Functional Role and Immunocytochemical Localization of the gamma a and gamma b Forms of the Na,K-ATPase gamma  Subunit*

Helen X. PuDagger , Francoise Cluzeaud§, Rivka Goldshleger, Steven J. D. Karlish, Nicolette Farman§||, and Rhoda BlosteinDagger ||**

From the Dagger  Department of Medicine, McGill University, Montreal, Quebec H3G1A4, Canada, § INSERM U478, Institut Federatif de Recherche 02, Faculte de Medecine X. Bichat, BP 416, 75870 Paris Cedex 18, France, and the  Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 761001, Israel

Received for publication, November 30, 2000, and in revised form, March 6, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The gamma  subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat gamma  is present primarily in the kidney as two main splice variants, gamma a and gamma b, which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between gamma a and gamma b. At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha 1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E1 left-right-arrow E2 conformational equilibrium and (ii) an increase in K+ antagonism of cytoplasmic Na+ activation. Antibodies against the C terminus common to both variants (anti-gamma ) abrogate the first effect but not the second. In contrast, gamma a and gamma b show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha  subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma  in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma a and gamma b. In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma  but at lower levels. Antibodies specific for gamma a and gamma b showed differences in their cortical location; gamma a is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma b but not gamma a is present in the cortical TAL only. Thus, the importance of gamma a and gamma b may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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A small membrane protein, gamma , first described over 20 years ago in purified kidney Na,K-ATPase preparations (1, 2) associates, in approximately equimolar amounts, with the alpha  and beta  subunits (3, 4). Molecular cloning of the gamma  subunits of rat, mouse, cow, and sheep indicated a molecular weight of ~6500 (5). Cloning and sequencing of the human (6) and Xenopus laevis (7) gamma  subunits have also been reported. Comparison of sequences shows ~75% homology among gamma  subunits of the aforementioned different species but is much higher (93%) for only mammalian sequences. Further structural analysis has shown that gamma  comprises a single transmembrane domain and has an N terminus-out, C terminus-in topology (7, 8). In addition, two major forms have been recently identified at the molecular level as described below.

On SDS-polyacrylamide gel electrophoresis, the gamma  subunit runs as a doublet (apparent molecular masses of ~8 and ~9 kDa) (5, 8), and a doublet is observed following expression in tissue culture cells (8, 9) and in in vitro expression in the presence (5) but not absence of pancreatic microsomes (5, 7). Recent mass spectrometry of the gamma  chains of rat kidney Na,K-ATPase showed that gamma a (upper band on SDS-polyacrylamide gel electrophoresis) has a mass of 7184.0 ± 1 Da (carbamidomethyl cysteine) (10), corresponding closely to that for the published sequence without the initiator methionine (11), while gamma b (lower band) has a mass of 7337.9 ± 1 Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma a, TELSANH, are replaced by Ac-MDRWYL in gamma b, but otherwise the two chains are identical. These sequences are identical to those obtained by searching the expressed sequence tag data base (12). Expression of gamma a or gamma b cDNAs in human embryonic kidney (HEK)1 as well as HeLa cells was analyzed by Western blotting using antiserum raised against a peptide representing the C-terminal 10 residues of the gamma  subunit. The results showed clearly that the major bands expressed correspond to gamma a or gamma b of the renal Na,K-ATPase. Additional minor bands seen after transfection, namely gamma a' in HEK and gamma b' in HeLa cells imply that these are cell-specific posttranslational modifications (10).

Although earlier studies showed that the gamma  subunit is co-expressed with alpha  and beta  ATPase subunits in kidney and not at the surface of Xenopus oocytes in the absence of alpha  and beta  subunits (7), Jones et al. (13) have reported expression of gamma  in the absence of the sodium pump on the apical surface of mouse blastocysts. In contrast with the ubiquitous localization of alpha  and beta  subunits, however, the gamma  subunit is expressed in a limited number of organs (8). Earlier studies showed identical expression patterns of alpha  and gamma  in renal proximal tubules and collecting ducts as well as co-immunoprecipitation of the gamma  subunit with both the alpha  and beta  subunits in kidney membranes (5).

The functional role of the gamma  subunit has only recently begun to be investigated. Although it was previously shown that the gamma  peptide is not necessary for function (4, 14, 15) and gamma  subunit mRNA could not be detected in many tissues in both mammals (5, 6) and amphibia (7), recent experiments have shown that gamma  has an important functional role in some systems. Thus, treatment of mouse blastocysts with gamma  subunit antisense oligodeoxynucleotide reduced the amount of expressed gamma  subunit and caused a reduction in ouabain-sensitive 86Rb+ transport as well as delayed blastocoel formation (13). A recent report describing a mutation in gamma  in a family with dominant renal hypomagnesemia suggests a role of gamma  in magnesium reabsorption (16).

Studies in our laboratory (8, 11) have shown that gamma  is a tissue-specific regulator of the Na,K-ATPase and that it causes an increase in the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum. The specific effect of anti-gamma on ATP affinity implies a specific structural interaction whereby the gamma  subunit counteracts short term changes in ATP concentration in kidney cells in which ATP utilization is high. A role of the gamma  subunit in interactions of the Na,K-ATPase with K+ was suggested on the basis of findings that the gamma  subunit is a component of the protein complex found in so-called "19-kDa membranes," the product of tryptic digestion following occlusion of K+ or Rb+ by the enzyme to form E2(K) (17), and partial protection by K+ ions against tryptic digestion of the gamma  subunit in renal microsomes (8). In addition, experiments on cRNA-injected Xenopus oocytes have shown that the gamma  subunit has an influence on the apparent affinity of the Na,K-ATPase for K+ in a complex Na+- and voltage-dependent fashion (7), although the interpretation of these results remains unclear. A recent report has attributed modulation of Na+ and K+ affinities to the gamma a subunit when expressed in kidney cells (9). The gamma  subunit has also been shown to induce ouabain-independent ion currents in injected Xenopus oocytes and 86Rb+ and 22Na+ influx in baculovirus-infected Sf-9 cells (18).

Based on the recent structural analysis of Kuster et al. (10) showing two main gamma  species in rat kidney (gamma a and gamma b), it is now clear that all aforementioned functional studies (7, 8, 9, 11, 14, 15) examined effects of only the gamma a variant, thus raising the critical issue of whether the two variants have distinct roles. The study described in this paper addresses two aspects of the role of gamma  in regulation of Na,K-ATPase. One set of experiments extends the functional studies and examines the question of whether, and to what extent, the two variants have distinct effects on Na,K-ATPase kinetic behavior. The other set of experiments concerns the localization of the two gamma  variants along the nephron.

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Microsome Preparation-- Rat kidney outer medulla microsomes (3-4 units/mg of protein) or partially purified Na,K-ATPase (10-15 units/mg of protein) were prepared as in Ref. 19.

Antibodies-- Two preparations of anti-gamma (C-terminal) were raised in rabbits. One is against both bands of the pig kidney gamma  subunit excised from a gel. The antibody was purified on an affinity column of the peptide KHRQVNEDEL corresponding to the last 10 residues of the gamma  subunit; thus, it is referred to as anti-gamma (C-terminal). The other is gamma C33, described in Ref. 8. It was raised against a synthetic peptide consisting of the last 10 residues and used for enzyme assays without further purification. Anti-gamma a and anti-gamma b antibodies were raised against N-terminal peptides TELSANHC and MDRWYLC, respectively, after coupling to keyhole limpet hemocyanin. Anti-gamma a was purified on an affinity column of TELSANHC. Attempts to affinity-purify anti-gamma b were unsuccessful. For affinity purification, peptides were coupled to Poros activated affinity chromatography columns (epoxide or amino), and the antibodies were purified on a BioCad Perfusion Chromatography apparatus. The antibody was eluted off the column in a solution of 0.2 M Tris-HCl, pH 2, neutralized immediately with Tris base, and diluted 1:1 with glycerol, and 0.02% sodium azide was added for preservation.

Immunocytochemical experiments were performed with affinity-purified anti-gamma (C-terminal) diluted 1:200 and anti-gamma a antibodies (1:100) and anti-gamma b antiserum (1:200). In addition, the following polyclonal antibodies (as described in Ref. 20) have been used for co-localization: (i) anti-aquaporin 2 (AQP2) antiserum, (1:100); (ii) anti-Tamm-Horsfall protein antibody (sheep anti-oromucoid from Biodesign International, Kennebunk, ME; 1:50); (iii) antibody against the alpha subunit of Na,K-ATPase (1:50).

Immunolocalization of the gamma  Subunit of Na,K-ATPase in the Kidney-- Rat kidneys were frozen in liquid N2. Immunolocalization was performed as previously described (20) by incubating cryostat sections with the different anti-gamma subunit antibodies (gamma -C-terminal, gamma a and gamma b) and with a secondary antibody (goat anti-rabbit Fab fraction, Jackson; 1:200) coupled to the fluorochrome CY3 (red fluorescence). For colocalization experiments (20), the CY3-labeled sections were incubated with antibodies against different protein markers (see above) and then overlaid with fluorescein isothiocyanate-labeled (green fluorescence) goat anti-rabbit IgG Fab fraction or donkey anti-sheep antibody. The specificity of the anti-gamma antibodies in immunofluorescence studies was established in competition experiments by including their respective peptides in the treatment of the kidney sections (data not shown).

Cloning and Transfection of Rat gamma a and gamma b cDNAs-- The cloning and transfection of rat gamma a and gamma b cDNAs were carried out exactly as described for wild-type HeLa cells (10), except that a stable cell line expressing rat alpha 1 was used (alpha 1-HeLa cells obtained as a gift from E. Jewell and J. B Lingrel; see Ref. 21). After transfection, the cells were cultured for 3 weeks in Dulbecco's modified Eagle's medium containing 10% newborn calf serum and selected by including 400 µg/ml hygromycin B and 1 µM ouabain (10) in the medium. Western blot analysis was carried out as described previously (8) except using antibodies against the C terminus (gamma C33) and against the N termini of gamma a and gamma b. Quantitative PhosphorImaging was carried out using a Storm PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Enzyme Assays and Kinetic Analysis of Data-- Membranes were prepared from HeLa cells as in Ref. 21. Unless indicated otherwise, Na,K-ATPase and Na-ATPase assays were carried out as described previously (11). Na-ATPase activity refers to activity measured at low (1 or 10 µM) ATP concentration, in the absence of K+. For experiments with HeLa cells expressing the ouabain-resistant rat alpha 1 catalytic subunit (alpha 1-HeLa), the cells are grown in 1 µM ouabain and assayed in medium containing a low (5 µM) ouabain concentration. Activities shown are the differences in ATP hydrolysis measured in the presence of low (5 µM) and high (5 mM) ouabain concentrations. For studies with wild-type HeLa cells, activities shown are differences measured in the absence and presence of 5 mM ouabain. For studies of sensitivity to vanadate, at each concentration of vanadate, Na-ATPase assays were carried out in sets of triplicates, without and with vanadate, with low and high ouabain added to one set of each (cf. Ref. 22). For practical purposes, either gamma a- or gamma b-transfected alpha 1-HeLa were compared, on the same day, with control alpha 1-HeLa membranes. Data for the vanadate sensitivity of Na-ATPase activity, expressed as a percentage of that obtained in the absence of vanadate, were analyzed by fitting the data to a one-site model using a nonlinear least squares fit (Kaleidagraph computer program) to a general logistic function as referred to previously (23).

Data for Na+ and K+ activation of Na,K-ATPase activity were expressed as percentages of Vmax using the Kaleidagraph computer program (Synergy Software) with the noninteractive model of cation binding described by Garay and Garrahan (24),
v=V/(1+K′<SUB><UP>cat</UP></SUB><UP>/</UP>[<UP>cat</UP>])<SUP>n</SUP> (Eq. 1)
where v represents the rate of the reaction, V represents the maximal rate, K'cat represents either K'Na or K'K (the apparent affinity for Na+ or K+, respectively), [cat] represents the concentration of cation (either Na+ or K+), and n represents the number of binding sites (either three in the case of the Na+ activation experiments or two in the case of K+). Values of Vmax and K'cat were obtained from this fitting procedure.

Evaluation of K+ antagonism of Na+ activation at cytoplasmic sites was determined by analyzing Na+ activation profiles as a function of K+ concentration. As in our previous study (26) and based on the Albers-Post model with the assumption that Na+ and K+ bind randomly at three equivalent (noninteractive) cytoplasmic sites, the data were analyzed using the relationship described by Garay and Garrahan (24),
v=V/(1+K<SUB><UP>Na</UP></SUB><UP>/</UP>[<UP>Na</UP>](1+[<UP>K</UP>]<UP>/</UP>K<SUB><UP>K</UP></SUB>)) (Eq. 2)
Where [Na] and [K] represent the cytoplasmic concentrations of Na+ and K+, respectively; v and V have their usual meaning; KNa is the affinity for Na+ binding at cytoplasmic activation sites in the absence of K+; and KK is the affinity for K+ acting as an antagonist of Na+ binding at cytoplasmic sites. This equation predicts a linear relationship between the apparent affinity constant for Na+, K'Na, and K+ concentration according to the following relationship.
K′<SUB><UP>Na</UP></SUB>=K<SUB><UP>Na</UP></SUB>(1+[<UP>K</UP>]/K<SUB><UP>K</UP></SUB>) (Eq. 3)

All experiments shown or described are representative of at least three similar experiments, except for experiments summarized in the inset of Fig. 1 and those shown in Fig. 6, in which cases the means of at least three independent experiments are shown. For the representative experiments shown, each data point is the mean ± S.D. of three replicate samples.

    RESULTS
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INTRODUCTION
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Functional Studies

The experiments described below were carried out to extend previous investigations of the functional role of gamma  and to compare the effects of the two variants, gamma a and gamma b. This was done primarily by comparing the kinetic behavior of gamma a- and gamma b-transfected cells with mock-transfected HeLa cells and, where indicated, by analyzing the effect of anti-gamma antibodies on the renal enzyme. Experiments were initially carried out with gamma -transfected wild-type HeLa cells and then repeated with gamma -transfected alpha 1-HeLa to assure that effects, or absence thereof, were not specific to the species of alpha , either rat or human. Similar findings were obtained with both cell lines. Only those from either wild-type or rat alpha 1-transfected HeLa cells are shown. Furthermore, we noted that selection and growth of alpha 1-HeLa cells in a low concentration of ouabain (cf. Ref. 21) down-regulates endogenous human alpha 1 such that expressed rat alpha 1 predominates (experiments not shown).

Expression in Cultured Cells

We showed recently that expression in cultured human cells (wild-type HeLa cells and HEK cells), of cDNA encoding the two individual gamma  variants revealed additional bands (see Fig. 5 of Ref. 10). That these additional bands, termed gamma a' and gamma b', are due to posttranslational modifications was evidenced by the cell-specific manner of their appearance following transfection into cultured cells. Thus, gamma a' appears primarily in HEK cells, and gamma b' appears primarily in HeLa cells. In the present study, identical results were obtained for gamma a and gamma b expressed in rat alpha 1-transfected HeLa cells (not shown). As in the case of wild-type HeLa, a conspicuous gamma b' is visible in gamma b-transfected alpha 1-HeLa, and only one band is visible in gamma a-transfected alpha 1-HeLa cells. Densitometry of the alpha  and gamma  subunits of membranes from clones of gamma a-HeLa, gamma a-alpha 1-HeLa, and gamma b-HeLa and gamma b-alpha 1-HeLa, in which expression is relatively high and used for functional analysis, showed that the gamma /alpha ratios are ~50% that of rat kidney (density of both gamma  bands).

Effects of gamma  on Apparent Affinity for ATP

Fig. 1 confirms the earlier finding that gamma a decreases K'ATP for ATP (low affinity binding) and shows further that a similar (~2-fold) decrease in K'ATP (low affinity binding) is effected by gamma b. Furthermore, this effect appears to be independent of the presence (HEK cells; Ref. 11) or absence (HeLa cells; this study) of gamma a'. It was also noted earlier that this effect of gamma  is abrogated by anti-gamma antibody as evidenced in the increase in K'ATP following preincubation of either gamma a-transfected HEK cells or the rat kidney enzyme with the antiserum gamma C33 raised against the last 10 residues of the C terminus. Similar effects of gamma a and gamma b were observed in other experiments (not shown) in which the cDNAs of these variants were transfected into alpha 1-HeLa cells. Although anti-gamma (C-terminal) decreases the Vmax of kidney enzyme (8), variation in specific activity among different membrane preparations from the HeLa cells (see the legend to Fig. 1) precluded the determination of gamma  effects on Vmax.


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Fig. 1.   Effects of gamma a and gamma b on ATP affinity. Membranes isolated from gamma a- and gamma b-transfected as well as control mock-transfected HeLa cells were assayed for Na,K-ATPase activity at varying ATP concentrations (50, 100, 200, 300, 500, and 1000 µM) in the presence of 100 mM NaCl, 10 mM KCl, and 40 mM choline chloride as described under "Experimental Procedures." Base-line values (5 mM ouabain added) were subtracted. Values ± S.D. shown are the differences in activities obtained after subtraction of the base-line values. Data were analyzed by the Michaelis-Menten formulation using the linear Lineweaver-Burk transformation. Inset, K'ATP values ± S.D shown are the averages of three or four separate experiments, each of which was carried out with membranes of either gamma a- or gamma b-transfected HeLa and control HeLa membranes. K'ATP values for both gamma a and gamma b compared with control are considered statistically significant (p < 0.01). Average Vmax values ± S.D. (nmol/mg/min) for control and gamma a- and gamma b-transfected cells were 254.5 ± 12.7, 168.5 ± 13.8, and 225.1 ± 10.1, respectively.

Effect of gamma  on the Steady-state Conformational Equilibrium: Studies with Vanadate

The question of whether the effect of gamma  on K'ATP is primarily on interaction of the enzyme with ATP and/or secondary to an alteration in the conformational equilibrium was tested by using inorganic orthovanadate as a probe of the E2 conformation. Accordingly, vanadate sensitivity of ATPase activity was tested under conditions in which its effect should not be secondary to an alteration in Kext interaction with the enzyme. This was done by measuring the steady-state hydrolysis of the Na-ATPase activity with ATP added at low concentration (1 µM) to assess turnover of the enzyme in the absence of K+ as in Ref. 22 with modifications described in the legend to Fig. 1. In one series of experiments, we tested the effect of anti-gamma (C-terminal) on the renal enzyme. As shown in Fig. 2A, the sensitivity to vanadate is increased; the I50 is decreased ~2-fold (see legend to Fig. 2). As predicted, the opposite effect was observed with the two gamma  variants. In the typical experiment shown for gamma a in Fig. 2B, it is evident that, compared with control (mock-transfected) alpha 1-HeLa cells (see "Experimental Procedures"), the sensitivity to vanadate is decreased. The I50 for vanadate was increased ~2-fold. Similar results were obtained for gamma b-transfected alpha 1-HeLa cells (not shown; p also <0.01). These results are consistent with the conclusion that both gamma a and gamma b shift the E1 left-right-arrow E2 equilibrium in favor of E1 form(s) as suggested in earlier studies (8, 11, 25). Those experiments showed that (i) inhibition of the kidney enzyme by anti-gamma is greatest at acidic pH (pH 6.2) and least at alkaline pH (pH 8.9), under which conditions the E2 (K) right-arrow E1 transition is either the major rate-limiting or a non-rate-limiting step of the reaction, respectively.


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Fig. 2.   Effects of gamma  on vanadate sensitivity of Na-ATPase of the rat alpha 1 pump. Assays of Na-ATPase were carried out at 1 µM ATP, 2 mM NaCl and 148 mM choline chloride. A, Effect of anti-gamma : comparison of control (nonimmune serum) and anti-gamma (gamma C33)-treated rat kidney membranes. From the least squares fits (see "Experimental Procedures"), I50 values ± S.E. are 15.4 ± 1.6 × 10-7 and 7.43 ± 0.9 × 10-7 M, respectively. In the absence of vanadate, activities were 34.3 ± 0.1 and 23.1 ± 0.1 nmol/mg/min, respectively. B, comparison of gamma a- and mock-transfected rat alpha 1-HeLa cells. I50 values ± S.E. are 5.80 ± 0.41 × 10-7 M and 2.87 ± 0.25 × 10-7 M, respectively. In the absence of vanadate, activities were 4.8 ± 0.02 and 5.9 ± 0.09 nmol/mg/min, respectively. At each vanadate concentration shown, the activity was measured in triplicate both in the presence of high (5 mM) and the presence of low (10 µM) ouabain concentrations. Values shown are differences ± S.D. Where not apparent, S.D. values are smaller than the symbols. (For both experiments, p < 0.01.)

Effects on Cation Interactions

K+ Interactions Relevant to the E2P right-arrow E2 (K) right-arrow E1 Pathway-- Treatment of the rat kidney enzyme with anti-gamma decreases the K'K for K+ activation ~25%, at least at suboptimal ATP concentration (25). Those results are consistent with the conclusion that effects on K'K are secondary to effects of gamma  on the E1/E2 conformational equilibrium. However, similar experiments aimed to show the presumably opposite effect (increase in K'K) of gamma a and gamma b on alpha 1-transfected HeLa cells were equivocal due to the large variances in small increases (<= 25%) in K'K values due, presumably, to much lower specific activity and higher nonspecific ATP hydrolysis of HeLa membranes. In other experiments (not shown) in which the ouabain-sensitive influx of 86Rb+(K+) was assayed as in Ref. 23, a significant difference in the apparent affinity for extracellular K+ between either gamma a- or gamma b-transfected and mock-transfected alpha 1-HeLa cells could not be detected (experiments not shown). Nonetheless, Fig. 3 shows clearly that when the ATP concentration was reduced to 10 µM so that the K+ deocclusion reaction becomes strongly rate-limiting, both gamma a and gamma b increased the extent of K+ activation of Na-ATPase of HeLa cells. Similar results were obtained with alpha 1-HeLa cells (experiments not shown). This result is consistent with the conclusion that both variants increase the rate of the rate-limiting E2(K) right-arrow E1 reaction, an effect abrogated by anti-gamma .


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Fig. 3.   Effects of gamma a and gamma b on K+ sensitivity of Na-ATPase at 10 µM ATP. Transfected HeLa cells were assayed at 10 µM ATP in the presence of 30 mM Na+ without and with varying concentrations of KCl as indicated, with choline chloride added so that the final chloride concentration was 40 mM. Base-line values (40 mM KCl) were subtracted. Values ± S.D. shown are the differences in activities obtained after subtraction of the base-line values measured in the absence of Na+ and presence of 40 mM KCl.

K+ Antagonism of Na+ Activation-- One of the notable differences between alpha 1beta 1 pumps of kidney compared with many other tissues is the notably lower apparent affinity for Na+ (increased K'Na) in kidney, which is readily seen at high K+ concentrations (>= 20 mM; Ref. 26). In that study, we showed that this increase in K'Na is accounted for by the higher apparent affinity for K+ (KK) as an antagonist at cytoplasmic Na+ activation sites rather than a difference in Na+ affinity, per se. At face value, the notion that this effect is due to interaction with gamma  seems unlikely, since this K+/Na+ antagonism is even more dramatic in heart tissue (26) that is devoid of gamma  (8). Nevertheless, to determine whether the gamma  subunit is relevant to this phenomenon in the kidney, the following experiments were carried out: (i) an analysis of the sensitivity of the enzyme to inhibition by K+ at low (5 mM) Na+ concentration (Fig. 4) and (ii) an extensive kinetic analysis to determine K'Na as a function of K+ concentration. The results for transfected alpha 1-HeLa cells are shown in Figs. 5 and 6 and Table I. Similar results were obtained with transfected wild-type HeLa cells (not shown). Fig. 4 shows that, compared with the control, cells transfected with either gamma a or gamma b are more inhibited by K+ at low Na+ concentration compared with mock-transfected control cells. Since stabilization of E1 by the gamma  subunit, described above, should itself have the effect of lowering the apparent affinity for K+ ions as antagonists of Na+ ions at cytoplasmic sites, the result in Fig. 4 shows that there must be an additional effect of gamma  on the cation sites.


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Fig. 4.   Effects of gamma a and gamma b on K+ sensitivity of Na,K-ATPase measured at 5 mM Na+. Membranes isolated from gamma a- and gamma b- as well as mock-transfected rat alpha 1-HeLa cells were assayed at varying concentrations of KCl in the presence of 5 mM NaCl at near optimal (1 mM) ATP. Data are expressed as percentages of the activity measured at 2 mM KCl. *, Significantly different versus mock-transfected (p < 0.01).


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Fig. 5.   Effects of gamma a and gamma b on Na+ activation of Na,K-ATPase at 20 and 100 mM K+. Membranes were isolated from gamma a- and gamma b-transfected as well as mock-transfected rat alpha 1-HeLa cells and assayed as described under "Experimental Procedures" at either 20 mM KCl (left panel) or 100 mM KCl (right panel) at varying concentrations of NaCl. ×---×, control, mock-transfected alpha 1-HeLa; open circle ------open circle , gamma a-alpha 1-HeLa; ------, gamma b-alpha 1-HeLa. Data points shown are means ± S.D. (triplicate determinations) of representative experiments, expressed as percentage of Vmax (see "Experimental Procedures"). The data were fitted to Equation 1. Values of K'Na for gamma a- and gamma b-transfected and control rat alpha 1-HeLa cells (mean ± S.D. of at least three independent experiments) were 2.3 ± 0.1, 2.3 ± 0.2, and 1.8 ± 0.1, respectively, at 20 mM KCl and 8.1 ± 0.3, 8.3 ± 0.1, and 5.3 ± 0.3, respectively, at 100 mM KCl. Differences between either gamma a- or gamma b-transfected and control cells are significant, i.e. p < 0.05 at 20 mM KCl and p < 0.01 at 100 mM KCl.


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Fig. 6.   Effects of gamma a and gamma b on K+/Na+ antagonism of rat alpha 1-HeLa cells. Lines shown are least squares fits of the values of K'Na determined at varying Na+ concentration as indicated in Fig. 5, with the K+ concentration kept constant at either 5, 10, 20, 50, or 100 mM. For each concentration of K+, the three types of membranes were assayed concurrently in the same experiment. Each point shown is the mean ± S.D. from at least three separate experiments. KNa and KNa/KK obtained from the least squares fits of the data to Equation 3 and values of KK calculated from the ratio of KNa/(KNa/KK) are shown in Table I. ×---×, control, mock-transfected alpha 1-HeLa; open circle ------open circle , gamma a-alpha 1-HeLa; ------, gamma b-alpha 1-HeLa.

                              
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Table I
Effects of gamma a and gamma b on affinities for Na+ and K+ binding to Na+ activation sites of the Na,K-ATPase

Fig. 5 is a kinetic analysis of the effect of varying Na+ as a function of K+. The results shown are representative comparisons of gamma a- and gamma b-transfected and control mock-transfected alpha 1-HeLa activity profiles determined at 20 and 100 mM K+. The data were analyzed by fitting the Na+ activation curves to a noncooperative model of Na+ activation (cf. Ref. 24). As indicated in the legend to Fig. 5, both gamma a and gamma b decrease the apparent Na+ affinity, and the decrease is greater at the higher K+ concentration. Fig. 6 shows the results of a large series of similar experiments carried out for both variants at varying K+ concentration. The plots show the relationship between K'Na and K+ concentration for control (mock-transfected) and gamma a- and gamma b-transfected alpha 1-HeLa cells. As described under "Experimental Procedures," the data were analyzed according to Equation 3, which predicts a linear relationship between K'Na and cytoplasmic [K+] whereby KNa and KK are the affinity constants for Na+ (extrapolated to [K+] = 0) and for K+ at cytoplasmic site(s), respectively. At each K+ concentration, the data point shown is the mean of at least three separate determinations of K'Na. The linearity of the plots indicates that this relationship provides a valid basis for using our data to estimate KNa and KNa/KK. As shown by the data summarized in Table I, both gamma a and gamma b have similar effects. Both increase KNa/KK with no significant effect on KNa, consistent with the conclusion that the effect of both variants is mainly due to a decrease in KK, i.e. an increase in the affinity for K+ acting as an antagonist of Na+ binding at cytoplasmic sites. It is noteworthy that (i) the kinetic constants for control alpha 1-HeLa taken from Fig. 6 are virtually identical to those reported earlier (26), indicating the high reproducibility of the assays, (ii) the same effects of the two variants were observed in a similar series of experiments carried out with wild-type HeLa cells (experiments not shown), and (iii) the magnitude of the change in KNa/KK (~50% increase) effected by gamma  is notably similar to the higher (50%) KNa/KK of kidney compared with alpha 1-HeLa cells reported earlier (26).

As summarized in Table II, antisera raised against the cytoplasmic C- and extracellular N-terminal regions of gamma  failed to abrogate the effect of gamma  on K+/Na+ antagonism. Thus, treatment of the rat kidney enzyme with antiserum gamma C33 raised against the C terminus failed to change the magnitude of K+ inhibition noted at low Na+ concentration (75% inhibition in both the presence and absence of 100 mM K+); the same holds true of anti-gamma a raised against the N terminus of gamma a (see below).

                              
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Table II
Effects of anti-gamma antibodies on kinetic behavior of the kidney enzyme

Are Interactions Involving the N Terminus Relevant to the Effects of gamma ?

This issue was addressed using anti-gamma a antiserum raised against the extracellular N terminus. As summarized in Table II, anti-gamma a failed to affect the activity at either limiting or nonlimiting concentrations of either ATP, Na+, or K+ or affect the concentration dependence of inhibition by ouabain (experiments not shown).

Immunolocalization

The intrarenal pattern of expression of the gamma  subunit of Na,K-ATPase was examined by immunofluorescence in the renal cortex and outer and inner medulla (Fig. 7). The anti-gamma (C-terminal) antibody alone (CY3, red fluorescence), decorates several tubules (Fig. 7, A-C). All regions show staining, but it is particularly strong in outer medulla. Double immunofluorescence experiments were performed using antibodies against the alpha  subunit of Na,K-ATPase (Fig. 7, D-F), against AQP2 (apical membrane of principal cells of the collecting duct) in G-I and against Tamm-Horsfall protein (apical membrane of the thick ascending limb of Henle's loop) in J-L, all evidenced by their fluorescein isothiocyanate-green fluorescence. Colocalization with the gamma  subunit appears in yellow. The alpha  subunit of Na,K-ATPase (Fig. 7, D-F) is apparent in all tubular segments, as expected: proximal tubules, loop of Henle, and distal nephron. In outer and inner medulla, there is extensive overlap of alpha  and gamma  subunits (E and F), but in the latter some nephron segments express alpha  but no gamma  subnit (F). In cortex, some cells (Fig. 7D) clearly show colocalization (arrow). Since they lie in close vicinity to the glomerulus, they may be macula densa cells; this is documented further in Fig. 8. Colocalization of AQP2 and the gamma  subunit (Fig. 7, G-I) was apparent in the cortex only, indicating that the collecting duct, in its cortical portion only, expresses the gamma  subunit. Tamm-Horsfall protein (Fig. 7, J-L) colocalizes with the gamma  subunit in medullary TAL (mTAL) (K, in yellow), with respective apical and basolateral expression. Thus, these experiments show that the gamma  subunit of Na,K-ATPase has predominant expression, at the protein level, in the mTAL, with few positive cortical TAL (cTAL), and faint labeling of proximal tubules and cortical collecting ducts. Its expression is more restricted than that of the alpha  subunit of the sodium pump, indicating that both subunits are not systematically coexpressed.


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Fig. 7.   Detection of the gamma  subunit of Na,K-ATPase within the kidney. Cryostat sections from rat kidney cortex (A, D, G, and J), outer medulla (B, E, H, and K), or the junction of outer and inner medulla (C, F, I, and L) were incubated with the affinity-purified anti-gamma raised against the whole gamma  subunit of Na,K-ATPase (in red), either alone (A-C) or in the presence of the following antibodies: against the alpha  subunit of Na,K-ATPase (1:50) (D-F), against AQP2 (1:100; to identify collecting ducts) (G-I), or against Tamm-Horsfall protein (1/50; to identify the thick ascending limb of Henle's loop) (J-L), all appearing in green. Colocalization generates yellow fluorescence. g, glomerulus; p, proximal tubule; cd, collecting duct; asterisk, thick ascending limb of Henle's loop. The arrow in D shows macula densa cells. Bar, 50 µm.


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Fig. 8.   Expression of the gamma a and gamma b forms of Na,K-ATPase within the kidney. Cryostat sections from cortex (A-H) and medulla (I-P) were used; this kidney zone is at the junction of most inner part of outer medulla (upper part of each image) and of the inner medulla or papilla (lower part of each image). Sections were incubated with antibodies raised against the gamma a (A-D and I-L) or the gamma b (E-H and M-P) subunit (appearing in red). These antibodies were used either alone (A, E, I, and M) or in the presence of antibodies (in green) against the alpha  subunit of Na,K-ATPase (B, F, J, and N), against AQP2 (to identify the apical membrane of collecting duct principal cells) (C, G, K, and O), or against Tamm-Horsfall protein (THP; to identify apical membrane of thick ascending limbs of Henle's loop) (D, H, L, and P). Colocalization generates yellow fluorescence. Conditions were as follows: gamma a alone (A and I); gamma b alone (E and M); gamma a + alpha 1 Na,K-ATPase (B and J); gamma a + AQP2 (C and K); gamma a + THP (D and L); gamma b + alpha 1 Na,K-ATPase (F and N); gamma b + AQP2 (G and O); gamma b + THP (H and P). g, glomerulus; p, proximal tubule; cd, cortical collecting duct; mcd, medullary and papillary collecting ducts; asterisk, Henle's loop. The arrow in B shows macula densa cells appearing at the very end of Henle's loop. Bar, 50 µm except in C and G (20 µ m).

The recent identification of a splice variant of the gamma  subunit of Na,K-ATPase leads to the notion that the two forms, gamma a and gamma b, may have different cellular expression and function. In order to establish whether gamma a and gamma b have distinct localizations, immunofluorescence experiments were performed using antibodies specific for each gamma  variant, namely anti-TELSANH, and anti-MDRWYL representing the two extracellular N-terminal sequences that differ in gamma a and gamma b, respectively. Fig. 8 documents the expression of gamma a and gamma b in the cortex (A-H) and medulla (I-P). These antibodies were used either alone (A, E, I, and M, in red) or together with antibodies (in green) raised against either the alpha  subunit, anti-AQP2, or anti-Tamm-Horsfall protein, as indicated in the legend to Fig. 8. In the cortex, gamma a is expressed in most cortical tubules (proximal tubules, cortical collecting ducts) together with the alpha  subunit of the pump (except in cTAL, appearing in green), while colocalization with gamma b is detectable only in few tubules (i.e. cTAL) in yellow. In particular, the gamma a form is clearly present together with the alpha  subunit in the macula densa (MD) cells (Fig. 8B, arrow) (i.e. at the very end of the cTAL (labeled by an asterisk)); both subunits colocalize in the MD basolateral membrane lining the glomerulus. In contrast, the gamma b form was never found in MD cells. In the cortical collecting duct (labeled cd), as identified by its apical expression of AQP2 in principal cells (in green in Fig. 8C), the gamma a variant is in the opposite membrane (i.e. in the basolateral membrane of principal cells), while these cells have only background fluorescence for gamma b (Fig. 8G); proximal tubules (labeled p) express both gamma  forms at low levels. Of particular interest is the observation that the cortical portion of the thick ascending limb of the loop of Henle (cTAL) expresses only gamma b (Fig. 8H), not gamma a (Fig. 8D), as shown by selective colocalization of gamma b with Tamm-Horsfall protein. Panels I-P illustrate the immunofluorescence for similar immunocytochemistry carried out with sections at the junction of the outer medulla and inner medulla. Antibodies specific for gamma a and gamma b show that each has a restricted pattern of expression. This expression is superimposed on that of the alpha  subunit of the Na,K-ATPase in some (mTAL), but not all tubules (Fig. 8, J and N), since medullary collecting tubules (mcd) are positive only for the alpha  subunit, while others (mTAL; labeled by an asterisk) express alpha  together with either gamma a (Fig. 8J) or gamma b (Fig. 8N) although with lower intensity. The absence of gamma a and gamma b in the medullary and papillary collecting duct is shown by the distinct localization of AQP2 and either gamma a or gamma b (Fig. 8, K and O). All along the medullary thick ascending loops (identified with anti-Tamm-Horsfall protein antibody) there is evidence of both gamma a (Fig. 8L) and gamma b (Fig. 8P).

It is relevant that other studies2 showed that (i) anti-alpha immunoprecipitated both gamma a and gamma b from C12E8-solublized membranes and (ii) either anti-gamma a or anti-gamma (C-terminal) immunoprecipitated the alpha  subunit. However, anti-gamma a did not immunoprecipitate gamma b. This result suggests that alpha /beta subunits are present either as the complex alpha /beta /gamma a or alpha /beta /gamma b, but not alpha /beta /gamma a/gamma b, but does not, of course, exclude the possibility of alpha /beta subunits without associated gamma  subunits.

On the whole, these results indicate that the gamma  subunit of Na,K-ATPase has a restricted pattern of expression along the nephron, as compared with the alpha  subunit, with prevalent expression in the mTAL, and, at lower levels, in the proximal tubule, and the collecting duct in its cortical part only. The gamma a form is in the basolateral membrane of MD cells and of cortical collecting duct principal cells as well as in the medullary portion of the thick ascending limb of Henle's loop. The gamma b form is selectively expressed all along the thick ascending limb of Henle's loop (Table III).

                              
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Table III
Expression of the gamma a and gamma b forms of the gamma  subunit along the nephron


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of gamma a and gamma b on Catalytic Functions-- In earlier studies we showed that anti-gamma antiserum raised against the C terminus of gamma  inhibits Na,K-ATPase of the renal enzyme but not of tissues that do not express gamma  (8). Further analysis showed that anti-gamma decreases the apparent affinity for ATP probably by stabilizing the E2 form(s) of the enzyme. Thus, the pH dependence of the anti-gamma -mediated inhibition of activity is consistent with a role of anti-gamma in shifting the equilibrium of the K+ deocclusion reaction (E2(K) left-right-arrow E1) toward E2(K). It was hypothesized that anti-gamma mediates its effects by disrupting interactions between the Na,K-ATPase complex and the gamma  subunit, such that the role of the gamma  subunit is to shift the equilibrium toward E1. By transfecting one of the two gamma  variants (gamma a) into HEK cells, it was then shown that this is indeed the case, at least for gamma a (11). The studies described in this paper show that the same holds true for the gamma b variant. Thus, as we show here, both variants decrease K'ATP to an equal extent, and furthermore this effect is abrogated by anti-gamma (C-terminal) for both variants (summarized in Table II). The present observation that anti-gamma treatment of the renal enzyme increases sensitivity to vanadate and, conversely, that gamma  transfection decreases sensitivity to vanadate, strongly support the conclusion that the change in K'ATP reflects primarily an effect on the E1/E2 conformational equilibrium. The result in Fig. 3 is consistent with the assumption that gamma a and gamma b stabilize E1 by accelerating the rate of the conformational transition E2(K) right-arrow E1.

The other new finding concerning the function of gamma  is that it has not one but at least two distinct effects on the catalytic function of the Na,K-ATPase. In addition to an increase in apparent ATP affinity, gamma  induced an increase in K+/Na+ antagonism, which results in a reduction in the effective affinity of Na+ ions for activating Na,K-ATPase. As mentioned above, the mechanism cannot be explained on the basis of stabilization of E1 and thus implies an additional effect of gamma  on intrinsic binding of K+ ions at cytoplasmic sites (perhaps on one of the two K+ sites). This effect of gamma  on K+/Na+ antagonism is seen equally with both gamma  variants, and, interestingly, this function of gamma  is not altered by antibodies raised against either the C terminus or N terminus (summarized in Table II). Overall, it is evident from this study that both gamma  variants alter the kinetics similarly, with no evidence of a significant difference between the two on catalytic function. It may also be noted that the functional effects do not depend on tissue-specific posttranslational modifications of the gamma  subunit, although such modifications can be observed in HeLa cells (gamma b') or HEK cells (gamma a'). Expression of gamma a in NRK-52E kidney cells has been reported to modulate (decrease) Na+ and K+ affinities (9). Although the effect on K'Na appears similar to that described here for gamma a, there are puzzling experimental differences. First, the level of gamma  expression was much lower in their experiments (15-20%) than in the present ones carried out with the HeLa transfectants (>= 50%) despite the similar functional effect. Second, the increase in K'Na was only observed in gamma a-transfected NRK-52E cells expressing a doublet, and not in clones expressing a single species the identity of which, in light of later studies (10), is not clear. These issues are difficult to reconcile with the present work with gamma a-transfected HeLa cells that express only a single gamma a species. As far as K'K is concerned, we could not detect a significant difference either with membrane fragments (ATPase assays at high ATP concentrations) or in 86Rb+(K+) influx studies in ATP-replete (millimolar ATP) cells, despite the higher level of expression in the HeLa cells. The reason for these discrepancies remains enigmatic.

The recognition that the gamma  subunit induces at least two functional effects, only one of which is abrogated by anti-gamma (C-terminal), suggests that there must be more than one region of interaction between the gamma  and alpha  subunits on which the functional sites reside. Presumably, the effect on the E1/E2 equilibrium and apparent ATP affinity involves the C-terminal sequence KHRQVNEDEL, and the K+/Na+ antagonism is mediated by other sequences in the molecule. Our observations that antibodies raised against the N-terminal sequence of gamma a, TELSANH, do not affect any kinetic function (summarized in Table II) imply that this sequence is not responsible for functional interactions. (Antibodies raised against the N terminus of gamma b are reactive only with SDS-denatured enzyme, which precludes meaningful interpretation of its failure to alter the effects of gamma b on alpha 1beta 1 pumps.) Nevertheless, the distinctness of the two effects is underscored by the observation (26) that polyethylene glycol-mediated fusion of kidney pumps into cells devoid of pumps (dog erythrocytes) abrogates the kidney-specific increase in K+/Na+ antagonism but not anti-gamma -mediated inhibition of overall activity. Definition of the regions of interaction of gamma  and alpha  subunits is a question to be addressed in the future.

Physiological Role-- The physiological significance of the gamma  subunit could be that it provides a self-regulatory mechanism for maintaining the steady-state activity of the pump in the kidney. This notion is underscored by its abundance in mTAL (see below), suggesting that its functional effects are tailored to meet the requirement of Na+ and K+ homeostasis in the prevailing environmental conditions and in particular by the observations of the dual effects on the kinetic properties, the one on K'ATP and the other on KK, the affinity of the pump for K+ acting as an antagonist of cytoplasmic Na+. The effect of gamma  on K'ATP was discussed recently in terms of its importance in maintaining pump activity under putative anoxic parts of the medulla, i.e. to increase ATP utilization and maintain optimally high intracellular K+ and low Na+ under energy-compromised conditions as discussed previously (11, 25, 27). Such a regulator of K'ATP should alter the pump's affinity for the nucleotide only moderately, for an excessive increase would affect even greater decreases in ATP concentration, thus leading to compromised cell viability. mTAL is characterized by a rapid transcellular Na+ flux, and the cellular Na+ concentration should reflect the balance of rates of passive Na+ entry and active Na+ efflux. The ability of gamma  to increase K+/Na+ antagonism at the cytoplasmic surface as shown in this study may provide a means of acute regulation of the steady-state Na+ concentration. A lowered effective cytoplasmic Na+ affinity for activating the pump, due to a regulatory interaction, may be tailored to fit cells in which the steady-state Na+ concentration is higher than in cells that lack the regulator, but that, nevertheless, must respond to changes in Na+ entry. Thus, the optimal affinity for cytoplasmic Na+ ions should be one at which there is plenty of reserve capacity for responding to changes in cell Na+ at the prevailing set point of Na+ concentration. The recent report of a putative dominant-negative mutation (G41R) in the gamma  subunit of the Na,K-ATPase may be relevant to a role of gamma  in maintaining intracellular Mg2+ secondary to elevating intracellular Na+. Such a relationship between intracellular Na+ and Mg2+ was seen not only in sublingual mucous acini (28), but also in renal tubular cells.3

Localization of gamma -- In this study, we have used immunocytochemistry to identify the tubular cells that express the gamma  subunit of the sodium pump in cortex and outer and inner medulla and to define the cellular localization of gamma . Interestingly, antibodies specific for the gamma a or gamma b forms of the gamma  subunit of Na,K-ATPase revealed clear differences in their expression (Table III). Both are in the mTAL (which reabsorbs sodium chloride at high rates) and to a lesser extent in the proximal tubules. The gamma a variant appears specific for cells in the region of the MD (a sensor of tubular sodium chloride) and for principal cells of the cortical collecting duct, but not cTAL, while the gamma b form is present in the cTAL and absent from MD and cortical collecting duct. Altogether, these findings extend the immunocytochemical observations of Arystarkova et al. (9) both spatially (providing information about gamma  expression in medulla and papilla, in addition to cortex, with identification of tubular segments) and in terms of specific expression of each gamma  form in tubules. They also show that both forms of the gamma  subunit are in the basolateral membrane, i.e. in close vicinity to the pump.

The gamma  subunits of Na,K-ATPase share homologies with other small molecules, such as CHIF (channel-inducing factor), phospholemman, and phospholamban, which are thought to be regulators of ion transporters or channels. Among these, CHIF also has a restricted pattern of expression, in the surface cells of the distal colon epithelium and in the terminal portions of the nephron (20, 29). More specifically, CHIF is found to some extent in the cortical collecting duct but essentially in its medullary and papillary portions. As for the gamma  subunit of Na,K-ATPase, CHIF is located essentially at the basolateral membrane. A specific interaction between CHIF and Na,K-ATPase in colon membranes has been demonstrated recently (30).

Although we cannot absolutely exclude the possibility of different functional effects of gamma a and gamma b, the lack of a notable difference between gamma a and gamma b with respect to their effects on pump kinetics may not be surprising. The two variants differ with respect to only the six or seven residues at the extracellular amino terminus. Accordingly, the two variants may influence differentially such properties as membrane targeting, pump turnover, or basolateral signaling and affect the rate of active Na+ and K+ transport by altering the density of pumps in the basolateral membrane. It is also plausible that each of these variants may influence differentially, in a cell-specific manner, some interactions of the pump with the extracellular matrix. Of particular relevance is the proposal that extracellular hensin influences directly polarity of collecting duct intercalated cells (31).

    ACKNOWLEDGEMENT

The excellent technical assistance of Rosemarie Scanzano and Ania Wilczynska is gratefully acknowledged. We thank Dr. J. B Lingrel for the rat alpha 1-transfected HeLa cells.

    FOOTNOTES

* This work was supported by grants from the Canadian Institutes for Health Research (to R. B.), the Kidney Foundation of Canada (to R. B.), INSERM (to N. F.) and the Weizmann Institute Renal Research Fund (to S. J. D. K.).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.

|| The last two authors contributed equally to this work.

** To whom correspondence should be addressed: Montreal General Hospital, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. Tel.: 514-937-6011 (ext. 4501); Fax: 514-934-8332; E-mail: Rhoda. Blostein{at}mcgill.ca.

Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M010836200

2 S. J. D. Karlish and R. Goldshleger, unpublished results.

3 G. Quamme, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: HEK, human embryonic kidney; TAL, thick ascending limb of Henle's loop; cTAL and mTAL, cortical and medullary portions, respectively, of the thick ascending limb of Henle's loop; MD, macula densa; AQP2, aquaporin 2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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