Modulation of Na,K-ATPase by the γ Subunit

The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the γ subunit, has been shown to decrease the apparent Na+ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of γ mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of γ effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing αβ pumps modulates Na+ affinity in a manner similar to transfected full-length γ subunit. Using mutated γ peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system.

The Na,K-ATPase or sodium pump is an integral membrane protein found in the cells of virtually all higher eukaryotes and is one of the most important systems involved in cellular energy transduction (1,2). It catalyzes the electrogenic exchange of three intracellular Na ϩ for two extracellular K ϩ ions energized by the hydrolysis of one molecule of ATP. The transporter plays a major role in ion homeostasis, and, in epithelia, the sodium gradient created by the pump also plays an important role in secondary active transport mechanisms that are necessary for Na ϩ -dependent reabsorption of a variety of solutes including sugars and amino acids.
There is an increasing body of evidence that members of a family of membrane proteins, the so-called FXYD family (3), associate with and modulate the kinetic behavior of the sodium pump (for a recent overview, see Ref 4). Members of this family of proteins are small, single transmembrane (TM) 1 proteins characterized by an N-terminal PFXYD motif that remains invariant in all mammals. There are at least seven known family members, of which several appear to modulate the kinetic behavior of the pump in a tissue-specific manner (5)(6)(7)(8). To date, the ␥ "subunit" of the renal Na,K-ATPase is the best characterized member (reviewed in Refs. 9 and 10). Gamma, or FXYD2, exists as two main splice variants, ␥a and ␥b, with distinct as well as overlapping localization along the nephron (11). Mass spectrometry of ␥a and ␥b indicate that they differ only in the N terminus; in rat ␥a, TELSANH is replaced by Ac-MDRWYL in ␥b (12). Previous studies using membrane fragments isolated from ␥-transfected rat ␣1-HeLa cells have shown that ␥ serves at least two distinct functions in regulating the pump, and the effects are similar for both variants (11,13). These distinct effects of ␥ include the following: (i) an increase in the apparent affinity for ATP; and (ii) an increase in K ϩ /Na ϩ competition at cytoplasmic Na ϩ activation sites, as seen by an increase in KЈ Na at high [K ϩ ]. Previous studies have suggested that the effect on ATP affinity is mediated by the cytoplasmic C-terminal domain of the protein (5,14), whereas effects on K ϩ /Na ϩ antagonism may be associated with the TM domain (15).
In this study, we have used a novel strategy to assess directly the functional effects of the TM region of FXYD2 whereby peptides corresponding to the TM domain of ␥ are added directly to membranes derived from cells devoid of the protein. In addition, we describe a mutagenesis approach to structure/ function analysis of ␥ using both transfected cells and peptides. A residue of particular interest is Gly-41, because it is replaced by Arg (G41R) in a familial form of renal magnesium wasting (16) and is invariant throughout the FXYD family. The other residue examined is Gly-35 because of its location at the alternate face of the membrane relative to Gly-41 (17). Effects of peptides with either of these Gly residues replaced by Arg or Leu are compared with those of the full-length transfected ␥ proteins (wild-type or mutants of the ␥b variant) expressed in cultured rat ␣1-transfected HeLa cells. This approach allows for assessment of the role of the TM region alone and provides an opportunity to distinguish the effects of mutations on biogenesis and routing to the plasma membrane from the direct effects on association and kinetic modulation of ␣␤ pumps.

EXPERIMENTAL PROCEDURES
Mutagenesis, Transfection, Tissue Culture, and Membrane Preparations-Point mutations were introduced into ␥b cDNA subcloned into * This work was supported, in part, by grants from the Canadian Institutes of Health Research (CIHR) and the Kidney Foundation of Canada (to R. B.), and CIHR and the Natural Sciences and Engineering Research Council of Canada (NSERC) (to C. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work. ** To whom correspondence should be addressed. E-mail: rhoda. blostein@mcgill.ca the pIRES expression vector and then transfected into HeLa cells stably expressing the rat ␣1 subunit of the Na,K-ATPase (␣1 HeLa cells were kindly provided by Dr. J. B. Lingrel) as described previously (14). Unless indicated otherwise, cellular membranes from the stably transfected cells were prepared as described by Jewell and Lingrel (18).
Polyacrylamide Gel Electrophoresis and Western Blotting-SDS-PAGE and Western blotting were carried out as described previously (11), and, following transfer to polyvinylidene difluoride membranes, the lower half was analyzed with a polyclonal ␥ antibody (␥C32 raised against the C-terminal KHRQVNEDEL peptide and essentially the same as ␥C33 described previously in Ref. 19), and the upper half was analyzed with anti-␣1 subunit A277 (Sigma).
Co-immunoprecipitation-The method used is a modification of Garty et al. (20). Briefly, 250 g of membranes, prepared from ␥b(WT)and ␥b(mutant)-transfected cells as described previously (18), were resuspended and incubated at room temperature for 30 min in immunoprecipitation buffer containing 50 mM imidazole, pH 7.5, 1 mM EDTA, 10 mM RbCl, and 5 mM ouabain in a final volume of 250 l. Polyoxyethylene 10 lauryl ether (C 12 E 10 ) (250 l of a 2 mg/ml solution in water) was then added (final concentration, 1 mg/ml), and the suspension was incubated for 30 min at 4°C with end-to-end rotation. Following centrifugation at 4°C for 30 min at 16,000 ϫ g, the supernatant was added to 50 l of bovine serum albumin (1 mg/ml)-treated protein A-Sepharose beads and incubated for 30 min at 4°C, and the beads were then centrifuged to remove non-specifically bound proteins. The supernatant was removed and a 10-l aliquot was taken for SDS-PAGE. To another 450-l aliquot, 90 l of RbCl (118 mM final concentration) and 30 l of ␥C32 anti-serum (stored at Ϫ20°C in 50% glycerol) were added, and the suspension was further incubated for 4 h at 4°C with end-to-end rotation. The suspension was then mixed with protein A-Sepharose beads (100 l) and incubated overnight at 4°C. Beads were washed six times with immunoprecipitation buffer without ouabain, and the immunoprecipitated proteins retained by the beads were eluted with 100 l of SDS-PAGE sample buffer containing 5% ␤-mercaptoethanol at 37°C for 30 min. A 20-l aliquot of the eluate and the 10-l sample removed before addition of antiserum were resolved by SDS-PAGE followed by Western blotting.
Cell Surface Biotinylation-Transfected ␣1-HeLa cells were grown to Ϸ80% confluence in 6-well plates. The surface biotinylation is a modification of Stephan et al. (21) Briefly, the cell surface biotinylation reaction was carried out on ice for one 20-min period using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) (Pierce). After biotinylation, each well was rinsed briefly twice with a phosphate-buffered saline (PBS), 0.1 mM CaCl 2 and 1 mM MgCl 2 (CM) solution containing 100 mM glycine and then treated with the same solution for 30 min to ensure complete quenching of unreacted sulfo-NHS-SS-biotin. The cells were then lysed for 45 min with 500 l of L1 buffer (1% Triton X-100/0.1% SDS in 150 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.5, containing 10 g/ml each leupeptin and pepstatin and 200 M phenylmethylsulfonyl fluoride). Each well was then scraped, and the sample was collected and centrifuged at 18,000 ϫ g for 10 min. One 20-l aliquot of supernatant (total fraction, T) was taken for protein determination (Lowry method) and another (5 l), for Western blotting. The biotinylated surface-exposed proteins were isolated by incubating a 150-g (Ϸ85 l) fraction in L2 buffer (L1 buffer without SDS; final volume, 500 l) with 100 l of streptavidin-agarose beads (Pierce) overnight at 4°C with gentle rotation. The samples were then centrifuged to separate beads and supernatant (unbound fraction, U), and an aliquot of U (15 l Х 2.5 l of T) was taken for Western blotting. The beads were washed three times with L2 buffer, twice with high salt L2 buffer (L2 buffer with 500 mM NaCl and 0.1% Triton X-100), and then once with 50 mM Tris-HCl, pH 7.5. The biotinylated proteins were eluted from the beads (Fraction B) by incubation in 100 l of SDS-PAGE sample buffer containing 5% ␤-mercaptoethanol at 85°C for 10 min and an aliquot (30 l Х 25 l of T) was taken for Western blotting.
Peptide Synthesis-Synthetic peptides corresponding to residues 23-48 of the TM segment of wild-type ␥b, mutant TM peptides with replacements G41R, G41L, G35R, G35L, and the scrambled peptide, all containing three lysine residues on the N and C termini to aid in synthesis and purification, were prepared as described previously (17,22). The sequences of these peptides are shown in Fig. 1.
Kinetic Assays and Data Analysis-Kinetic assays of Na,K-ATPase were carried out in triplicate as described previously (11) using membranes isolated from mock-transfected and either mutant or WT ␥btransfected rat ␣1-HeLa cells. For studies of the kinetic effects of the various peptides, the permeabilized membranes were incubated in buffered medium (15 mM Tris-HCl, pH 7.4 containing 1 mM EDTA) with peptide such that the peptide concentration was 16.7 M (3.3 M in the final assay mixture) and the membrane protein concentration was 0.15-0.2 mg/ml. Following incubation for 10 min at room temperature and then for 1 h on ice, the membranes were preincubated for 10 min at 37°C with 5 M ouabain and assayed for Na,K-ATPase activity with the data analyzed as described (11).

RESULTS
Studies with Transfected Cells-Several approaches were used to gain insight into the role of two glycine residues, Gly-41 and Gly-35, on ␥ processing, trafficking, and association with the Na,K-ATPase on the one hand and the functional effect on K ϩ /Na ϩ antagonism on the other.
To assess ␥ association with the catalytic ␣ subunit and ␥ surface expression, we tested ␥/␣ co-immunoprecipitation and cell surface biotinylation followed by streptavidin bead isolation and Western blotting of ␥ and ␣, respectively (see Ref. 14). The co-immunoprecipitation experiment ( Fig. 2A) shows that ␥b and ␥b-G35L associate with ␣, whereas ␥bG41L shows weak association, and ␥b-G41R and ␥b-G35R show little, if any, association. In additional experiments (not shown), ␥/␣ association seen at lower detergent concentration (0.4 mg/ml) is increased for ␥b-G41L but not for ␥b-G41R. The surface biotinylation results (Fig. 2B) show that ␥b and ␥b-G35L and, to some extent, the ␥b-G41L mutant, traffic to the surface. As shown earlier (13), little if any ␥b-G41R appears at the surface. Although the low expression of ␥b-G35R precluded assessment of its surface expression, these results indicate that the appearance of the WT and mutant ␥ chains at the cell surface parallels their ability to associate with ␣. A surprising finding is the low level of ␥b-G41L relative to ␣ in the Triton X-100-solubilized cells (total fraction, Fig. 2B) compared with the relative amounts seen in Western blots of total membranes that were not subjected to this detergent treatment (see Fig. 2A). The reason for this apparent discrepancy is under investigation, although this observation does not detract from the conclusion that this mutant does, in fact, traffic to the cell surface.
Further evidence for altered ␥ trafficking in the Gly 3 Arg mutants is the failure to detect post-translational modification of ␥. Thus, in Western blots of multiple clones of rat ␣1-HeLa cells transfected with WT ␥b and each of the above-mentioned mutants (experiments not shown), the ␥b chain appeared as a doublet (␥b and ␥bЈ; cf. Ref. 11) in WT ␥b and the conservative mutants ␥bG41L and ␥bG35L but not in the ␥bG41R and ␥bG35R mutants. We then looked at the functional properties of the various ␥b-transfected cells. Table I summarizes measurements of apparent Na ϩ -affinity of Na,K-ATPase measured at high K ϩ concentration (100 mM) of membranes isolated from the mock-transfected (control) as well as the WT and mutant ␥b-transfected cells. The results indicate that ␥bG35L behaves like WT ␥ in that it decreases the apparent affinity for Na ϩ ; KЈ Na is increased 2.5-fold by ␥bG35L and nearly 2-fold by ␥b. In contrast, ␥bG41L, like ␥bG41R, does not increase KЈ Na . Unfortunately, ␥bG35R mutants were expressed at levels too low for meaningful kinetic analysis.
Effects of Transmembrane Mimetic Peptides-Studies of the effects of peptides comprising the TM sequence of ␥ (␥-TM), G41R (␥G41R-TM), G41L (␥G41L-TM), G35R (␥G35R-TM), and G35L (␥G35L-TM) ␥ as well as a "scrambled" peptide ("␥Scr-TM") were carried out with synthetic peptides containing "lysine tags" at both the N and the C termini. These tags overcome the inherent insolubility of these ␣-helical TM peptides, thus aiding in their synthesis and purification, but retain their native TM oligomeric states (17,22). Fig. 3 shows the Na ϩ activation profile measured at high (100 mM) K ϩ concentration without and with the various TM peptides. Compared with ␥-Scr-TM, the addition of either ␥-TM or ␥G35L-TM peptide to ␥-devoid membranes isolated from rat ␣1-HeLa cells increases KЈ Na . The increase effected by G35L is even greater than seen with the WT peptide, similar to the greater effect of ␥bG35L compared with ␥b in ␥-expressing cells (described above). In contrast, ␥G41R-TM, ␥G41L-TM, and ␥G35R-TM did not increase KЈ Na above that observed with the control scrambled peptide (␥Scr-TM). A nonspecific effect of peptide addition is evident in a small but consistent increase in KЈ Na seen irre-spective of whether ␥Scr-TM or any of the three mutants, ␥G35R-TM, ␥G41R-TM and ␥G41L-TM, is added. The other notable kinetic effect of the full-length ␥ chain, the decrease in KЈ ATP , was not seen with ␥-TM (experiments not shown), consistent with earlier evidence (14) that this effect is mediated primarily by the cytoplasmic C terminus.
We showed previously that KЈ Na is a linear function of K ϩ concentration, approximating a simple competitive model of cytoplasmic K ϩ /Na ϩ competition, i.e. KЈ Na ϭ K Na (1 ϩ [K ϩ ]/K K ) (19,23,24), and that the main effect of either ␥a or ␥b was not due to an increase in K Na , but rather to a decrease in K K . To further assess the specificity of the mimicry of the TM peptides, we determined KЈ Na as a function of K ϩ concentration and, thus, values of K Na (KЈ Na when [K ϩ ] ϭ 0) and K K (the apparent K ϩ affinity for competition with cytoplasmic Na ϩ ). The results shown in the inset to Fig. 3 support the conclusion that ␥-TM and ␥G35L-TM peptides, like their full-length counterparts (see table in Ref. 11), decrease K K with little effect on K Na .
As shown in Fig. 4, in control experiments carried out with the ␥-TM peptide and/or the ␥G35L-TM peptide, an increase in KЈ Na seen with mock-transfected cell membranes is seen also with ␥bG41R-transfected but not ␥b-transfected membranes. Thus, the kinetic effects of the mimetic peptides are highly specific in that they are seen only with pumps not already associated (mock transfected) or minimally associated (␥b-G41R) with full-length ␥ chains. As expected, ␥G41L-TM and ␥G41R-TM peptides had no effect on the KЈ Na values of pumps in any of the transfected systems. DISCUSSION The experiments described herein unequivocally show that the effect of ␥ (FXYD2) on the KЈ Na values of Na,K-ATPase is mediated solely by the TM domain of the protein, as suggested recently (15). Thus, peptides comprising only this region of ␥ simulate functional effects obtained in assays performed on membranes isolated from wild-type and mutant ␥b-transfected ␣1-HeLa cells. The mimetic effects of the peptides underscore    (3), respectively, and, in the absence of an added peptide, 4.78 Ϯ 0.18 (10). Inset, K Na , and K K were estimated from values of KЈ Na determined at various concentrations of K ϩ using the relationship KЈ Na ϭ K Na (1 ϩ [K ϩ ]/K K ), where (KЈ Na ϭ K Na at [K] ϭ 0) (23). the feasibility of using TM peptides to evaluate kinetic effects of TM helices, including instances in which adequate expression is problematic, as is the case of the ␥b-G35R mutant. TM mimetic peptides offer the unique advantage of examining the function of the TM domain without the complexity of longrange effects of extramembranous regions. In fact, concern over this complexity was mentioned by Lindzen et al. (15) who raised the possibility that a change in KЈ ATP effected by the cytoplasmic domain may mask an apparent effect of ␥-TM on K ϩ /Na ϩ antagonism, which may account for the larger decrease in K K effected by ␥-TM and ␥G35L-TM compared with the analogous transfectants (compare inset of Fig. 3 with Table  I).
The results of the present study provide insight into the role of two Gly residues, Gly-35 and Gly-41, which reside on opposite faces of the helical TM domain of ␥. The observation that both the ␥b-G35R (Fig. 1) and ␥b-G41R (11) mutants show either minimal expression or processing and trafficking led us to consider more conservative mutations in our attempts to understand the role of these residues in modulating pump function. We therefore studied G35L and G41L mutants in experiments with both transfected cells and synthesized peptides. These mutants circumvent the energetic cost associated with inserting a positively charged residue within the hydrophobic core of the membrane and are therefore more relevant in studying the role of the native glycine residues. This consideration may not only explain the low processing/trafficking of Arg mutants but also the lack of effect of the ␥G35R-TM peptide on sodium pump kinetics (Fig. 3).
The studies with both transfected cells and peptides provide evidence that disruption of a Gly residue (Gly-41) on one face of the ␥ helix but not on the other (Gly-35) abrogates the increased K ϩ /Na ϩ antagonism effected by ␥ as seen by a notable increase in KЈ Na at a high K ϩ concentration. Thus, the abolition of the kinetic effect is seen not only with the disrupting mutation G41R replacement associated with autosomal dominant renal magnesium wasting (16) but also with the conservative mutation, G41L. Previous results from our laboratory (14) suggested that Gly at position 41 is essential for normal trafficking to the cell surface and association with the pump. However, even when the cellular processing and trafficking problems are circumvented, as is the case with the G41L mutant and the peptide experiments presented herein, mutating this residue fails to increase KЈ Na above levels seen with a control scrambled peptide. Importantly, ␥b-G41L appears to be expressed at the surface (Fig. 2B) and to associate, at least to an extent, with the ␣ subunit ( Fig. 2A), confirming a role for Gly-41 in mediating one of the kinetic effects of ␥ but not primarily in ␣␤-␥ association. Such a dissociation between residues important for the functional effects of ␥ and its interaction with the sodium pump is consistent with the findings of Lindzen et al. (15).
In contrast to Gly-41, Gly-35 on the opposite face of the helix does not appear to be functionally important, because the G35L mutation does not abrogate kinetic function. Our results also suggest that Gly-35 is not involved in association of ␥ with the ␣␤ complex, because ␥b-G35L was able to co-immunoprecipitate with the ␣ subunit ( Fig. 2A). The latter finding is consistent with recent experiments showing that the residues important for this association are on a different side of the helix (see Fig. 5 in Ref. 15).
As described previously (17), ␥-TM and ␥G35R-TM peptides form oligomers in the weak detergent PFO, but mutants of Gly-41 (␥G41R-TM and ␥G41L-TM) do not. Using PFO-PAGE analysis (see Ref. 17), we have confirmed that the ␥G35L-TM peptide can form oligomers in PFO (not shown), revealing a potential correlation between the effects of ␥ peptides on KЈ Na and their ability to form oligomers in PFO. The physiological relevance of this observation remains to be investigated.
Mimetic TM peptides reconstituted in detergent micelles have been used previously in structural studies of membrane proteins (22,25,26) and have been shown to inhibit membrane protein function (27)(28)(29). However, to our knowledge, the results reported here constitute an unique example of a TM peptide that can display specific kinetic effects attributable to the TM domain of a membrane protein. The remarkable mimetic properties of the TM peptides and the expressed ␥ mutants have particular significance. The failure of the G41R and G41L mutants to affect KЈ Na , whether added as TM peptides to cell membranes or expressed as full-length proteins in transfected cells, underscores the conclusion that Gly-41, which is associated with renal magnesium wasting when mutated to Arg, is important for the effect of the ␥ subunit on K ϩ /Na ϩ antagonism and possibly for oligomerization of the protein, whereas Gly-35 is not.