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J. Biol. Chem., Vol. 281, Issue 23, 15790-15799, June 9, 2006

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Functional Interactions of Phospholemman (PLM) (FXYD1) with Na⁺,K⁺-ATPase

PURIFICATION OF 1/1/PLM COMPLEXES EXPRESSED IN PICHIA PASTORIS^*

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, March 2, 2006 , and in revised form, April 3, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human FXYD1 (phospholemman, PLM) has been expressed in Pichia pastoris with porcine ₁/His₁₀-₁ subunits of Na⁺,K⁺-ATPase or alone. Dodecyl--maltoside-soluble complexes of ₁/₁/PLM have been purified by metal chelate chromatography, either from membranes co-expressing ₁,His₁₀-₁, and PLM or by in vitro reconstitution of PLM with ₁/His₁₀-₁ subunits. Comparison of functional properties of purified ₁/His₁₀-₁ and ₁/His₁₀-₁/PLM complexes show that PLM lowered K_0.5 for Na⁺ ions moderately (30%) but did not affect the turnover rate or K_m of ATP for activating Na⁺,K⁺-ATPase activity. PLM also stabilized the ₁/His₁₀-₁ complex. In addition, PLM markedly (>3-fold) reduced the K_0.5 of Na⁺ ions for activating Na⁺-ATPase activity. In membranes co-expressing ₁/His₁₀-₁ with PLM the K_0.5 of Na⁺ ions was also reduced, compared with the control, excluding the possibility that detergent or lipid in purified complexes compromise functional interactions. When expressed in HeLa cells with rat ₁, rat PLM significantly raised the K_0.5 of Na⁺ ions, whereas for a chimeric molecule consisting of transmembranes segments of PLM and extramembrane segments of FXYD4, the K_0.5 of Na⁺ ions was significantly reduced, compared with the control. The opposite functional effects in P. pastoris and HeLa cells are correlated with endogenous phosphorylation of PLM at Ser⁶⁸ or unphosphorylated PLM, respectively, as detected with antibodies, which recognize PLM phosphorylated at Ser⁶⁸ (protein kinase A site) or unphosphorylated PLM. We hypothesize that PLM interacts with ₁/His₁₀-₁ subunits at multiple locations, the different functional effects depending on the degree of phosphorylation at Ser⁶⁸. We discuss the role of PLM in regulation of Na⁺,K⁺-ATPase in cardiac or skeletal muscle cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Na⁺,K⁺-ATPase actively transports Na⁺ and K⁺ ions across cell membranes, thus maintaining the normal transmembrane gradients of Na⁺ and K⁺ ions. As could be expected, Na⁺,K⁺-ATPase is closely regulated at several levels. A unique mode of regulation of the Na⁺,K⁺-ATPase has been described recently, involving interactions between the / complex and the FXYD proteins (1–4). The FXYD family consists of seven short single span transmembrane proteins (N terminus extracellular), named after the conserved FXYD motif in the extracellular segment (5). The FXYD proteins show a tissue-specific expression pattern. For example, FXYD2 (-subunit) is expressed in kidney (6–8), and FXYD4 (CHIF)³ also in kidney, but largely in other nephron segments, as well as in colon (9–12). FXYD1 (phospholemman or PLM), the topic of this article, is expressed abundantly in heart and skeletal muscle (13–15), and has also been detected in cerebellum and choroid plexus (16), as well as in renal extra glomerular mesangium and afferent arterioles of the juxtaglomerular apparatus, but not in nephrons (or macula densa) (17).

Six members of the family, FXYD1 (PLM), FXYD2 (), FXYD3 (Mat-8), FXYD4 (CHIF), FXYD5 (RIC), and FXYD7, are now known to interact specifically with the Na⁺,K⁺-ATPase, and modulate the functional properties (1–4). The FXYD proteins are not required for basic pump function, but they act as accessory subunits to the - and -subunits, and modulate the kinetic properties in a tissue-specific fashion. PLM was discovered originally as a major substrate for PKC and for PKA accompanying - and -adrenergic stimulation of heart (18–20), and it is also a substrate of other kinases (21, 22). Although expression of PLM was shown previously to induce chloride currents in Xenopus oocytes, and taurine efflux and regulatory volume decrease in mammalian cells (23–25), in regard to its physiological role in cardiac contraction, more attention has been paid recently to the functional interaction of PLM with Na⁺,K⁺-ATPase.

After co-expression in Xenopus oocytes together with rat -isoforms, PLM was co-immunoprecipitated with either 1 or 2 and 3, and co-immunoprecipitation with either 1 or 2 was also seen for native rat heart sarcolemma, albeit more weakly with 2 (26). In Xenopus oocytes The PLM induced an almost 2-fold decrease in apparent affinity for cytoplasmic Na⁺, a small decrease in apparent affinity for extracellular K⁺, and little or no effect on the maximal pump current or on ouabain binding. As mentioned above, PLM has been detected in bovine choroid plexus membranes, and an anti-PLM antibody reduces V_max without affecting the apparent Na⁺ affinity (16). This result does not necessarily contradict the findings with Xenopus oocytes because the antibody can neutralize interactions only at the C terminus of PLM, whereas the effect on apparent Na⁺ affinity may be mediated by other segments of PLM. However, a recent study using sarcolemma membrane obtained from hearts of PLM knock-out mice showed a lower V_max of Na⁺,K⁺-ATPase, even after taking into account a 20% lower expression of the -subunit, but again, no difference in apparent Na⁺ affinity (27). The implication is that PLM stimulates Na⁺,K⁺-ATPase activity, with no change in Na⁺ affinity. This does not fit well with the observations in Xenopus oocytes.

There is some evidence that phosphorylation of PLM affects its functional interactions. For example, Na⁺,K⁺-ATPase in sarcolemma membranes isolated from ischemic rat hearts has been found to be strongly stimulated compared with the controls, and this is accompanied by activation of PKA and PKC and phosphorylation of PLM (but not of the -subunit) ((28)). Two recent studies have concluded that, in cardiac myocytes, phosphorylation of PLM at the PKA site (Ser⁶⁸) mediates the stimulatory effects of -adrenergic agonists on Na⁺/K⁺-pump function (29, 30). It was proposed that PLM raises the K_0.5 of Na⁺ ion for activating Na⁺,K⁺-ATPase and phosphorylation relieves this effect. Similarly in an experimental model of heart failure, phosphorylation of rabbit cardiac myocyte PLM is greatly increased (31). It has also been demonstrated recently that phosphorylation of PLM is required for translocation of PLM from the endoplasmic reticulum to the plasma membrane in mammalian cell (32).

An FXYD protein in shark rectal gland membranes, which co-purifies with the - and -subunits, is termed PLMS because of its PLM-like phosphorylation sites, although the closest mammalian sequence homolog is in fact Mat-8 (33–35). Phosphorylation of PLMS by PKC or selective proteolysis of the C terminus, which removes the phosphorylated serine, increases Na⁺,K⁺-ATPase activity. Thus, the Na⁺,K⁺-ATPase activity is partially inhibited when the enzyme interacts with intact or unphosphorylated PLMS, and inhibition is relieved upon proteolysis or PKC phosphorylation (1).

Structural interactions of FXYD proteins with / subunits are not well understood, but information is accumulating (3). There is now good evidence that the transmembrane segments of FXYD1 (PLM), FXYD2 (), FXYD4 (CHIF), and probably all other FXYD proteins lie within a groove bounded by M2, M6, and M9 of the -subunit (36–39). There is also evidence for multiple interactions in the case of FXYD2, which imply that extramembrane domains also interact with the -subunit and affect its function (39, 40). It is an open question whether the extramembrane domains of all other FXYD proteins interact with the same or different domains of the -subunit (see Ref. 41). Both FXYD2 and FXYD4 have also been shown to be in proximity to the -subunit (38, 39).

The present work makes use of the P. pastoris expression system for an initial characterization of functional interactions between the Na⁺,K⁺-ATPase and FXYD1 (PLM). Recently, we have described expression of Na⁺,K⁺-ATPase (porcine ₁₁ subunits) in the methanotrophic yeast P. pastoris (42) and purification of functional detergent-soluble / subunit complexes (43). This system offers an opportunity to look at interactions of FXYD proteins with the purified / complexes in vitro and, in principle, could allow extensive studies of functional and structural interactions of FXYD proteins under well controlled conditions. Here, we describe functional interactions detected either by co-expression of PLM with / subunits, or after in vitro reconstitution of separately expressed / subunits and PLM to form //PLM complexes. The results show some interesting differences from those observed in Xenopus oocytes (26). Therefore, for comparison with the P. pastoris system, we have also looked at effects of PLM expressed in HeLa cells, which have been used extensively to examine functional and structural interactions of FXYD proteins, particularly FXYD2 () and FXYD4 (CHIF) (7, 38, 40, 44–46). A preliminary account of this work has appeared in Ref. 47.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of the Clones pHIL-D2(/His₁₀-), pHIL-D2(PLM), and pGAPzA(PLM)—A pHIL-D2 vector (Invitrogen) construct containing cDNAs encoding porcine ₁ (GenBank^TM accession X03938 [GenBank] ) and porcine ₁ (GenBank^TM accession no. X04635 [GenBank] ) with a 10x histidine tag at its N terminus (/His₁₀-) has been described previously (43).

Human PLM (GenBank^TM accession H23593 [GenBank] ) was subcloned either into the EcoRI site of pHIL-D2, or into the EcoRI/NotI site of pGAPZa (Invitrogen). In the pGAPZ vector PLM is constitutively expressed in P. pastoris under the GAPDH promoter.

Yeast Transformation, Selection, and Growth—SMD1165, a protease-deficient strain (his4, prb1) of P. pastoris was transformed with 10 µg of NotI-linearized pHIL-D2(/His₁₀-) construct. Preparation of spheroplasts and selection for His+,Mut^s transformants were done as described (42, 43). The selected Mut^s clone showing the highest expression level of the enzyme was further transformed by electroporation with AvrII-linearized pGAPZ vector containing human PLM. Clone selection was done by Zeocin resistance and by DNA Dot Blot screening. The resulting positive clones showed methanol-inducible expression of /His₁₀- and constitutive expression of PLM in the same cell (co-expression of /His₁₀- with PLM). Independently, linear cDNA of PLM in pHIL-D2 (without and His₁₀- cDNA) was used to transform spheroplasts, similarly to the pHIL-D2(/His₁₀-) vector. This clone was used to express PLM alone and reconstitute it in vitro with the /His₁₀- complex.

Standard large scale 3-liter cultures were grown in Bellco Spinner Flasks^TM with air supplement, magnetic stirring, and temperature maintained at 25 °C. Growth media were prepared from YNB medium (Difco) as described (42).

DNA Dot Blot Screen—Dot blot analysis has been used to scan for maximal copy number of integrated PLM, as described (42). A radioactive probe was synthesized from PLM cDNA using a random prime labeling kit (rediprime, Amersham Biosciences, cat. no. RPN 1633).

P. pastoris Membrane Preparations—Cells were broken with glass beads, and membranes were prepared as described (43), including a step involving treatment with 2 M urea. Membranes were stored at –80 °C in MOPS-Tris, 10 mM, pH 7.2; EDTA, 1 mM; glycerol, 25% with protease inhibitors. Roughly, 1 g of membrane protein was obtained per 3-liter culture. Renal Na⁺,K⁺-ATPase was prepared as described (48).

Purification of Recombinant Na⁺,K⁺-ATPase—Purification of /His₁₀- co-expressed with PLM was done as described before (43). Synthetic DOPS (cat. no. 830035) was obtained from Avanti, and stored as a chloroform solution.

Urea-treated membranes were homogenized with n-dodecyl--maltoside (DDM:protein, 2:1 w/w) (cat. no. D310, Anatrace) in a medium containing NaCl, 250 mM; Tris-HCl, 20 mM, pH 7.4; imidazole, 5 mM; phenylmethylsulfonyl fluoride, 0.5 mM; and glycerol, 10%. The unsolubilized material was removed by ultracentrifugation. The soluble material was incubated for 6 h (/His₁₀-, as control) or overnight (/-reconstituted PLM) at 4 °C with Ni²⁺-NTA beads (Qiagen), at a ratio of 100 µl of beads per 10 mg of soluble membrane protein. For standard preparations, 100-µl beads were washed with 1 ml of buffer containing NaCl, 100 mM; Tris-HCl, 20 mM, pH 7.4; DDM, 0.2 mg/ml; DOPS 0.05 mg/ml; glycerol, 10%, and imidazole, 30 mM (first wash) or imidazole, 60 mM (second wash). The recombinant enzyme was eluted by mixing the beads for 1 h on ice with 100 µl of imidazole, 250 mM; NaCl, 100 mM; Tris-HCl, 20 mM. pH 7.4, DDM, 0.2 mg/ml; DOPS, 0.05 mg/ml; glycerol, 40%. The eluted protein was stored at 0 °C. Protein concentration was determined by comparative gel densitometry with pig kidney Na⁺,K⁺-ATPase.

In later experiments, purification was done using Co²⁺-chelate chromatography (BD-Talon Biosciences), which will be described fully in a forthcoming article.⁴ Briefly, the DDM-solubilized membranes were incubated with BD-Talon beads in the presence of 50 µM EDTA for the same period of time as with Ni²⁺-NTA beads and with the same beads: supernatant ratio. The beads were washed twice with NaCl, 100 mM; Tris-HCl, 20 mM, pH 7.4; DDM, 0.2 mg/ml; DOPS 0.05 mg/ml; glycerol, 10%, and imidazole, 10 mM, and eluted with imidazole, 150 mM; NaCl, 100 mM; Tris-HCl, 20 mM pH 7.4, DDM, 0.2 mg/ml; DOPS, 0.05 mg/ml; glycerol, 40%. For preparations to be used for Na⁺,K⁺ ATPase assay, washing and protein elution were done in the presence of choline chloride, 100 mM instead of NaCl.

In Vitro Reconstitution of the /His₁₀-/PLM Complex—Membranes expressing PLM in pHIL-D2 were solubilized in DDM (DDM:protein, 2:1 w/w), and concentrated by ultrafiltration (Centriprep YM-30 cat. no. 4322 by Amicon, Millipore) at 1500 x g for 2 h at 4 °C. The retentate fraction containing PLM in micelles was then incubated overnight at 4 °C with Ni²⁺-NTA beads or BD-Talon beads pre-bound with the pig ₁/His₁₀-₁ complex. The //PLM complex formed was then eluted as described above.

SDS-PAGE, Western Blots, and Immunoprecipitation—2–5 µg of recombinant enzyme and 40 µg of yeast membranes were separated on 7.5%, or 10% polyacrylamide SDS-Tricine gels (49). Gels were stained with Coomassie, scanned with an imaging densitometer (GS-690, Bio-Rad), and analyzed using the Multi-analyst software (Bio-Rad). Immunoblots were blotted with anti-KETYY antibody that recognizes the C terminus of the -subunit, or with anti- antibodies raised against the extracellular domain of the -subunit (50). Two antibodies were used to recognize PLM: anti-PLM C terminus raised against the C-terminal sequence CRSSIRRLSTRRR (26), and anti-CP-68, kindly provided by Prof. Donald Bers, which recognizes the phosphorylated Ser⁶⁸ of the cytosolic domain of PLM (51).

Immunoprecipitation of , His₁₀-, and PLM subunits from the yeast membranes dissolved in C₁₂E₁₀ was done essentially as described (45) using a monoclonal antibody directed against the N terminus of the -subunit (6H).

Biochemical Assays—Na⁺,K⁺-ATPase assays were done as described (42, 43). Na⁺,K⁺-ATPase assays of membranes (P. pastoris or HeLa cell) were carried out at 37 °C, and of purified complexes at 25 °C, both in a final volume of 100 µl, using [-³²P]ATP. P. pastoris membranes were assayed for Na⁺,K⁺-ATPase activity at 0.5–0.8 mM ATP, and HeLa cells membranes at 1 mM ATP, with varying Na⁺ concentrations in the presence of 100 mM KCl. Assays using membrane preparations were done with or without 5 mM ouabain, and the Na⁺,K⁺-ATPase activity was calculated from the ouabain inhibitable fraction of ATP hydrolysis.

For purified recombinant enzyme, 1 µl(0.2–0.4 µg of protein) was added to 100 µl of the reaction medium. DDM and lipid concentrations in the reaction medium were the same as in the elution buffer. The Na⁺,K⁺-ATPase activity of purified complexes was measured at 25 °C and 100 µM ATP in the presence of 100 mM KCl and different Na⁺ concentrations (0–100 mM). The ionic strength was maintained constant at 250 mM with choline chloride. Na⁺-ATPase activity was performed at 20 nM ATP in medium containing 250 mM NaCl plus choline chloride.

Rates of ATP hydrolysis at different Na⁺ activities were fitted to the following form of the Hill equation: v = V_max x [Na]^nH/([Na]^nH + K ^nH_0.5) using Kaleidagraph software (Synergy Software). In this equation n_H is the Hill coefficient and K_0.5 is the concentration of Na⁺ ions required for half-maximal stimulation of ATP hydrolysis. For each curve the ratio of v/V_max was then calculated and, where available, values of v/V_max for replicate experiments were averaged for each Na⁺ concentration. Then the best-fit average parameters K_0.5 ± S.E. and n_H ± S.E. were recalculated.

Ouabain binding to membranes was assayed using [³H]ouabain, and phosphoenzyme (EP) levels were measured as ATP-dependent phosphorylation, essentially as described (52).

PKA Phosphorylation and Dephosphorylation—50 µg of P. pastoris membranes expressing PLM, or 5 µg of HeLa cell membranes, or 1 µgof purified //PLM complexes were treated with either 20 units of alkaline phosphatase (AP) for 2 h at 37°C or with 2500 units of the catalytic subunit of PKA in the presence of 10 mM MgCl₂ and 1 mM ATP, for 2 h at 30 °C. Recombinant PKA (2,500,000 units/ml, cat. no. P6000L), and calf intestinal alkaline phosphatase (CIP) (10,000 units/ml, cat. no. M0290S) were obtained from Bio Labs.

Expression of PLM and PLM/CHIF Chimera in HeLa Cells—CHIF/PLM chimera were constructed by exchanging the extracellular (Met¹–Gln³⁸), transmembrane (Leu³⁹–Leu⁵⁷), and cytoplasmic (Ser⁵⁸–Thr⁸⁷) domains of rat CHIF with the corresponding PLM sequences. Constructs were prepared by PCR using overlapping primers and verified by sequencing. The coding regions of CHIF, PLM, and CHIF/PLM chimera were subcloned into the BamHI/BstXI site of the mammalian expression vector pIRES-hyg (Clontech). This vector has an internal initiation site which enables translation of the cloned cDNA and the hygromycin resistance gene from a single RNA species. Thus, a better correlation is achieved between hygromycin resistance and high levels of the desired protein.

HeLa cells overexpressing the rat ₁-subunit of Na⁺-K⁺-ATPase (HeLa-₁ cells, kindly provided by Dr. J. B. Lingrel, University of Cincinnati College of Medicine) were transfected with the above cDNAs using Polyfect (Qiagen) according to the manufacturer's instructions. Colonies expressing the FXYD constructs were selected in 400 µg/ml hygromycin B, and tested by Western blotting using antibodies directed to the C termini of CHIF and PLM (26, 45).

Isolation of Crude Plasma Membranes from HeLa Cells—Five confluent 150-cm² tissue culture plates were washed twice with phosphate-buffered saline, and cells were scraped with a rubber policeman. The cells were lysed in a 20-min incubation at 4 °C in 10 mM Tris plus 1 mM MgCl₂ at 4 °C for 20 min, and the lysate was subjected to 20 strokes of a Teflon-glass hand homogenizer. Intact cells, debris, and nuclei were removed in a 3-min centrifugation at 3,000 x g. Membranes were then pelleted at 40,000 x g for 50 min and subsequently washed twice in 1 mM EDTA (pH 7.4).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
P. pastoris cells expressing - and His₁₀--subunits, which we have described recently (43), were transfected with the pGAPZ vector containing human PLM (FXYD1), under control of the GAPDH promoter. Selection of transformed yeast is based on the dominant selectable marker, Zeocin, and transformants should constitutively express the recombinant PLM. Recombinant clones were screened by dot blot analysis for their content of PLM cDNA, as described previously (42). Three colonies with the highest signals, marked in Fig. 1A with asterisks, were then grown in small volumes, membranes prepared, and Western blots run to detect PLM and the -subunit. Fig. 1B shows that all three clones expressed easily detectable amounts of PLM together with the -subunit. Although the molar ratio of PLM: expressed in the yeast membranes cannot be estimated from the immunoblots, the ratio appears to be quite similar to that found for PLM: in native rat sarcolemma vesicle membranes (lane marked SLV). Fig. 1C shows results of an immunoprecipitation experiment. The membranes were solubilized with C₁₂E₁₀ under conditions known to maintain //FXYD complexes intact and optimize co-immunoprecipitation of FXYD proteins with - and -subunits (45). Immunoprecipitation was done with a monoclonal anti- antibody (6H), and the blots were probed with anti-KETYY (anti-), anti-, and anti-PLM. The solubilized membrane protein (S) contains , the two characteristic bands of the -subunit, and PLM, expressed in this preparation as two bands. The immunoprecipitated protein (Co-IP) contains the -subunit, the -subunit, and the upper band of the PLM, demonstrating that the , , and the upper band of PLM interact in a detergent-soluble complex, and presumably also in the yeast cell membranes. The lower band of PLM, which was not seen in all preparations, could be a proteolytic fragment of PLM, and does not appear to interact with - and -subunits.

View larger version (54K): [in this window] [in a new window]   FIGURE 1. Co-expression and co-immunoprecipitation of PLM with / subunits. P. pastoris cells harboring - and -subunits (42) were transformed with the pGAPz vector containing PLM, and recombinant clones were selected in Zeocin as described under "Materials and Methods." A, dot blot analysis of incorporated PLM cDNA. B, immunoblot detecting protein expression of -subunit (anti-KETYY) and PLM (anti-PLM, C terminus). SLV, rat sarcolemma vesicles. C, co-immunoprecipitation. P. pastoris membranes expressing , , and PLM were dissolved in C12E10 in the presence of 5 mM RbCl and 10 mM ouabain and co-immunoprecipitation was done with the monoclonal antibody 6H (45). S, soluble protein. Co-IP, co-immunoprecipitated protein. Detection with anti-KETYY, anti-, and anti-PLM, respectively, is shown.

View larger version (70K): [in this window] [in a new window]   FIGURE 2. Purification of the //PLM complex. PLM, and His10- subunits were co-expressed, and the complex was purified on Ni2+-NTA beads. Left, Coomassie stain; PKE, pig kidney enzyme. Right, immunoblots probed with anti-KETYY or anti-PLM. mem, crude P. pastoris membranes.

As a test of the hypothesis, and a way around the problem just discussed, we have developed a procedure for in vitro reconstitution of the //PLM complex. PLM was expressed in P. pastoris separately from the - and -subunits and a clone expressing maximal amounts of PLM was selected for further study. Reconstitution depends on spontaneous and specific association of uncomplexed PLM with the / subunits bound on the beads and co-elution of a //PLM complex. This technique should allow experimental control of the relative amounts of / subunits and PLM exposed to each other in vitro, and thus maximization of the ratio of PLM:/ subunits in the eluted complex (see Fig. 4). Fig. 4A shows an immunoblot of PLM in membranes of the clone co-expressing PLM with / subunits, or the clone expressing PLM alone, which appears as two bands, and in significantly higher amounts. For the reconstitution experiment in Fig. 4B membranes from the clone expressing PLM alone were dissolved in DDM, protein was concentrated by ultrafiltration, and the soluble membrane protein was incubated overnight with Ni²⁺-NTA beads already bound with /His₁₀- subunits. The amount of the soluble membrane protein was varied between 1-, 2-, or 5-fold the amount of membrane protein used for binding / subunits to the beads. The beads were then washed twice with 60 mM imidazole to remove contaminant proteins and, finally, the /His₁₀-/PLM complex was eluted with 250 mM imidazole. The blot in Fig. 4B shows that PLM was indeed eluted off the beads together with the -subunit, and the amount of eluted PLM was similar whether 1-, 2-, or 5-fold of the soluble membrane protein was used for the reconstitution. The blot of the PLM remaining in the supernatant after reconstitution shows clearly that a significant fraction of PLM remained unbound whether 1-, 2-, or 5-fold of the soluble membrane protein was used. In short, the experiment shows that the lowest amount of total PLM applied to the beads represents a large excess and suffices for optimal reconstitution. Usually a 2-fold excess of membrane protein containing PLM was used for subsequent experiments. Because the PLM is only a minor component of the added membrane protein, one obvious conclusion from the reconstitution is that PLM must interact rather specifically with the / subunits attached to the beads.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Activation of Na+,K+-ATPase activity by Na+ ions. / and //PLM complexes were purified as in Fig. 2. The data in the upper figure show the Na+,K+-ATPase activity at 25 °C and varying Na+ concentrations, with curves fitted to the Hill equation. The lower figure shows the same data and fitted curves for the calculated values of v/Vmax. See also Table 1.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Expression of PLM alone and in vitro reconstitution with / subunits. A, immunoblot of PLM co-expressed with / subunits or expressed alone. B, reconstitution. BD-Talon beads bound with /His10- subunits were incubated with DDM-solubilized membranes expressing PLM alone, in 1-, 2-, or 5-fold amounts compared with the membrane protein used for binding /His10- subunits to the beads (overnight at 4 °C). The figure shows an immunoblot of proteins eluted from the beads incubated without or with the PLM, and unbound PLM in the supernatant removed from the beads after the overnight incubation.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Comparison of effects of PLM on activation of Na+,K+-ATPase activity by Na+ ions for either co-expressed or reconstituted //PLM complexes. / and co-expressed //PLM complexes or reconstituted //PLM complex were purified as in Figs. 2 and 4, respectively. The experiment and data analysis were done exactly as in Fig. 2. See also Table 1.

Fig. 6 demonstrates the effect of PLM on the slow Na⁺-ATPase activity of the protein observed in the absence of K⁺ ions (53, 54). By looking at the Na⁺ dependence of Na⁺-ATPase activity a relatively direct measure of effects of PLM on interaction of Na with the enzyme is obtained and, for reasons given in the "Discussion," the activity was measured in a reaction medium of high ionic strength (250 mM choline chloride plus NaCl) and at a very low concentration of ATP (20 nM). To visualize the effect of PLM on the K_0.5 for activation by Na⁺ ions, the data were plotted as v/V_max versus Na⁺ concentration, averaged for five (control) or six (+PLM) experiments. Evidently, under these conditions, the K_0.5 of Na⁺ ions for the //PLM complex was reduced more than 3-fold lower compared with the / control. The difference in K_0.5 values (control 3.12 ± 0.49 versus +PLM 0.92 ± 0.15) is highly significant (p = 0.0009). This finding has an interesting mechanistic implication and is significant, also, because the magnitude of the effect is much greater than that observed for activation of Na⁺,K⁺-ATPase activity by Na⁺ ions (see "Discussion").

It is striking that, whereas the present results show that PLM lowers K_0.5 of Na⁺ ions for activation of Na⁺,K⁺-ATPase activity in the purified complexes isolated from P. pastoris (Figs. 3, 5, and 6), in Xenopus oocytes PLM has the opposite effect, and raises the K_0.5 of cytoplasmic Na⁺ ions for activation of the active pump current (26). One possible explanation of the difference is that interactions of PLM with / subunits in P. pastoris are non-native or are not fully native. However, the following data makes this quite unlikely. One could imagine, for example, that PLM-/ interactions in purified complexes, exposed to detergent and phospholipids, are altered compared with native PLM-/ interactions in intact membranes or cells. Accordingly, activation of Na⁺,K⁺-ATPase activity by Na⁺ ions was looked at in crude membranes prepared from P. pastoris expressing either / or / plus PLM. Fig. 7 presents the data as a plot of v/V_max averaged for three experiments and shows that PLM reduces K_0.5 by comparison with the control (24.8 ± 1.08 versus 32.1 ± 0.6, p = 0.019), similar to the result with purified detergent-soluble /His₁₀- complexes prepared from those membranes. The somewhat higher K_0.5 values for Na⁺, compared with those in Table 1, could be related to the fact that Na⁺,K⁺-ATPase activity of these membranes was measured at 37 °C and at 1 mM ATP in order to optimize the ouabain-inhibitable fraction of ATP hydrolysis. However, this does not alter the conclusion of the experiment. Another possibility we have considered, and can now exclude, is that a 2 M urea treatment during membrane preparations, described in Ref. 43, partially denatures the PLM and precludes a fully native interaction with the - or -subunits. An essentially identical effect of PLM on the K_0.5 of Na⁺ for activating Na⁺,K⁺-ATPase activity was observed in an experiment using urea-treated or untreated membranes expressing PLM for purification of the //PLM complex (K_0.5 of Na⁺ for control is 17.76 ± 0.93 mM, plus PLM from untreated membranes K_0.5 = 12.66 ± 1.21 mM, plus PLM from urea-treated membranes K_0.5 = 11.52 ± 1.42 mM). Finally, since we are looking at an interaction of human PLM with porcine / subunits one could also hypothesize that some interactions are missing because of the species difference. However, recent work has shown essentially identical effects on the K_0.5 for Na⁺ ions of human PLM interacting with human ₁ in ₁₁ complexes purified from P. pastoris.⁴

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Effect of PLM on activation of Na+-ATPase activity by Na+ ions for the reconstituted //PLM complex. The figure shows Na+-ATPase activity at 20 nM ATP and varying Na+ concentrations for /His10- or reconstituted /His10-/PLM complexes purified on BD-Talon beads. The Na+-ATPase activities in different experiments were in the range 10–30 nmol/mg protein/min without or with PLM.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Effect of PLM on activation of Na+,K+-ATPase activity by Na+ ions in P. pastoris membranes. The data show the Na+,K+-ATPase activity at 37 °C for membranes expressing either - and His10--subunits or , His10-, and PLM. Data were analyzed as in Fig. 2. The values of Na,K-ATPase activity in different experiments were in the range 0.07–0.18 µmol/min/mg protein.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Effects of PLM, CHIF, and a CPC chimera on activation of Na+,K+-ATPase activity by Na+ ions in HeLa cell membranes. Na+,K+-ATPase of HeLa cell membranes was assayed at 37 °C and varying Na+ concentrations as described under "Materials and Methods." The table shows best-fit values of K0.5 ± S.E. of Na+ ions for the average values of v/Vmax at the different Na+ concentrations. The maximal Na+,K+-ATPase activities in different preparations were in the range 40–100 nmol/minute/mg protein. EV, empty vector. CPC-CHIF-PLM-CHIF.

View larger version (59K): [in this window] [in a new window]   FIGURE 9. PLM expressed in P. pastoris is phosphorylated at Ser68. Immunoblot probed with the CP68 antibody: A, P. pastoris membranes expressing PLM not incubated, or incubated with alkaline phosphatase, or alkaline phosphatase in the presence of 0.2 mM -glycerophosphate (Inh). B, as in A, but for the reconstituted /His10-/PLM complex. C, as in B, including also incubation of the /His10-/PLM complex with PKA+ATP/Mg. D, immunoblot probed with the anti-PLM (C terminus) antibody. Conditions were as in C, but included also incubation with alkaline phosphatase after incubation with PKA+ATP/Mg.

View larger version (51K): [in this window] [in a new window]   FIGURE 10. PLM expressed in HeLa cells is not phosphorylated at Ser68. HeLa cell membranes were treated with alkaline phosphatase or with PKA+ATP/Mg. Immunoblot probed with the CP68 antibody.

An appealing hypothesis is that the opposite effects of PLM on the K_0.5 for Na⁺ in P. pastoris and HeLa cells are caused by the different degrees of PLM phosphorylation in these preparations. We attempted to test this hypothesis directly by assaying Na⁺,K⁺-ATPase activity of / complexes purified with PLM that was either phosphorylated or dephosphorylated in vitro. However, the protocols involving additional manipulations and long incubations at 0 °C of //PLM complexes bound to the beads with alkaline phosphatase or PKA, led to variable degrees of inactivation of the enzyme, variable efficiencies of phosphorylation and dephosphorylation of PLM, and inconclusive results. Thus, direct assessment of the role of PLM phosphorylation on K_0.5 for Na⁺ awaits future experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The work in this article demonstrates the isolation and initial functional characterization of partially purified complexes consisting of porcine / subunits of Na⁺,K⁺-ATPase and human FXYD1 (PLM) expressed in P. pastoris, and also a comparison with functional effects of PLM expressed in HeLa cells.

One effect of the PLM is stabilization of the / complex. This is seen as a 20–30% increase in V_max of Na⁺,K⁺-ATPase and phosphoenzyme level with no change in the turnover number (Table 2). Because this feature is observed even in reconstitution experiments, it shows that PLM protects against partial inactivation occurring during the purification procedure.

A possible mechanism of action of PLM in the P. pastoris system is suggested by a comparison of its effects on Na⁺,K⁺-ATPase (Figs. 3 and 5) and Na⁺-ATPase activity (Fig. 6). From basic enzyme kinetic principles a PLM-induced reduction of K_0.5 of Na⁺ ions for activating Na⁺,K⁺-ATPase activity, without simultaneous effects on the Na⁺,K⁺-ATPase turnover rate or K_m for ATP, is compatible with an increased intrinsic binding affinity of Na⁺ ions. In this respect the observed effect of PLM on activation of Na⁺-ATPase activity by Na⁺ ions is interesting. In open membrane preparations, with access of the Na⁺ ions to both cytoplasmic and extracellular sites, the Na⁺-ATPase activity shows a rather complex dependence on Na⁺ ion concentration (55). Activation of phosphorylation at 0–5 mM Na⁺ at normal cytoplasmic sites is followed by partial inhibition at 5–10 mM Na⁺ and then further activation at 10–150 mM Na⁺, because of Na⁺ binding at extracellular sites and effects on hydrolysis of the E₂-P intermediate (53, 54, 56). To avoid these complexities the Na⁺-ATPase activity of the detergent soluble complexes was measured at a high ionic strength, which strongly stabilizes the E₁ conformation, and at a very low ATP concentration (20 nM), which should make the phosphorylation of E₁ rate-limiting. In this situation one could expect the Na⁺ dependence of the Na⁺-ATPase to reflect primarily binding of Na⁺ ions to the cytoplasmic sites. The observed Na⁺ activation curves are indeed quite simple, reaching saturation at about 5 mM Na⁺. The relatively large difference in the K_0.5 for Na⁺ without and with PLM, greater than 3-fold under these conditions, is consistent with an increased binding affinity of Na⁺ ions induced by PLM. By contrast, the more modest effect observed in the Na⁺,K⁺-ATPase reaction conditions is expected, because the K_0.5 for Na⁺ ions is a less direct measurement, reflecting not only Na⁺ binding but also competition with K⁺ ions and rate constants of other steps in the catalytic cycle.

The effect of PLM to reduce K_0.5 of Na⁺ for activating Na⁺,K⁺-ATPase in the P. pastoris system is a striking difference from that observed previously in Xenopus oocytes, in which PLM significantly raises K_0.5 for Na⁺ (26) and, as shown here, also in the HeLa cell system. By contrast, for the chimeric protein CPC, a reduced K_0.5 for Na⁺ was observed, compared with the control, similar to the effect of full-length PLM in the P. pastoris (Fig. 8). Thus, the comparison with HeLa cell experiments shows that the observed functional effect depends on the cell type. An economical explanation of the opposite effects of full-length PLM and the CPC chimera on the K_0.5 of Na⁺ in the HeLa cell membrane is the existence of at least two types of structural and functional interaction of the full-length PLM. For example, the interaction of the transmembrane segment of PLM with M2, M6, and M9 of the -subunit could raise binding affinity for Na⁺ ions in the Na⁺ occlusion sites, thus reducing K_0.5 of Na⁺ for activation of ATPase activity, while interactions of the extramembrane sectors of PLM could stabilizes E₂ conformations, thus indirectly raising the K_0.5 of Na⁺. The overall effect on the K_0.5 of Na⁺ of full-length PLM would reflect the balance of these interactions, and in the CPC chimera only the transmembrane interaction would persist. Of course, this explanation is both hypothetical and is not exclusive, but there is a convincing precedent for multiple structural and functional interactions of at least one other FXYD proteins, the -subunit (FXYD2) see Ref. 57.

A second striking difference between the PLM expressed in P. pastoris and HeLa cells is that the PLM in P. pastoris is significantly phosphorylated at the PKA site (Ser⁶⁸) (Fig. 9) whereas that in HeLa cells is hardly phosphorylated at all (Fig. 10). Obviously, the opposite functional effects of PLM on K_0.5 for Na⁺ ions in P. pastoris and HeLa could be related to this different state of phosphorylation. In principle, one could envisage that PKA-dependent phosphorylation of Ser⁶⁸ disrupts the interaction between the cytoplasmic C terminus of PLM and / subunits, leaving intact the interaction between the transmembrane segments of PLM and . This is similar in concept to one proposed for the structural effects of PKC-dependent phosphorylation of the PLM-like protein (PLMS) in shark rectal gland Na⁺,K⁺-ATPase, (1, 33, 34).

How does the information in this article add to and fit in with the reported physiological effects of PLM and modulation of PLM-/ interactions by phosphorylation? The reduced Na⁺ affinity observed upon expression of PLM in HeLa cells (Fig. 8) is attributable to the non-phosphorylated form of PLM, shown in Fig. 10. The same may be true also for Xenopus oocytes (26). As argued recently in reviews (3, 57), a reduced Na⁺ affinity ensures that the pumping rate is not saturated with respect to cytoplasmic Na⁺ concentration, and may be required for regulation of pumping during basal electrical and mechanical activity in heart cells. During -adrenergic activation the increased cardiac contractility is associated with an increased Na⁺ concentrations and a large transient rise in cytoplasmic Ca²⁺ ion. The increase in cytoplasmic Na⁺ concentrations must be limited during -adrenergic activity, and restored to the basal level after activation, by increased Na⁺ extrusion via the Na⁺,K⁺-ATPase (58). By functional coupling with the 3 Na⁺/Ca²⁺ exchange mechanism the Ca²⁺ ion concentration then returns to the basal level. There are two relevant recent observations on intact ventricular myocytes. It has been shown that activation of PKA induced by forskolin (29) or isoproterenol, a -adrenergic agonist, (30) is associated with phosphorylation of PLM at S68. In (29) the Na⁺,K⁺-pump current was elevated and this was restricted specifically to the 1 and not the 2 isoform. In Ref. 30, it was found that in myocytes from wild-type mice the curve for activation of the pump current by Na⁺ is shifted to the left upon treatment with isoproterenol, while in myocytes obtained from PLM knock-out mice (see Ref. 27), no effect of isoproterenol was observed. This led to the suggestion that phosphorylation of PLM at Ser⁶⁸ raises the apparent affinity of Na⁺ by neutralizing the modulatory effect of non-phosphorylated PLM, which acts to reduce the apparent affinity for Na⁺ ions. The final result is activation of active Na⁺ and K⁺ transport during the -adrenergic stimulation, accelerated restoration of Na⁺ and Ca²⁺ gradients, and cardiac muscle relaxation. The present results are completely consistent with this scenario at the physiological level, but there is an important difference in interpretation at the level of PLM-/ interactions. As discussed above, the model best compatible with our finding is that phosphorylation at the PKA site abrogates one type of interaction, but leaves another interaction intact. The elevated Na affinity is the result of this interaction with phosphorylated PLM, and is not due merely to abrogation of the interaction with non-phosphorylated PLM. Although the native myocyte is obviously superior for defining the physiological role of the PLM, the different expression systems allow one to detect effects of PLM compared with the control without PLM, and thus better define the interactions with the / subunits.

In PLM knock-out mice the V_max of the Na⁺,K⁺-ATPase in isolated heart sarcolemma membranes was 50% lower than in the wild type, even allowing for lower expression levels of both 1 and 2 (27). The implication is that PLM increases the turnover rate of the Na⁺,K⁺-ATPase. This is different from observations in Xenopus oocytes, mouse myocytes, and the P. pastoris system, which did not show effects of PLM on V_max, but rather on the apparent Na⁺ affinity. Conceivably the PLM is in a different state in the wild-type mouse heart to that in the expression systems or isolated mouse myocytes. In this regard, it is important to remember that PLM has both PKA sites (at Ser⁶⁸) and a PKC site (Ser⁶³ and Ser⁶⁸) and is also the substrate of other kinases (21, 22). The state of phosphorylation of PLM at Ser⁶³ in the Xenopus oocytes, or P. pastoris membranes or mouse myocyte membranes is unknown. There might be complex interactions between PLM phosphorylated at say Ser⁶³ and Ser⁶⁸, and the / subunits, and thus multiple functional effects.

Perspective—Expression of / subunits and PLM in P. pastoris, combined with purification of / and reconstitution of //PLM complexes, provides a well controlled system for looking at interactions between / subunits and PLM. Detailed investigation of the mechanism of action of PLM, by direct measurement of cation occlusion or conformational changes detected with fluorescence probes should become possible, after upscaling of the amounts of the purified / and //PLM complexes. Upscaling is now being undertaken.⁶ Eventually, structural studies may become feasible.

	FOOTNOTES

 
^* This work was supported by the Weizmann Institute Renal Research Fund, the Josef Cohen Minerva center for Biomembranes, and the Israel Science Foundation. 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.

¹ Incumbent of the Hella and Derrick Kleeman Chair of Biochemistry.

² Incumbent of the William Smithburg Chair of Biochemistry. To whom correspondence should be addressed: Dept. of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel. Tel.: 972-8-934-2278; Fax: 972-8-934-4118; E-mail: Steven.Karlish{at}weizmann.ac.il.

³ The abbreviations used are: CHIF, corticosteroid hormone-induced factor; PLM, phospholemman; DOPS, 1,2-oleoyl-sn-glycero-3-[phospho-L-serine] or dioleoyl phosphatidylserine; C₁₂E_10, polyoxyethylene(10)dodecyl ether; Ni-NTA, nickel-trinitriloacetic acid; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AP, alkaline phosphatase.

⁴ E. Cohen, H. Haviv, R. Goldshleger, and S. J. D. Karlish, manuscript in preparation.

⁵ Because of variation of expression levels of the -subunit in the different clones, it is not possible to reliably detect effects of expression of the FXYD proteins on the V_max values.

⁶ E. Cohen and H. Haviv, unpublished experiments.

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