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA). Slides were examined, and images were collected on a Nikon TE300 fluorescence microscope equipped with a Bio-Rad MRC 1024 scanning laser confocal system (version 3.2).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mobility of gamma a- and gamma b-subunits on SDS gels. To assess distribution of the gamma -subunit splice variants in the kidney, splice-specific antibodies were developed. The difference between the splices is restricted to the short amino-terminal segment [8 amino acids in the gamma a-subunit are replaced with 6 residues in the gamma b-subunit (55)], and mass spectroscopy indicates that the initiation methionine is removed from the gamma a-subunit (35; Arystarkhova E and Sweadner KJ, unpublished observations). Thus the following sequences were chosen as the antigens: TELSANHGGC (gamma a-subunit) and MDRWYLGGC (gamma b-subunit).

The specificity of the antibodies for the gamma -subunit of Na+-K+-ATPase was first tested by Western blot analysis. Figure 1A demonstrates immunostaining of rat kidney microsomes. Unfortunately, RNGA (gamma a-subunit-specific antibody) failed to recognize the SDS-denatured protein, although it was active in other assays. RNGB clearly recognized the lower band in the gamma -subunit doublet, implying that the gamma b-subunit had a faster electrophoretic mobility on SDS gels than the gamma a-subunit, despite the calculated molecular masses (7,126 for the gamma a-subunit without methionine and 7,238 for the gamma b-subunit, with the assumption of no chemical modification of the primary structure). Specific staining of the gamma a-subunit with the Karlish gamma a-subunit antibody is shown in Fig. 1B. This antibody, like RNGA, was also weak on blots, and to detect stain it was necessary to load a large amount of purified enzyme, instead of microsomes and to use the antibody at a high concentration. Heavy gel loading precluded good resolution of the doublet; nonetheless, the gamma a-subunit migrated more slowly than the gamma b-subunit. The relative gel mobilities are in agreement with results of Küster et al. (35), who also measured mass values (with carboxyamidomethyl cysteine) of 7,184.0 for the gamma a-subunit (no methionine) and 7,337.9 for the gamma b-subunit (N-acetylated), and also with in vitro synthesis experiments of Beguin et al. (10). This suggests that factors other than peptide molecular mass (such as net charge or labile posttranslational modification) influence electrophoretic mobilities of the gamma -subunit splice variants on SDS gels. Posttranslational modification of the gamma -subunit, detected in transfectants or by in vitro translation with microsomes, has been reported to generate doublets (5, 10, 42, 60). However, in our hands and in the report of Beguin et al. (10), these migrate closer together than gamma a- and gamma b-subunits, and no one has reported resolving four such bands in preparations from the kidney. The gamma a-subunit from renal cortex migrated more slowly than the gamma a-subunit from the medulla (see below), and so much is yet to be determined about the factors that influence gel migration.

Immunoprecipitation of a Na+-K+-ATPase macromolecular complex. Both antibodies were effective in immunoprecipitation and coprecipitated the Na+-K+- ATPase alpha -subunit, validating the specificity of RNGA as well as RNGB. (Coprecipitation with the beta -subunit was not investigated, because it migrates too close to immunoglobulin heavy chain on Tricine gels, but it would be expected to be present on the basis of the literature.) Surprisingly, evidence was found that the RNGA and RNGB antibodies could precipitate each other's antigen as well. Rat renal medulla microsomes were first solubilized with the nonionic detergent C12E8, and then RNGA or RNGB serum was applied. Immune complexes were collected and resolved on SDS gels, and the blots were stained with the antibody against the shared COOH terminus, RCT-G1. As shown in Fig. 2A, addition of RNGA or RNGB antibody resulted in precipitation of a complex containing alpha 1-, gamma a-, and gamma b-subunits of Na+-K+-ATPase. The ratio of one splice variant to another was similar in the starting material and the final precipitates as judged by densitometry analysis. The reaction was specific, since none of the subunits was precipitated with preimmune serum (control lanes in Fig. 2A). Additional bands in the top portion of the gel represent heavy and light chains of immunoglobulins, since these bands were also seen in the control samples containing no rat kidney microsomes (not shown). Figure 2B shows a similar experiment, but precipitation was with RNGA compared with precipitation with an alpha -subunit-specific antibody, and for final detection RNGB and RCT-G1 antibodies were used. Detection of the gamma b-subunit in the RNGA precipitate strongly suggests that the gamma -subunit splice variants in medullary membranes associate in the same complex. Similar results were obtained with low-salt buffers (Fig. 2A) and NaCl-containing buffers (Fig. 2B), suggesting that ionic strength was not a major factor.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 2.   Coimmunoprecipitation of gamma -subunit splice variants. A: rat kidney outer medulla microsomes were treated with n-dodecyl octaethylene glycol monoether (C12E8) detergent in buffer A containing 25 mM imidazole and 1 mM EDTA, pH 7.3. Immunoprecipitation was carried out with splice-specific antibodies RNGA (IP-gamma a) or RNGB (IP-gamma b) or with preimmune sera (pre-gamma a and pre-gamma b). The blot was cut horizontally in the middle, and the lower portion was stained with RCT-G1 antibody (gamma a + gamma b) and the upper portion with McK1 (alpha 1); only the relevant portions of the gel are shown. B: rat kidney outer medulla microsomes were solubilized with C12E8 in buffer B containing 20 mM HEPES, pH 7.3, and 100 mM NaCl and precipitated with RNGA or the monoclonal antibody VG4 (alpha -subunit specific). Identical blots were stained with McK1 (alpha 1) and RCT-G1 (gamma a + gamma b) or McK1 (alpha 1) and RNGB (gamma b). The pellet represents unsolubilized material, which was minimal. C: rat kidney outer medulla microsomes were solubilized with 1 or 0.3% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in buffer B and precipitated with RNGA or RNGB antibody. Blots were stained with McK1 (alpha 1) and RCT-G1 (gamma a + gamma b). Results illustrate coprecipitation of gamma a- with gamma b-subunit in several conditions, notably low salt and NaCl.

A similar composition of complexes was seen after solubilization with a charged detergent, CHAPS. A proportional amount of the alpha 1-subunit and both splice variants of the gamma -subunit were recovered with RNGA or RNGB antibodies (Fig. 2C). If the coprecipitation of subunits were due to incomplete dissociation of supermolecular assemblies (such as complexes with cytoskeletal components) by the detergent, one might expect it to be enhanced by reducing the concentration of detergent. Reducing the CHAPS concentration from 1 to 0.3%, from above to below its critical micelle concentration, however, just reduced the solubilization of the enzyme and the efficiency of immunoprecipitation, while the ratio of one subunit to another in the immunoprecipitates was not changed. Thus the data suggest a strong interaction between the alpha 1- (or beta 1-) and gamma -subunits within the ATPase complex in rat kidney outer medulla membranes. The results do not rule out association within one functional unit (i.e., alpha beta gamma 2), but it is more likely that it requires more complex multimeric assembly, for example, a dimer or tetramer of the alpha -subunits with associated beta - and gamma -subunits, such as the (alpha beta gamma )2- or (alpha beta gamma )4-subunit.

Further evidence that gamma -subunit association with the other Na+-K+-ATPase subunits is specific is that the stability of the complex proved to be sensitive to the enzyme ligands present during the immunoprecipitation. Na+-K+-ATPase goes through a series of conformation changes during its turnover: in simplified form, E1 right-arrow E1-P right-arrow E2-P right-arrow E2 right-arrow E1, in which P represents phosphate (transferred from ATP) covalently bound to an aspartate in the active site. The best precipitation by RNGA and RNGB antibodies was obtained in buffers with NaCl (Fig. 3, A and C). This condition favors the E1 conformation. Buffers with KCl, which favors E2, showed greatly reduced precipitation at the same ionic strength. In tests of enzyme stability in the conditions used for precipitation, we found that, in Na+, 45% of the activity was retained after 30 min and 36% after 3 h. In K+, 75% was retained after 30 min and 59% after 3 h. This should eliminate the hypothesis that precipitation is reduced in K+ because the enzyme is less stable. Greater stability in K+ has been reported elsewhere (24, 32).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Ligand-dependent association of gamma -subunit with Na+-K+-ATPase. Rat kidney outer medulla microsomes were solubilized with C12E8 in 20 mM HEPES, pH 7.3, supplemented with 100 mM NaCl (A), 20 mM KCl (B), 100 mM NaCl, 5 mM MgCl2, and 5 mM ATP (C), or 5 mM MgCl2, 5 mM Pi, and 1 mM ouabain (D) and precipitated with RNGA (IP-gamma a) or RNGB (IP-gamma b) antibody. "Pellet" is shown to demonstrate efficiency of solubilization. As in Fig. 2, the blot was cut so that alpha - and gamma -subunits could be stained separately. HC, heavy chain; LC, light chain. Results illustrate coprecipitation of gamma -subunit in conditions that favor Na+-K+-ATPase E1 conformation and lability of the complex in conditions that favor the E2 or E2-P conformation.

The E2 conformation can also be obtained in the presence of NaCl by including ATP and Mg2+, which causes the enzyme to be phosphorylated and adopt the E2-P conformation without greatly changing the precipitation conditions. This caused a substantial reduction in precipitation and an apparent loss of coprecipitation of the gamma a-subunit with the gamma b-subunit, as judged by gel mobility (Fig. 3C). Another way to arrive at the E2-P conformation, the so-called "backdoor" way, is to incubate the enzyme with its product, Pi, in the presence of Mg2+ and ouabain and in the absence of Na+ or K+. In this case, while precipitation of the gamma -subunits alone was reasonable, the coprecipitation of alpha - with gamma -subunit antibodies appeared to be reduced, as expected if the complex is more labile. Coprecipitation of gamma b- with the gamma a-subunit antibody was again reduced compared with that in NaCl (Fig. 3D), as it was in Na+, Mg2+, and ATP. As shown in Fig. 2A, low ionic strength alone did not cause the same effect. As discussed below, these conformation-specific effects on complex stability are evidence that the gamma -subunits are functionally integrated into Na+-K+-ATPase and that the coprecipitation of the two splice forms was not an artifact or the result of gamma -gamma aggregation.

Table 1 shows an analysis of the immunoprecipitation results. The amount of precipitated protein obtained was approximated by measuring the density of stain on X-ray film, and interexperiment variation in stain intensities was corrected by comparing the ratios of gamma - to alpha -subunit stain in the precipitate with that in the starting material. This should not be regarded as true quantification, because it does not take into account the lower overall precipitation seen in E2 conditions. We also, of course, do not assume that equal stain intensity with two different antibodies means equal yield of antigen. The derived average ratios show that ratios of gamma - to alpha -subunit stain in precipitates with anti-gamma a- and anti-gamma b-subunit antibodies in Na+ were similar (~0.7) and that ratios of gamma - to alpha -subunit were larger by 4- to 15-fold in all E2 conditions. This indicates less recovery of alpha - than of gamma -subunit, consistent with dissociation of the complex in E2. The yield of precipitate with the RNGA antibody in Na+, Mg2+, and ATP was too low to quantify.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of E1-E2 conformation states on immunoprecipitation of alpha - with gamma -subunit

Expression of gamma -subunit splice variants in the kidney. The first indication of differential distribution of the gamma -subunit splice variants in the kidney was obtained by ELISA. Because activity of Na+-K+-ATPase varies significantly along the nephron, ELISA with membrane preparations from the medulla and cortex were first normalized for the amount of alpha 1-subunit with anti-KETYY antibody as the reference. This antibody recognizes the COOH terminus of the alpha -subunit (9). As shown in Fig. 4A, binding of KETYY appeared similar when the amount of cortical membranes in each well was almost doubled compared with medullary membranes. Comparable results were found for RNGA binding to adjusted amounts of cortical and medullary membranes, a difference of ~1.6-fold (Fig. 4B). Unexpectedly, in these conditions, RNGB binding was ~3.5 times weaker to cortical than to medullary membranes (3- to 4-fold in different experiments; Fig. 4C). This implies that expression of the gamma b-subunit should be much lower in cortical segments of the rat nephron than in the medulla, if it is assumed that all the gamma -subunit was in a complex with the alpha -subunit. Alternatively, although less likely, accessibility of the RNGB epitope may be more masked in one type of membrane than in the other, or the epitope could be structurally modified.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   ELISA-detected difference in proportions of gamma a- and gamma b-subunits. Microsomes from medulla (1 µg/well) or cortex (2 µg/well) were adsorbed to Nunc immunoplates and then incubated with different dilutions of KETYY (the COOH terminus of the alpha -subunit, A), RNGA (gamma a-subunit, B), and RNGB (gamma b-subunit, C) antibody. Data represent the difference between total and nonspecific binding, which was evaluated on BSA-coated plates. There was much less gamma b-subunit immunoreactivity in the cortex than in the medulla. OD450, optical density at 450 nm.

To evaluate whether expression of the gamma b-subunit in rat cortex was indeed lower than in the medulla, Western blot analysis was performed on isolated membrane fractions. Although only RNGB antibody proved to be suitably sensitive for Western blots, combined use of RNGB and the RCT-G1 antibody against the COOH terminus of the gamma -subunit allowed us to monitor the expression pattern of splice variants of the gamma -subunit in the kidney. Figure 5A shows representative blots stained with McK1 (alpha 1-subunit), RCT-G1, and RNGB antibodies. Staining for the gamma b-subunit was significantly weaker in the cortex than in the medulla, similar to our ELISA observation. Although we did observe some batch-to-batch variations, we always saw much less gamma b-subunit expressed in the cortex. Figure 5B represents a summary of the densitometric analysis of four independent experiments. Identical blots were stained with McK1 and either RCT-G1 or RNGB antibodies and then scanned. To normalize the data to the amount of alpha -subunit loaded, the ratio was taken between either gamma  (i.e., gamma a + gamma b) or gamma b and the alpha 1 staining, and the data were expressed in arbitrary units. There was some (20%) decrease in the ratio of gamma - to alpha 1-subunit staining in the cortex compared with the medulla and a significant reduction (~70-80%) in the ratio of gamma b- to alpha 1-subunit staining in the cortex compared with the medulla. This indicates that 1) some cortical segments must express alpha  without gamma  and 2) some must express gamma a without (or with much less) gamma b. The former conclusion is in agreement with our previous findings that the gamma -subunit is not detectable in some cortical segments, whereas expression of the alpha 1-subunit is ubiquitous along the nephron (5, 62). A similar decrease in the level of gamma b-subunit expression in cortical vs. medullary segments was observed in membrane preparations from mouse kidney (not shown).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5.   Expression of the gamma b-subunit was significantly lower in the cortex than in the medulla. A: microsomes from rat kidney outer medulla or cortex were separated on SDS-Tricine gels and transferred to nitrocellulose, and blots were stained with McK1 (alpha 1), RCT-G1 (gamma a + gamma b), and RNGB (gamma b) antibody. B: densitometric analysis. Blots were scanned, and the ratio of the "gamma "- and/or gamma b-subunit to the alpha 1-subunit stain in cortical (C) and medullary (M) segments is expressed in arbitrary units. Data represent a summary from 4 independent experiments. The proportion of gamma b-subunits was significantly lower (P < 0.00005 by Student's t-test) than "gamma "-subunits in the cortex.

Selective expression of the gamma a-subunit in proximal tubules. Because the great majority of renal cortex volume is represented by proximal tubules, next we isolated individual cells from the cortex by procedures commonly used to prepare proximal tubule cells (PTC) and checked them for expression of gamma -subunit splice variants. Cortical tissue (the very superficial cortex) was dissected and dissociated with collagenase, and the cell suspension was gradually poured through molecular sieves with different opening diameters. Expression of gamma - and/or gamma b-subunits was analyzed on blots. As shown in Fig. 6, a substantial amount of gamma -subunit with low mobility was detected in PTC, similar to rat kidney outer medulla membranes. At the same time, expression of the gamma b-subunit was not detected in these samples. The gamma -subunit band in PTC appeared fuzzy, and it had a slightly slower electrophoretic mobility on SDS gels than the gamma -subunit in control samples; the appearance of the cortical sample in Fig. 5A was similar. This may indicate a structural peculiarity of the gamma a-subunit in cortical membranes, such as posttranslational modification, compared with medullary segments of the nephron. Objectively, the gamma b-subunit could be present in sieve-isolated PTC of rat nephron but with a modified amino-terminal sequence (e.g., with oxidized methionine and/or tryptophan residues) that would have made it undetectable by the RNGB antibody. Consequently, to interpret the results correctly, the distribution of gamma -subunit splice variants was next evaluated on kidney sections using confocal immunofluorescence microscopy.


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 6.   Selective expression of the gamma a-subunit in proximal tubule cells. Microsomes from rat kidney outer medulla and proximal tubule cells were separated on SDS-Tricine gels and transferred to nitrocellulose, and blots were stained with McK1 (alpha 1; A), RCT-G1 (gamma a + gamma b; B), and RNGB (gamma b; C) antibodies. The gamma b-subunit was undetectable in isolated proximal tubule cells; the gamma a-subunit was found in proportion to the alpha -subunit.

Immunolocalization of splice variants of the gamma -subunit in the kidney. Figures 7 and 8 show low-magnification images of the rat kidney in which distribution of gamma -, gamma a-, or gamma b-subunit stain can be seen throughout the four major layers (cortex, outer and inner stripes of the outer medulla, and inner medulla). The boundary between the cortex and the outer stripe is the point at which glomeruli (faint dark holes) can no longer be seen, and the boundary between the outer stripe and the inner stripe is the point at which proximal straight tubules (PST) terminate and the packing of medullary thick ascending limbs (MTAL) becomes much more dense. The boundary between the inner stripe of the outer medulla and the inner medulla is very obvious because of the sharp drop in stain (for any Na+-K+-ATPase subunit). The red stain shows the monoclonal anti-gamma -subunit COOH terminus antibody McG-H11, and the green stain shows the rabbit anti-TELSANHC or RNGB. Total gamma -subunit stain [as described in more detail and shown at higher magnification elsewhere (62)] was brightest in the MTAL in the inner and outer stripes and in the distal convoluted tubule (DCT) and connecting tubule (CNT) in the cortex. This gamma -subunit antibody also stained the proximal convoluted tubule (PCT) and PST in proportion to alpha -subunit stain of these structures, which is much lighter than alpha -subunit stain of the thick ascending limb (TAL), DCT, or CNT. Our laboratory also previously reported finding no gamma -subunit stain in the CTAL (5), cortical collecting duct (CCD), or outer medullary collecting duct (62).


View larger version (122K):
[in this window]
[in a new window]
 
Fig. 7.   Localization of the gamma a-subunit in rat kidney by double-label confocal immunomicroscopy with antibodies against the gamma -subunit COOH terminus (gamma -subunit) and against the gamma a-subunit. Layers of the kidney are designated as follows: cortex (C), outer stripe of the outer medulla (OS), inner stripe of the outer medulla (IS), and inner medulla (IM). The odd structure to the right of the inner medulla is a fold in the section. Red segments in the superficial cortex are distal convoluted tubule and connecting tubule; in the outer stripe and base of the cortex they are the medullary thick ascending limb (and the initial portion of the cortical thick ascending limb). Segments that appear red in the combined image did not stain well for the gamma a-subunit. Stain for the gamma a-subunit was fairly uniform in the cortex, with the exception of the blank glomeruli and unstained cortical thick ascending limb. Brightly stained segments in the inner stripe are the medullary thick ascending limb. Scale bar, 100 µm.



View larger version (98K):
[in this window]
[in a new window]
 
Fig. 8.   Localization of the gamma b-subunit in rat kidney. With this combination of antibodies, the red segments (fainter stain) are the proximal convoluted tubule in the cortex and the proximal straight tubule in the outer stripe. Brightly stained distal convoluted tubule and connecting tubule in the cortex and thick ascending limb in outer and inner stripes of the medulla expressed the gamma b-subunit abundantly. Light gamma b-subunit stain can also be seen in the thick ascending limb in the lowest portions of the cortex, but not in the superficial cortex. Scale bar, 100 µm.

The low-power images show the uniformity of the differences in splice form distribution. Figure 7 compares stain for the gamma a-subunit with total gamma -subunit stain. Although it is true that gamma a-subunit stain was much stronger in the inner stripe than elsewhere, it was proportional to the amount of alpha -subunit there (62). Stain for the gamma a-subunit was light but fairly uniform in the cortex, apparently absent from glomeruli (round black holes), but present clearly in the majority of tubules. We have shown that the segments in the cortex that were stained brightly red by the gamma -subunit COOH terminus antibody are DCT and CNT (62), and gamma a-subunit stain of these was reduced (perhaps absent) compared with the rest of the stain. That the rest of the stain was in the PCT was supported by the light level of stain (like alpha -subunit stain, not shown) and by the large numbers of stained tubules. We have also seen gamma a-subunit stain of initial segments of the PCT emerging from glomeruli (not shown). Interestingly, a similar segregation of red and green stain was observed in the outer stripe of the outer medulla. The PST were stained lightly for the gamma a-subunit, while the MTAL segments that ascend between them were predominantly red, not green or yellow, suggesting little expression of the gamma a-subunit.

Figure 8 is the complementary image in which stain for gamma b- and gamma -subunits is compared. Stain for the gamma b-subunit was prominent in the DCT and CNT of the cortex and in the MTAL of the outer and inner stripes of the outer medulla. The gamma b-subunit stain was undetectable in the PCT. The distinction between the gamma b- and all-gamma -subunit stain is shown at higher magnification for the cortex in Fig. 9. In the combined image, the colocalization of the all-gamma - and gamma b-subunit stain in the DCT and CNT is apparent as yellow, while the PCT are red, which indicates that they contain the gamma -subunit, but not the gamma b-subunit, and, therefore, should have the gamma a-subunit. The CNT can be tentatively identified by the presence of unstained cells (intercalated cells). Elsewhere, we used specific DCT, CNT, and CCD markers to discriminate among these distal cortical segments with the all-gamma -subunit antibody, and so it is used as the reference antibody here (62).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 9.   Apparent absence of the gamma b-subunit from the proximal tubule. Experiment is similar to that in Fig. 8, but at higher magnification in the superficial cortex. In the combined image, the red stain by the gamma -subunit COOH terminus antibody in the proximal convoluted tubule (PCT) is clear, confined to the basolateral membrane. Stain for the gamma b-subunit (green; yellow in the combined image) was undetectable in the PCT. DCT, distal convoluted tubule; G, glomerulus. Scale bar, 100 µm.

The TAL showed a gradient of differences in the expression of gamma a- and gamma b-subunits. In the MTAL in the inner stripe of the outer medulla, both were abundant. In the outer stripe, stain for the gamma a-subunit was greatly reduced relative to stain for the gamma b-subunit. These gamma b-subunit-stained segments penetrated slightly into the cortex but soon lost any detectable stain. No gamma -subunit stain was seen with any of the antibodies in the CTAL in the superficial cortex.

We observed light stain for gamma a- and gamma b-subunits in two other structures (data not shown). Macula densa cells stain prominently for the gamma -subunit on the basal surface facing the juxtaglomerular apparatus (48, 62). Here we observed stain of the macula densa by gamma a- and gamma b-subunit antibodies. Stain for the gamma b-subunit seemed qualitatively more prominent, but this may be due to a higher background with the gamma a-subunit antibody. In the deepest regions of the inner medulla (papilla), Na+-K+-ATPase alpha -subunit stain was higher than in the more superficial region visible in Figs. 7 and 8. We observed stain for gamma a- and gamma b-subunits there as well.

In summary, there were notable differences in gamma a- and gamma b-subunit distribution. In the cortex, the gamma a-subunit predominated in the PCT and the gamma b-subunit in the DCT and CNT. This pattern was also obtained in the outer stripe of the outer medulla, where PST expressed the gamma a-subunit, while this portion of the MTAL expressed predominantly the gamma b-subunit. In contrast, both gamma -subunit splice variants were expressed abundantly in the portion of the MTAL in the inner stripe of the outer medulla. The TAL in the superficial cortex (CTAL) were not detectably labeled by gamma a- or gamma b-subunit antibodies, just as they were not labeled by the all-gamma -subunit antibody. Table 2 summarizes the distributions.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Distribution of gamma -subunit and its splice variants in the kidney


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Segment-specific differences in gamma -subunit distribution. In the kidney, different segments of the nephron carry out different specific tasks and are subject to complex regulation. It is well established that Na+-K+-ATPase distribution and activity vary markedly along the nephron (17, 18, 33, 40). Along with unequal representation of Na+-K+-ATPase along the nephron, functional differences in intrinsic properties of the pump have been reported, including segment-specific differences in the affinity of Na+-K+-ATPase for Na+ (8, 13, 21, 23). Furthermore, there is also much evidence for segment-specific regulation of Na+-K+- ATPase activity (22, 49). The molecular basis of these differences has been highly controversial, and little consensus has emerged on the subject. Although there was general agreement that alpha 1- and beta 1-subunits predominated in the renal medulla, the cortex was less well studied, and it was at one time hypothesized that other isoforms were expressed in more distal segments. However, no one has reported segment-specific localization of any isoforms of Na+-K+-ATPase other than alpha 1- and beta 1-subunits at the mRNA or protein level. We and others have reported that expression of the gamma -subunit reduced the apparent affinity for Na+ in transfected cells (3, 5) and in Xenopus oocytes (10) or increased K+ antagonism of Na+ binding (48), and there were effects on apparent K+ affinity (5) and ATP affinity (59, 60) as well. The presence or absence of the gamma -subunit appears to correlate roughly with the differences in apparent affinities for Na+ in successive segments of the nephron. Localization of the gamma -subunit and its variants provides a new framework for considering apparent segment-specific differences.

We previously described the distribution of gamma -subunit stain in the rat kidney (62). Using immunofluorescence with the RCT-G1 antibody, which recognizes an epitope shared by the splice variants, and an antibody against the alpha 1-subunit of Na+-K+-ATPase, we demonstrated simultaneous expression of alpha - and gamma -subunits in many tubule segments. The gamma -subunit stain was brightest in the MTAL and DCT, moderate in CNT, low in PCT, and undetectable in glomeruli and collecting ducts. However, the alpha 1-subunit, but not the gamma -subunit, was found in the CTAL and in some segments of the CCD. Here we extended these studies by examining localization of gamma -subunit splice variants in medullary and cortical segments with splice-specific antibodies.

The major outcome is that we found colocalization of gamma a- and gamma b-subunits in medullary tubules, whereas their distribution in the cortex appeared to be strongly biased to different segments. As in our previous work, we observed light stain for the gamma -subunit in the PCT that colocalized with the alpha 1-subunit. This form of the gamma -subunit appeared to be gamma a. The bright gamma -subunit stain in the DCT and CNT was apparently predominantly gamma b. No staining with RNGA or RNGB antibody was detected in the upper CTAL or CCD, whereas the alpha 1-subunit was found there. The gradient of gamma a- and gamma b-subunit expression in the TAL is intriguing in view of this segment's physiological role as the diluting segment. If absence of the gamma -subunit results in higher ion affinities, this correlates with the presence of lower interstitial ion concentrations as the tubule ascends into the cortex.

While this report was in preparation, Pu et al. (48) reported on immunocytochemical localization of gamma -subunit splice variants in the rat kidney. Although much of their staining was similar to ours, there were some differences in results or interpretation. They concluded that both splice variants of the gamma -subunit were expressed in proximal tubules at low levels. Here, we did not detect gamma b-subunit in the proximal tubule in kidney sections or in isolated PTC. Whether this discrepancy originates in technical details (e.g., affinity or purity of the antibodies or fixation method) or reflects potential differences in physiological status of the animals used in the studies remains to be clarified; completely different fixation protocols were used. Pu et al. also detected the gamma b-subunit in the CTAL, but we detected it only close to the MTAL of the adjacent outer stripe of the outer medulla, and all the rest of our cortical gamma -subunit stain was in Tamm-Horsfall-negative segments. Pu et al. detected only the gamma a-subunit in the macula densa, while we have detected both gamma a- and gamma b-subunits there. Finally, they reported gamma b-subunit expression in the CCD on the basis of finding it in segments containing aquaporin 2, a distal tubule marker. We hypothesize, however, that this apparent discrepancy is due only to the difficulty of distinguishing the CNT and CCD without the use of specific markers. In rodents, the CNT has been shown to express aquaporin 2 (14), although this is not true of rabbits (37). In our studies with the gamma -subunit COOH terminus antibody, the brightest cortical stain colocalized with the thiazide-sensitive carrier (a DCT marker) or with calbindin (which stains CNT brightly), while thin-celled, weakly calbindin-stained CCD did not show detectable gamma -subunit (62). All these interpretations are, of course, subject to the general caveat that experimental conditions determine the limit of sensitivity of immunofluorescence.

CHIF is a homolog of the gamma -subunit that is expressed chiefly in the kidney and distal colon; its gene name is FXYD4. It has recently been demonstrated that CHIF expression is confined to the collecting duct (in the cortex and outer and inner medulla) (54). This distribution is complementary to that of the gamma -subunit, in that we did not detect the gamma -subunit in the collecting duct until deep in the papilla. Very recent evidence indicates that CHIF increases Na+-K+-ATPase apparent affinity for Na+, the opposite of the effect of the gamma -subunit (10).

Oligomeric assembly of Na+-K+-ATPase. Coexpression of gamma a- and gamma b-subunits in a majority of the MTAL in the inner stripe of the outer medulla was unambiguous. Coimmunoprecipitation of gamma a- and gamma b-subunits along with the alpha -subunit from medullary membranes raises the issue of the multimeric architectural organization of the Na+-K+-ATPase complex in those segments. Many reports support the idea that Na+-K+-ATPase exists as an oligomer of two to four alpha -subunits, recently reviewed by Taniguchi et al. (58). If this is so, in the MTAL a random association of gamma a- and gamma b-subunits with dimers or tetramers of the alpha -subunit would result in coimmunoprecipitation. Coprecipitation of gamma a and gamma b could in principle reflect gamma -gamma association but was more likely random assembly of the gamma -subunits with dimers or tetramers of alpha beta protomers, because the alpha -subunit was also coprecipitated. This is consistent with a 1:1 ratio of alpha - to gamma -subunit (gamma a + gamma b) (35) or, at a lower limit, 1:0.5 (36), as observed in trypsin-digested purified renal medullary Na+-K+-ATPase.

Ligand effects on the ability of anti-gamma -subunit antibodies to precipitate Na+-K+-ATPase complexes support the specificity of the coprecipitation. It seems unlikely that gamma -subunit aggregation into gamma -subunit-only oligomers (the only other way to explain coprecipitation) would be affected by specific conditions that are known to affect alpha -subunit conformation. The interpretation of ligand effects relative to the literature on Na+-K+-ATPase oligomer association has some interesting features, however. First, C12E8 can be used to solubilize the Na+-K+-ATPase as protomers of alpha beta (and presumably gamma ) (12, 28). These protomers soon oligomerize, however, either as diprotomers in equilibrium or as higher aggregates with time and various conditions (12), including ionic strength (15, 16) and the presence of lipid and ATP (43). Studies with membrane-associated enzyme indicated that formation of E2-P promotes formation of alpha -alpha - and alpha -beta -subunit links with several bifunctional cross-linking reagents or copper-phenanthroline (6, 47). The above literature suggests a closer association between alpha -alpha - and alpha -beta -subunits in E2 than in E1 conformations. Here, precipitation by anti-gamma -subunit antibodies was antagonized by formation of E2-P. If we consider what is likely to be happening on the molecular level, however, there may be no contradiction. First, the stability of gamma -subunit interaction with the rest of the complex may differ from that of alpha -alpha -, alpha -beta -, or even beta -beta -subunit associations, inasmuch as the gamma -subunit may lose part of a favorable binding contact when the other subunits are rearranged relative to one another. Ligand effects on cross-linking are more pertinent to the orientation of reactive groups than to complex stability per se. There are good examples of subtle ligand effects on the accessibility of sites near the membrane that support the importance of conformational changes for Na+-K+-ATPase intersubunit organization and function (30, 38, 39, 53).

Implications for a physiological role for the gamma -subunit. We observed previously that expression of the gamma a-subunit in stable renal cell transfectants can reduce Na+-K+-ATPase apparent Na+ affinity to a level similar to that observed in kidney membranes (5). The kinetic differences we observed were seen in partially purified enzyme, which indicates a very stable functional modification of enzyme properties. Qualitatively similar findings were reported by Pu et al. (48), who additionally concluded that the reduction in apparent Na+ affinity could be best interpreted as an increase in K+ competition for Na+ at the Na+ binding site. Selective expression of splice variants in cortical segments of the nephron provides another potential layer for gamma -subunit-mediated regulation of Na+-K+-ATPase.

The present data do not address whether and how gamma a- and gamma b-subunits differently regulate Na+-K+-ATPase properties. With mammalian cell transfectants, we have observed that expression of gamma a- or gamma b-subunits reduces apparent affinities for Na+ and/or K+ but that posttranslational modifications, revealed by shifts in gel mobility, complicate the functional effects (3, 5, 63). Others have reported equivalent functional effects of gamma a- or gamma b-subunits in transfectants on K+ antagonism of Na+ activation (and on affinity for ATP) in the presence of mixed levels of potential posttranslational modifications detected by gel mobility (48, 60) and equivalent effects of unmodified gamma a- and gamma b-subunits in Xenopus oocyte pump currents (10). Much remains to be learned about whether and where gamma -subunit posttranslational modifications occur in the kidney, since they have not yet been detected by mass spectroscopy of purified protein (35). It is clear that expression of unmodified gamma -subunit of either splice variant reduces the manifest affinity for Na+ in in vitro conditions that resemble physiological conditions. Even if both gamma -subunit splice variants had the same functional effect most of the time, the two FXYD2 gene alternative transcripts with their different 5'-untranslated regions could be subject to different transcriptional or translational expression control.

In this work, we observed exclusive expression of the gamma a-subunit in PCT, where >70% of filtered Na+ is reabsorbed. The process is under complex control of many hormones and neurotransmitters, including parathyroid hormone, dopamine, epinephrine and norepinephrine, angiotensin II, insulin, and glucocorticoids (22). Although apparent affinity of Na+-K+-ATPase for Na+ is lowest in PCT among analyzed microdissected nephron segments, it can be altered in response to some physiological stimuli. At low concentrations, the stimulatory effect of angiotensin II on Na+-K+-ATPase results at least partially from increased apparent affinity for Na+ (2). Dopamine, the major negative regulator of Na+ reabsorption, not only inhibits Na+-K+-ATPase in proximal tubules, but it also alters apparent affinity of the enzyme for cations (decreases apparent affinity for K+ and increases that for Na+) (1, 29). In proximal tubules, insulin induces an increase in the Na+ sensitivity without altering the maximal reaction velocity (21). Similarly, stimulation of Na+-K+-ATPase activity by protein kinase C in the proximal tubule occurs through enhancement of the affinity for Na+ (20). Thus apparent segment-specific regulation suggests the presence of different isoforms of Na+-K+-ATPase (which is not the case) or a specific modification of the intrinsic properties of the pump in the proximal tubules, such as, hypothetically, a posttranslational modification of the gamma a-subunit. Conversely, the gamma -subunit may be inactivated by suppression of gene expression. In both cases, the net effect would be antinatriuretic. Whether any of these mechanisms is indeed involved in regulation of Na+ readsorption in proximal tubules requires further investigation.


    ACKNOWLEDGEMENTS

We thank Drs. S. J. D. Karlish, N. Modyanov, and J. Kyte for their generosity with antibodies, Drs. D. Brown and M. S. Feschenko for useful discussions, and N. K. Asinovski for technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-36271 to K. J. Sweadner.

Address for reprint requests and other correspondence: K. J. Sweadner, 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129 (E-mail: sweadner{at}helix.mgh.harvard.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.

10.1152/ajprenal.00146.2001

Received 9 May 2001; accepted in final form 3 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aperia, A, Bertorello A, and Seri I. Dopamine causes inhibition of Na+-K+-ATPase activity in rat proximal convoluted tubule segments. Am J Physiol Renal Fluid Electrolyte Physiol 252: F39-F45, 1987[Abstract/Free Full Text].

2.   Aperia, A, Holtback U, Syren ML, Svensson LB, Fryckstedt J, and Greengard P. Activation/deactivation of renal Na,K-ATPase: a final common pathway for regulation of natriuresis. FASEB J 8: 436-439, 1994[Abstract/Free Full Text].

3.   Arystarkhova, E, Asinovski NK, and Sweadner KJ. Splice variants of the gamma-subunit differentially influence Na+ and K+ affinities of the Na,K-ATPase (Abstract). J Am Soc Nephrol 11: 24A, 2000.

4.   Arystarkhova, E, Gasparian M, Modyanov NN, and Sweadner KJ. Na,K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding. J Biol Chem 267: 13694-13701, 1992[Abstract/Free Full Text].

5.   Arystarkhova, E, Wetzel RK, Asinovski NK, and Sweadner KJ. The gamma -subunit modulates Na+ and K+ affinity of the renal Na,K-ATPase. J Biol Chem 274: 33183-33185, 1999[Abstract/Free Full Text].

6.   Askari, A, Huang WH, and Antieau J. Na+,K+-ATPase: ligand-induced conformational transitions and alterations in subunit interactions evidenced by cross-linking studies. Biochemistry 19: 1132-1140, 1980[ISI][Medline].

7.   Attali, B, Latter H, Rachamim N, and Garty H. A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes. Proc Natl Acad Sci USA 92: 6092-6096, 1995[Abstract/Free Full Text].

8.   Barlet Bas, C, Cheval L, Khadouri C, Marsy S, and Doucet A. Difference in the Na+ affinity of Na+-K+-ATPase along the rabbit nephron: modulation by K+. Am J Physiol Renal Fluid Electrolyte Physiol 259: F246-F250, 1990[Abstract/Free Full Text].

9.   Bayer, R. Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na+ + K+)-ATPase. Biochemistry 29: 2251-2256, 1990[ISI][Medline].

10.   Beguin, P, Crambert G, Guennoun S, Garty H, Horisberger JD, and Geering K. CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma -subunit. EMBO J 20: 3993-4002, 2001[Abstract/Free Full Text].

11.   Beguin, P, Wang X, Firsov D, Puoti A, Claeys D, Horisberger JD, and Geering K. The gamma -subunit is a specific component of the Na,K-ATPase and modulates its transport function. EMBO J 16: 4250-4260, 1997[Abstract/Free Full Text].

12.   Brotherus, JR, Jacobsen L, and Jorgensen PL. Soluble and enzymatically stable (Na+ + K+)-ATPase from mammalian kidney consisting predominantly of protomer alpha beta units. Preparation, assay and reconstitution of active Na+,K+ transport. Biochim Biophys Acta 731: 290-303, 1983[ISI][Medline].

13.   Buffin-Meyer, B, Marsy S, Barlet-Bas C, Cheval L, Younes-Ibrahim M, Rajerison R, and Doucet A. Regulation of renal Na+,K+-ATPase in rat thick ascending limb during K+ depletion: evidence for modulation of Na+ affinity. J Physiol (Lond) 490: 623-632, 1996[Abstract].

14.   Coleman, RA, Wu DC, Liu J, and Wade JB. Expression of aquaporins in the renal connecting tubule. Am J Physiol Renal Physiol 279: F874-F883, 2000[Abstract/Free Full Text].

15.   Craig, WS. Determination of the distribution of sodium and potassium ion-activated adenosine triphosphatase among the various oligomers formed in solutions of nonionic detergents. Biochemistry 21: 2667-2674, 1982[ISI][Medline].

16.   Craig, WS. Monomer of sodium and potassium ion-activated adenosine triphosphatase displays complete enzymatic function. Biochemistry 21: 5707-5717, 1982[ISI][Medline].

17.   Doucet, A. Na-K-ATPase in the kidney tubule in relation to natriuresis. Kidney Int 37: S118-S124, 1992.

18.   Farman, N. Na,K-pump expression and distribution in the nephron. Miner Electrolyte Metab 22: 272-278, 1996[ISI][Medline].

19.   Felsenfeld, DP, and Sweadner KJ. Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K- ATPase catalytic subunit. J Biol Chem 263: 10932-10942, 1988[Abstract/Free Full Text].

20.   Feraille, E, Carranza ML, Buffin-Meyer B, Rousselot M, Doucet A, and Favre H. Protein kinase C-dependent stimulation of Na,K-ATPase in rat proximal convoluted tubules. Am J Physiol Cell Physiol 268: C1277-C1283, 1995[Abstract/Free Full Text].

21.   Feraille, E, Carranza ML, Rousselot M, and Favre H. Insulin enhances sodium sensitivity of Na,K-ATPase in isolated rat proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 267: F55-F62, 1994[Abstract/Free Full Text].

22.   Feraille, E, and Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345-418, 2001[Abstract/Free Full Text].

23.   Feraille, E, Rousselot M, Rajerison R, and Favre H. Effect of insulin on Na+,K+-ATPase in rat collecting duct. J Physiol (Lond) 488: 171-180, 1995[Abstract].

24.   Fischer, TH. The effect of Na+ and K+ on the thermal denaturation of Na+ + K+-dependent ATPase. Biochem J 211: 771-774, 1983[ISI][Medline].

25.   Forbush, B, III, Kaplan JH, and Hoffman JF. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na,K-ATPase. Biochemistry 17: 3667-3676, 1978[ISI][Medline].

26.   Fu, X, and Kamps MP. E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts. Mol Cell Biol 17: 1503-1512, 1997[Abstract].

27.   Hardwicke, PMD, and Freytag JW. A proteolipid associated with Na,K-ATPase is not essential for ATPase activity. Biochem Biophys Res Commun 102: 250-257, 1981[ISI][Medline].

28.   Hayashi, Y, Mimura K, Matsui H, and Takagi T. Minimum enzyme unit for Na+/K+-ATPase is the alpha beta -protomer. Determination by low-angle laser light scattering photometry coupled with high-performance gel chromatography for substantially simultaneous measurement of ATPase activity and molecular weight. Biochim Biophys Acta 983: 217-229, 1989[ISI][Medline].

29.   Ibarra, F, Aperia A, Svensson LB, Eklof AC, and Greengard P. Bidirectional regulation of Na,K-ATPase activity by dopamine and an alpha -adrenergic agonist. Proc Natl Acad Sci USA 90: 21-24, 1993[Abstract].

30.   Ivanov, A, Zhao H, and Modyanov NN. Packing of the transmembrane helices of Na,K-ATPase: direct contact between beta -subunit and H8 segment of alpha -subunit revealed by oxidative cross-linking. Biochemistry 39: 9778-9785, 2000[ISI][Medline].

31.   Jørgensen, PL. Isolation of (Na + K)ATPase. Methods Enzymol 32: 277-290, 1974[Medline].

32.   Jørgensen, PL, and Andersen JP. Thermoinactivation and aggregation of alpha beta units in soluble and membrane-bound (Na,K)-ATPase. Biochemistry 25: 2889-2897, 1986[ISI][Medline].

33.   Kashgarian, M, Biemesderfer D, Caplan M, and Forbush B, III. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int 28: 899-913, 1985[ISI][Medline].

34.   Küster, B, Shainskaya A, Mann M, and Karlish SJD Mass spectrometric analysis of the gamma -subunit of Na,K-ATPase. In: Na/K-ATPase and Related ATPases, edited by Taniguchi K, and Kaya S.. Amsterdam: Elsevier, 2000, p. 599-602.

35.   Küster, B, Shainskaya A, Pu HX, Goldshleger R, Blostein R, and Karlish SJD A new variant of the gamma -subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding and expression in cultured cells. J Biol Chem 275: 18441-18446, 2000[Abstract/Free Full Text].

36.   Liu, L, and Askari A. Evidence for the existence of two ATP-sensitive Rb+ occlusion pockets within the transmembrane domains of Na+/K+-ATPase. J Biol Chem 272: 14380-14386, 1997[Abstract/Free Full Text].

37.   Loffing, J, Loffing-Cueni D, Macher A, Hebert SC, Olson B, Knepper MA, Rossier BC, and Kaissling B. Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex. Am J Physiol Renal Physiol 278: F530-F539, 2000[Abstract/Free Full Text].

38.   Lutsenko, S, Daoud S, and Kaplan JH. Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha -subunit. J Biol Chem 272: 5249-5255, 1997[Abstract/Free Full Text].

39.   Lutsenko, S, and Kaplan JH. Molecular events in close proximity to the membrane associated with the binding of ligands to the Na,K-ATPase. J Biol Chem 269: 4555-4564, 1994[Abstract/Free Full Text].

40.   McDonough, AA, Magyar CE, and Komatsu Y. Expression of Na+,K+-ATPase alpha - and beta -subunits along rat nephron: isoform specificity and response to hypokalemia. Am J Physiol Cell Physiol 267: C901-C908, 1994[Abstract/Free Full Text].

41.   Meij, IC, Koenderink JB, van Bokhoven H, Assink KFH, Tiel Groenestege W, De Pont JJHHM, Bindels RJM, Monnens LAH, van den Heuvel LPWJ, and Knoers NVAM Dominant isolated renal magnesium loss is caused by misrouting of the Na+,K+-ATPase gamma -subunit. Nat Genet 26: 265-266, 2000[ISI][Medline].

42.   Mercer, RW, Biemesderfer D, Bliss DP, Jr, Collins JH, and Forbush B, III. Molecular cloning and immunological characterization of the gamma -polypeptide, a small protein associated with the Na,K-ATPase. J Cell Biol 121: 579-586, 1993[Abstract].

43.   Mimura, K, Matsui H, Takagi T, and Hayashi Y. Change in oligomeric structure of solubilized Na+/K+-ATPase induced by octaethylene glycol dodecyl ether, phosphatidylserine and ATP. Biochim Biophys Acta 1145: 63-74, 1993[ISI][Medline].

44.   Morrison, BW, Moorman JR, Kowdley GC, Kobayashi YM, Jones LR, and Leder P. Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes. J Biol Chem 270: 2176-2182, 1995[Abstract/Free Full Text].

45.   Munzer, JS, Daly SE, Jewell-Motz EA, Lingrel JB, and Blostein R. Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase. J Biol Chem 269: 16668-16676, 1994[Abstract/Free Full Text].

46.   Palmer, CJ, Scott D, and Jones LR. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem 266: 11126-11130, 1991[Abstract/Free Full Text].

47.   Periyasamy, SM, Huang WH, and Askari A. Subunit associations of (Na+ + K+)-dependent adenosine triphosphatase. Chemical cross-linking studies. J Biol Chem 258: 9878-9885, 1983[Abstract/Free Full Text].

48.   Pu, HX, Cluzeaud F, Goldshleger R, Karlish SJD, Farman N, and Blostein R. Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma -subunit. J Biol Chem 276: 20370-20378, 2001[Abstract/Free Full Text].

49.   Satoh, T, Cohen HT, and Katz AI. Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron. Am J Physiol Renal Fluid Electrolyte Physiol 265: F399-F405, 1993[Abstract/Free Full Text].

50.   Schagger, H, and von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379, 1987[ISI][Medline].

51.   Scheiner-Bobis, G, and Farley RA. Subunit requirements for expression of functional sodium pumps in yeast cells. Biochim Biophys Acta 1193: 226-234, 1994[ISI][Medline].

52.   Seri, I, Kone BC, Gullans SR, Aperia A, Brenner BM, and Ballermann BJ. Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells. Am J Physiol Renal Fluid Electrolyte Physiol 255: F666-F673, 1988[Abstract/Free Full Text].

53.   Shainskaya, A, Schneeberger A, Apell HJ, and Karlish SJD Entrance port for Na+ and K+ ions on Na+,K+-ATPase in the cytoplasmic loop between transmembrane segments M6 and M7 of the alpha -subunit. J Biol Chem 275: 2019-2028, 2000[Abstract/Free Full Text].

54.   Shi, H, Levy-Holzman R, Cluzeaud F, Farman N, and Garty H. Membrane topology and immunolocalization of CHIF in kidney and intestine. Am J Physiol Renal Physiol 280: F505-F512, 2001[Abstract/Free Full Text].

55.   Sweadner, KJ, and Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 68: 41-56, 2000[ISI][Medline].

56.   Sweadner, KJ, Rael E, Wetzel RK, and Arystarkhova E. Splice variants of the Na,K-ATPase gamma -subunit. In: Na/K-ATPase and Related ATPases, edited by Taniguchi K, and Kaya S.. Amsterdam: Elsevier, 2000, p. 543-546.

57.   Sweadner, KJ, Wetzel RK, and Arystarkhova E. Genomic organization of the human FXYD2 gene encoding the gamma -subunit of the Na,K-ATPase. Biochem Biophys Res Commun 279: 196-201, 2000[ISI][Medline].

58.   Taniguchi, K, Kaya S, Abe K, and Mardh S. The oligomeric nature of Na/K-transport ATPase. Biochem J 129: 335-342, 2001.

59.   Therien, AG, Goldshleger R, Karlish SJD, and Blostein R. Tissue-specific distribution and modulatory role of the gamma -subunit of the Na,K-ATPase. J Biol Chem 272: 32628-32624, 1997[Abstract/Free Full Text].

60.   Therien, AG, Karlish SJD, and Blostein R. Expression and functional role of the gamma -subunit of the Na,K-ATPase in mammalian cells. J Biol Chem 274: 12252-12256, 1999[Abstract/Free Full Text].

61.   Therien, AG, Nestor NB, Ball WJ, and Blostein R. Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase. J Biol Chem 271: 7104-7112, 1996[Abstract/Free Full Text].

62.   Wetzel, RK, and Sweadner KJ. Immunocytochemical localization of the Na+-K+-ATPase alpha - and gamma -subunits in the rat kidney. Am J Physiol Renal Physiol 281: F531-F545, 2001[Abstract/Free Full Text].

63.  Arystarkhova E, Donnet C, Asinovski NK, and Sweadner KJ. Differential regulation of renal Na,K-ATPase by splice variants of the gamma subunit. J Biol Chem 10.1074/jbc.M111552200, 2001.


Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F393-F407