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 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 (1Kaplan J.H. Ann. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (893) Google Scholar, 2Scheiner-Bobis G. Eur. J. Biochem. 2002; 269: 2424-2433Crossref PubMed Scopus (185) Google Scholar). It catalyzes the electrogenic exchange of three intracellular Na+ for two extracellular K+ 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+-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 (3Sweadner K.J. Rael E. Genomics. 2000; 68: 41-56Crossref PubMed Scopus (354) Google Scholar), associate with and modulate the kinetic behavior of the sodium pump (for a recent overview, see Ref 4Cornelius F. Mahmmoud Y.A. News Physiol. Sci. 2003; 18: 119-124PubMed Google Scholar). Members of this family of proteins are small, single transmembrane (TM) 1The abbreviations used are: TM, transmembrane; PFO, perfluorooctanoate; Scr, scrambled (peptide); WT, wild-type. 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 (5Therien A.G. Goldshleger R. Karlish S.J. Blostein R. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 6Beguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google Scholar, 7Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 275: 35969-35977Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 8Beguin P. Crambert G. Guennoun S. Garty H. Horisberger J.D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (134) Google Scholar). To date, the γ “subunit” of the renal Na,K-ATPase is the best characterized member (reviewed in Refs. 9Therien A.G. Blostein R. Amer. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar and 10Therien A.G. Pu H.X. Karlish S.J. Blostein R. J. Bioenerg Biomembr. 2001; 33: 407-414Crossref PubMed Scopus (50) Google Scholar). Gamma, or FXYD2, exists as two main splice variants, γa and γb, with distinct as well as overlapping localization along the nephron (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Mass spectrometry of γa and γb indicate that they differ only in the N terminus; in rat γa, TELSANH is replaced by Ac-MDRWYL in γb (12Kuster B. Shainskaya A. Pu H.X. Goldshleger R. Blostein R. Mann M. Karlish S.J. J. Biol. Chem. 2000; 275: 18441-18446Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Previous studies using membrane fragments isolated from γ-transfected rat α1-HeLa cells have shown that γ serves at least two distinct functions in regulating the pump, and the effects are similar for both variants (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 13Therien A.G. Karlish S.J. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). These distinct effects of γ include the following: (i) an increase in the apparent affinity for ATP; and (ii) an increase in K+/Na+ competition at cytoplasmic Na+ activation sites, as seen by an increase in K′Na at high [K+]. Previous studies have suggested that the effect on ATP affinity is mediated by the cytoplasmic C-terminal domain of the protein (5Therien A.G. Goldshleger R. Karlish S.J. Blostein R. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 14Pu H.X. Scanzano R. Blostein R. J. Biol. Chem. 2002; 277: 20270-20276Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), whereas effects on K+/Na+ antagonism may be associated with the TM domain (15Lindzen M. Aizman R. Lifshitz Y. Lubarski I. Karlish S.J. Garty H. J. Biol. Chem. 2003; 278: 18738-18743Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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 γ are added directly to membranes derived from cells devoid of the protein. In addition, we describe a mutagenesis approach to structure/function analysis of γ 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 (16Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F. Groenestege W.T. de Pont J.J. Bindels R.J. Monnens L.A. van den Heuvel L.P. Knoers N.V. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (220) Google Scholar) 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 (17Therien A.G. Deber C.M. J. Mol. Biol. 2002; 322: 583-590Crossref PubMed Scopus (44) Google Scholar). Effects of peptides with either of these Gly residues replaced by Arg or Leu are compared with those of the full-length transfected γ proteins (wild-type or mutants of the γb variant) expressed in cultured rat α1-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 αβ pumps. Mutagenesis, Transfection, Tissue Culture, and Membrane Preparations—Point mutations were introduced into γb cDNA subcloned into the pIRES expression vector and then transfected into HeLa cells stably expressing the rat α1 subunit of the Na,K-ATPase (α1 HeLa cells were kindly provided by Dr. J. B. Lingrel) as described previously (14Pu H.X. Scanzano R. Blostein R. J. Biol. Chem. 2002; 277: 20270-20276Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Unless indicated otherwise, cellular membranes from the stably transfected cells were prepared as described by Jewell and Lingrel (18Jewell E.A. Lingrel J.B. J. Biol. Chem. 1991; 266: 16925-16930Abstract Full Text PDF PubMed Google Scholar). Polyacrylamide Gel Electrophoresis and Western Blotting—SDS-PAGE and Western blotting were carried out as described previously (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and, following transfer to polyvinylidene difluoride membranes, the lower half was analyzed with a polyclonal γ antibody (γC32 raised against the C-terminal KHRQVNEDEL peptide and essentially the same as γC33 described previously in Ref. 19Therien A.G. Nestor N.B. Ball W.J. Blostein R. J. Biol. Chem. 1996; 271: 7104-7112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and the upper half was analyzed with anti-α1 subunit A277 (Sigma). Co-immunoprecipitation—The method used is a modification of Garty et al. (20Garty H. Lindzen M. Scanzano R. Aizman R. Fuzesi M. Goldshleger R. Farman N. Blostein R. Karlish S.J.D. Amer. J. Physiol. Renal Physiol. 2002; 283: F607-F615Crossref PubMed Scopus (80) Google Scholar). Briefly, 250 μg of membranes, prepared from γb(WT)and γb(mutant)-transfected cells as described previously (18Jewell E.A. Lingrel J.B. J. Biol. Chem. 1991; 266: 16925-16930Abstract Full Text PDF PubMed Google Scholar), 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 μl. Polyoxyethylene 10 lauryl ether (C12E10) (250 μl 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 × g, the supernatant was added to 50 μl 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-μl aliquot was taken for SDS-PAGE. To another 450-μl aliquot, 90 μl of RbCl (118 mm final concentration) and 30 μl of γC32 anti-serum (stored at -20 °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 μl) 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 μl of SDS-PAGE sample buffer containing 5% β-mercaptoethanol at 37 °C for 30 min. A 20-μl aliquot of the eluate and the 10-μl sample removed before addition of antiserum were resolved by SDS-PAGE followed by Western blotting. Cell Surface Biotinylation—Transfected α1-HeLa cells were grown to ≈80% confluence in 6-well plates. The surface biotinylation is a modification of Stephan et al. (21Stephan M.M. Chen M.A. Penado K.M. Rudnick G. Biochemistry. 1997; 36: 1322-1328Crossref PubMed Scopus (66) Google Scholar) 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 CaCl2 and 1 mm MgCl2 (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 μl of L1 buffer (1% Triton X-100/0.1% SDS in 150 mm 5 mm EDTA, and 50 mm pH 7.5, containing 10 each and and well was then and the sample was and centrifuged at × for 10 min. 20-μl aliquot of supernatant was taken for protein and another for Western blotting. The proteins were isolated by a μl) in buffer buffer without final 500 μl) with 100 μl of beads overnight at 4 °C with rotation. The were then centrifuged to beads and supernatant and an aliquot of μl μl of was taken for Western blotting. The beads were washed three times with twice with high buffer buffer with 500 mm and Triton and then with 50 mm pH The proteins were eluted from the beads by incubation in 100 μl of SDS-PAGE sample buffer containing 5% β-mercaptoethanol at °C for 10 min and an aliquot μl μl of was taken for Western blotting. peptides corresponding to residues of the TM segment of γb, TM peptides with and the scrambled all containing three residues on the N and to in and were prepared as described previously (17Therien A.G. Deber C.M. J. Mol. Biol. 2002; 322: 583-590Crossref PubMed Scopus (44) Google Scholar, Deber C.M. Biochemistry. 2001; PubMed Scopus Google Scholar). The of these peptides are shown in and of Na,K-ATPase were carried out in as described previously (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) using membranes isolated from and either or rat α1-HeLa cells. studies of the kinetic effects of the the membranes were incubated in mm pH containing 1 mm with peptide that the peptide was in the final and the membrane protein was Following incubation for 10 min at room temperature and then for 1 h on the membranes were for 10 min at 37 °C with 5 ouabain and for Na,K-ATPase activity with the analyzed as described (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). with were used to into the role of two glycine Gly-41 and Gly-35, on γ and association with the Na,K-ATPase on the one and the functional effect on K+/Na+ antagonism on the To assess γ association with the subunit and γ surface we and cell surface biotinylation followed by and Western blotting of γ and Ref. 14Pu H.X. Scanzano R. Blostein R. J. Biol. Chem. 2002; 277: 20270-20276Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The that γb and associate with whereas and and show In association seen at lower is for for The surface biotinylation show that γb and and, to the to the shown A.G. Karlish S.J. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), at the the expression of assessment of its surface these indicate that the of the and γ at the cell surface to associate with A is the of relative to in the Triton cells compared with the relative seen in Western of membranes that were to this The for this apparent is this from the that this in to the cell evidence for γ in the Gly Arg mutants is the to modification of in Western of of rat α1-HeLa cells transfected with γb and each of the mutants the γb as a and Ref. H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) in γb and the mutants and in the and then at the functional of the cells. of apparent of Na,K-ATPase at high K+ (100 of membranes isolated from the as well as the and cells. The indicate that γ in that it the apparent affinity for K′Na is by and by In increase mutants were expressed at for kinetic of effects of and on apparent affinity for Na+ at 100 at 100 for γb and compared to to for γb and compared to for γb and compared to to expression in a Effects of of the effects of peptides the TM of γ and γ as well as a peptide were carried out with synthetic peptides containing at both the N and the These the of these TM in and TM (17Therien A.G. Deber C.M. J. Mol. Biol. 2002; 322: 583-590Crossref PubMed Scopus (44) Google Scholar, Deber C.M. Biochemistry. 2001; PubMed Scopus Google Scholar). the Na+ activation at high (100 K+ without and with the TM peptides. with the addition of either or peptide to membranes isolated from rat α1-HeLa cells The increase by is seen with the similar to the effect of compared with γb in cells In and increase K′Na that with the scrambled peptide A effect of peptide addition is in a increase in K′Na seen of or of the three and is The other kinetic effect of the full-length γ the decrease in was seen with with evidence (14Pu H.X. Scanzano R. Blostein R. J. Biol. Chem. 2002; 277: 20270-20276Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) that this effect is mediated by the cytoplasmic previously that K′Na is a of K+ concentration, a of cytoplasmic K+/Na+ K′Na (1 A.G. Nestor N.B. Ball W.J. Blostein R. J. Biol. Chem. 1996; 271: 7104-7112Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, J. Physiol. PubMed Scopus Google Scholar, J. Physiol. PubMed Scopus Google Scholar), and that the main effect of either γa or γb was to an increase in to a decrease in K. To further assess the of the of the TM we K′Na as a of K+ and, of when and apparent K+ affinity for competition with cytoplasmic The shown in the to the that and full-length in Ref. H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), decrease with effect on shown in in carried out with the peptide the an increase in K′Na seen with cell membranes is seen also with the kinetic effects of the mimetic peptides are specific in that they are seen only with pumps associated or associated with full-length γ and peptides had effect on the K′Na of pumps in of the transfected The described unequivocally show that the effect of γ on the K′Na of Na,K-ATPase is mediated by the TM domain of the protein, as suggested (15Lindzen M. Aizman R. Lifshitz Y. Lubarski I. Karlish S.J. Garty H. J. Biol. Chem. 2003; 278: 18738-18743Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). peptides only this region of γ functional effects in on membranes isolated from and α1-HeLa cells. The mimetic effects of the peptides the of using TM peptides to kinetic effects of TM including in which expression is as is the of the TM mimetic peptides the of the of the TM domain without the of effects of In this was by Lindzen et al. (15Lindzen M. Aizman R. Lifshitz Y. Lubarski I. Karlish S.J. Garty H. J. Biol. Chem. 2003; 278: 18738-18743Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) raised the that a in by the cytoplasmic domain may an apparent effect of on K+/Na+ which may for the decrease in by and compared with the of with The of the into the role of two Gly Gly-35 and Gly-41, which on of the TM domain of The that both the and (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) mutants show either expression or and to mutations in to the role of these residues in pump and mutants in with both transfected cells and peptides. These mutants the associated with a residue the of the membrane and are in the role of the glycine This may only the of Arg mutants also the of effect of the peptide on sodium pump The studies with both transfected cells and peptides evidence that of a Gly residue on one face of the γ on the other the K+/Na+ antagonism by γ as seen by a increase in K′Na at a high K+ the of the kinetic effect is seen only with the associated with renal magnesium wasting (16Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F. Groenestege W.T. de Pont J.J. Bindels R.J. Monnens L.A. van den Heuvel L.P. Knoers N.V. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (220) Google Scholar) also with the Previous from (14Pu H.X. Scanzano R. Blostein R. J. Biol. Chem. 2002; 277: 20270-20276Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) suggested that Gly at is for to the cell surface and association with the pump. when the cellular and are as is the with the and the peptide this residue to increase K′Na seen with a scrambled to be expressed at the surface and to at least to an with the subunit a role for Gly-41 in one of the kinetic effects of γ in a residues important for the functional effects of γ and its with the sodium pump is with the of Lindzen et al. (15Lindzen M. Aizman R. Lifshitz Y. Lubarski I. Karlish S.J. Garty H. J. Biol. Chem. 2003; 278: 18738-18743Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In to Gly-41, Gly-35 on the face of the appear to be because the kinetic also suggest that Gly-35 is involved in association of γ with the αβ because was to with the subunit The is with recent that the residues important for this association are on a of the 5 in Ref. M. Aizman R. Lifshitz Y. Lubarski I. Karlish S.J. Garty H. J. Biol. Chem. 2003; 278: 18738-18743Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). described previously (17Therien A.G. Deber C.M. J. Mol. Biol. 2002; 322: 583-590Crossref PubMed Scopus (44) Google Scholar), and peptides form in the PFO, mutants of Gly-41 and not. Using analysis Ref. A.G. Deber C.M. J. Mol. Biol. 2002; 322: 583-590Crossref PubMed Scopus (44) Google Scholar), we have that the peptide form in a the effects of γ peptides on K′Na and to form in The of this remains to be TM peptides in have been used previously in studies of membrane proteins Deber C.M. Biochemistry. 2001; PubMed Scopus Google Scholar, J.H. 1997; 276: PubMed Scopus Google Scholar, H. D. G. B. E. F. Biochemistry. 2000; PubMed Scopus Google Scholar) and have been shown to membrane protein D. Deber C.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). to the an of a TM peptide that specific kinetic effects to the TM domain of a membrane protein. The mimetic of the TM peptides and the expressed γ mutants have particular The of the and mutants to added as TM peptides to cell membranes or expressed as full-length proteins in transfected the that Gly-41, which is associated with renal magnesium wasting when mutated to Arg, is important for the effect of the γ subunit on K+/Na+ antagonism and for of the protein, whereas Gly-35 is not. and for with peptide

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