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J. Biol. Chem., Vol. 283, Issue 1, 476-486, January 4, 2008

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Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes^*

From the Department of Pharmacology and Toxicology, University of Lausanne, 27 Rue du Bugnon, 1005 Lausanne, Switzerland

Received for publication, July 16, 2007 , and in revised form, November 6, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K⁺ and Na⁺ affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481[Abstract/Free Full Text] ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K⁺ affinity of Na,K-ATPase 1/β1 and 2/β1 isozymes but increases their apparent Na⁺ affinity, dependent on phospholemman phosphorylation at Ser⁶⁸. Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of 1/β1 isozymes nor the K⁺ affinity of 1/β1 and 2/β1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of 2/β1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase 1/β and 2/β isozymes and increases their apparent Na⁺ affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase 2/β but not of 1/β isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phospholemman (PLM)² or FXYD1 is one of the seven members of the FXYD protein family (1), which are small single span membrane proteins exposing their C terminus to the cytoplasmic side. Based on the observation that PLM can form ion-selective channels, it was suggested that PLM might be implicated in cell volume regulation (2). More recently, however, PLM, similar to other FXYD proteins, was identified as a tissue-specific regulator of Na,K-ATPase (3, 4), a ubiquitous cation transporter responsible for the maintenance of the Na⁺ and K⁺ gradients of animal cells. PLM is abundantly expressed in brain (5) and in cardiac and skeletal muscle (6), where it is associated with Na,K-ATPase 1 and to a lesser extent with 2 isoforms (7-9). Expressed together with Na,K-ATPase in Xenopus oocytes (7) or in HeLa cells (10), PLM significantly reduces the Na⁺ affinity and to a lesser extent the K⁺ affinity of Na,K-ATPase (7) but has no effect on its maximal transport rate. These results suggested that regulation of Na,K-ATPase by PLM might be important to control intracellular Na⁺ concentrations and in turn intracellular Ca²⁺ concentrations necessary for appropriate muscle contractility.

The reduced Na⁺ affinity of Na,K-ATPase induced by PLM in certain expression systems indicates an inhibitory effect on Na,K-ATPase. This is supported by the observation that Na,K-ATPase activity increases in myocytes of PLM knock-out mice (11-13) and decreases in cardiac myocytes overexpressing PLM (14). In some reports, however, inhibition of Na,K-ATPase by PLM is attributed to an effect of PLM on the apparent Na⁺ affinity (13), whereas in other studies, PLM produced no effect on the apparent Na⁺ affinity but only on maximal pump rates (V_max) of Na,K-ATPase (14). Moreover, when expressed in Pichia pastoris together with Na,K-ATPase, PLM increases the apparent Na⁺ affinity of Na,K-ATPase (10). The matter is complicated further by reports that show a reduced rather than increased Na,K-ATPase activity in PLM-deficient cardiac sarcolemma (15).

A possible explanation for the apparently conflicting results on the effect of PLM on Na,K-ATPase activity may lie in the fact that PLM is subjected to phosphorylation by different protein kinases, which may differ in different experimental conditions. PLM has long been known as the main sarcolemmal substrate for protein kinases A (PKA) and C (PKC) in heart and skeletal muscle (16). PKA phosphorylates Ser⁶⁸, and PKC phosphorylates both Ser⁶³ and Ser⁶⁸ in the cytoplasmic domain of PLM (17). In myocytes, PLM becomes phosphorylated on Ser⁶⁸ upon forskolin treatment (9, 13) and on Ser⁶³ and Ser⁶⁸ upon PKC activation (11).

Regulation of Na,K-ATPase activity by activation of PKA and PKC has been described in various tissues (18), but it has remained unclear whether this occurs by direct phosphorylation of Na,K-ATPase or of an accessory protein. Recent experimental evidence suggests that PKA-dependent phosphorylation of PLM rather than of Na,K-ATPase 1 subunits stimulates 1-specific Na,K-ATPase activity in cardiac myocytes (9). Similarly, isoproterenol treatment phosphorylates PLM and increases Na,K-ATPase activity in cardiac myocytes of wild type mice but not of PLM-deficient mice (13). The increase in Na,K-ATPase activity is mainly attributed to an increase in the apparent Na⁺ affinity of Na,K-ATPase with little effect on V_max (13). On the other hand, Pavlovic et al. (12) reported that phosphorylation of PLM by PKA stimulates Na,K-ATPase to a level above that seen in the absence of PLM, suggesting an important effect on V_max.

Sympathetic stimulation of cardiac myocytes not only involves PKA activation via β-adrenergic receptors but also PKC activation via -adrenergic receptors. A recent study reports that PKC activation of myocytes leads to phosphorylation of PLM at Ser⁶³ and Ser⁶⁸ and increases the maximal Na,K-ATPase-mediated Na⁺ extrusion rate without changing the apparent Na⁺ affinity of Na,K-ATPase (11). PKC activation of myocytes from PLM-deficient mice had no effect on Na,K-ATPase activity, suggesting that PKC-dependent effects are due to PLM rather than phosphorylation of Na,K-ATPase. Interestingly, the effects on Na,K-ATPase of PKA and PKC phosphorylation of PLM are additive (11), opening the possibility that PKA and PKC phosphorylation of PLM regulates different Na,K-ATPase isozymes. Finally, studies on a shark phospholemman-like protein (FXYD10) also show that its phosphorylation by PKC stimulates Na,K-ATPase activity (19).

To contribute to the clarification of the role of PKA and PKC phosphorylation of PLM in the regulation of Na,K-ATPase activity, we have used, in this study, the Xenopus oocyte expression system to address some open questions, which have not been or cannot easily be answered in cardiac myocytes or have produced contradictory results. In particular, we have investigated the effect of PKA and PKC phosphorylation of PLM on the apparent Na⁺ and/or K⁺ affinity and on V_max of Na,K-ATPase and have explored the possibility that PLM phosphorylation might have differential effects on different Na,K-ATPase isozymes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Forskolin and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Calbiochem, and epinephrine and 4-β-phorbol-12,13-dibutyrate (PDBu) were from Sigma.

Mutagenesis and cDNAs—Point mutations were introduced into cDNAs of dog PLM (7) by the PCR-based method. The mutated PLM cDNAs were amplified from dog PLM using a sense primer tailed with an EcoRI site (5'-GCCGAATTCATGGCACCTCTCCACCACATCTTGGTTCTC-3') and antisense primers tailed with a NotI restriction site. The single S68A mutation and double mutations S63A/S68A were introduced using the antisense mutagenic synthetic oligonucleotide primers (5'-GCGGCCGC CTA CCG CCT GCG GGT CGC CAG ACG GCG GAT GGA GCT GCG-3'/5'-GCGGCCGC CTA CCG CCT GCG GGT CGC CAG ACG GCG GAT CGC GCT GCG). The double mutations S63D/S68D were introduced using the antisense mutagenic synthetic oligonucleotide primers (5'-GCGGCCGC CTA CCG CCT GCG GGT ATC CAG ACG GCG GAT GGA GCT GCG-3'/5'-GCGGCCGC CTA CCG CCT GCG GGT ATC CAG ACG GCG GAT ATC GCT GCG). Mutated nucleic acids are underlined. All products were cloned into a pSD5 vector and sequenced. cDNAs of rat Na,K-ATPase 1 (20), rat ouabain-resistant 2 (21), and rat β1 subunits (22) (all kindly provided by J. Lingrel; University of Cincinnati, Cincinnati, OH); human Na,K-ATPase 1, 2, and β1 subunits (23); human β2 adrenergic receptor (24) (kindly provided by S. Cotecchia, University of Lausanne, Switzerland); and dog PLM (23) were subcloned into a pSD5 vector. cRNAs were prepared by in vitro translation (25).

Expression and Association of Wild Type and Mutant PLM in Xenopus Oocytes—Stage V-VI oocytes were obtained from Xenopus laevis as described (26). Oocytes were injected with rat 1 or 2 subunit cRNA (10 ng) and β1 subunit cRNA (1 ng) alone or together with wild type or mutant phospholemman cRNAs (3 ng). To study protein expression and PLM association, cRNA-injected oocytes were incubated in modified Barth's solution in the presence of 1 mCi/ml [³⁵S]methionine (Easy Tag Express [³⁵S]Protein Labeling Kit; PerkinElmer Life Sciences) for 6 h and subjected to 24- and 48-h chase periods in modified Barth's solution containing 10 mM unlabeled methionine. After the pulse and chase periods, microsomes were prepared and subjected to immunoprecipitations under nondenaturing conditions as described (26) with an antibody against the Na,K-ATPase subunit (27), and proteins were resolved by SDS-PAGE and revealed by fluorography.

Phosphorylation by PKA and PKC and Detection of PLM—Phosphorylation of wild type and mutant PLM after PKA or PKC activation was studied after incubation of cRNA-injected oocytes for 48 h at 19 °C. PKA was activated by incubation of oocytes in modified Barth's solution containing 10 µM forskolin and 1 mM IBMX for 4 h or by incubation of oocytes expressing the β2-adrenergic receptor in modified Barth's solution containing 0.1 mM epinephrine for 2 min. Preliminary experiments showed that removal of PKA activators led to dephosphorylation of PLM within 1 h (data not shown). Therefore, long term experiments, such as measurements of the apparent Na⁺ affinity (see below), were performed in the continuous presence of epinephrine, which maintained phosphorylation. PKC was activated by incubation of oocytes in modified Barth's solution containing 100 nM PDBu for 3 or 10 min. In some experiments, oocytes were continuously incubated with PDBu over a period of 15 min.

The phosphorylation state of PLM was assessed before and after PKA or PKC activation and at the end of electrophysiological measurements by preparing oocyte microsomes with buffers containing 100 mM NaF, 10 mM sodium pyrophosphate, and 10 mM sodium orthovanadate to inhibit phosphatases. The protein content was determined by the method of Lowry. Microsomal proteins were subjected to SDS-PAGE (10-50 µg) or to immunoprecipitations (100 µg) under nondenaturing conditions with a Na,K-ATPase subunit antibody and then transferred overnight at 40 V to nitrocellulose membranes. Membranes were blocked with 10% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 and incubated with primary antibody. The following primary antibodies were used: 1) a PLM antibody recognizing PLM, which is not phosphorylated at Ser⁶⁸ (10) (1:500) (kindly provided by H. Garty and S. Karlish, Weizmann Institute, Israel) (we show in this study that this antibody recognizes the S68A and S68D PLM mutants, suggesting that it is the phosphate moiety that impedes recognition of PLM by this antibody); 2) a PLM antibody CP68 recognizing PLM phsphorylated at Ser68 (28) (1/10'000) (kindly provided by D. M. Bers (Loyola University, Chicago, IL)); and 3) a Na,K-ATPase subunit antibody (1:10,000) (27). After binding of the primary antibody, peroxidase-coupled secondary antibodies (1:10,000; Amersham Biosciences) were bound, and the complex was revealed with the ECL chemiluminescence kit (Amersham Biosciences) according to the manufacturer's protocol. Quantification of nonphosphorylated and phosphorylated PLM was performed with an ultrascan laser densitometer (LKB Bromma).

Determination of the Apparent K⁺ and Na⁺ Affinity of Na,K-ATPase—Electrophysiological measurements were performed 2 days after oocyte injections with rat 1 (10 ng) or ouabain-resistant 2 (12 ng) and β1 (1 ng) cRNAs with or without wild type (3 ng) or mutant PLM (3 ng) cRNAs by using the two-electrode voltage clamp technique. Oocytes were loaded with Na⁺ by overnight incubation in a K⁺-free medium (30). Measurements of the apparent external K⁺ affinity were carried out as described (29) in the presence of 1 µM ouabain that inhibits the endogenous oocyte Na,K-ATPase but not the expressed ouabain-resistant rat Na,K-ATPase. The apparent K⁺ affinity (KK⁺) measured in the presence of external Na⁺ (100 mM) was obtained by fitting the Hill equation to the data using a Hill coefficient of 1.6 (30). Measurements of the apparent Na⁺ affinity of Na,K-ATPase were performed as described (31) after co-expressing rat 1 or 2 and β1 cRNAs together with rat epithelial Na⁺ channel , β, and subunit (0.5 ng each) cRNAs in the presence or absence of PLM cRNAs. The apparent Na⁺ affinity (KNa⁺) was obtained by fitting the Hill equation by using a Hill coefficient of 3 (31). Statistical analysis was performed by unpaired Student's t test.

For the determination of effects of PKA stimulation on the apparent external K⁺ affinity of Na,K-ATPase, oocytes were incubated for 4 h in modified Barth's solution containing 10 µM forskolin and 1 mM IBMX before measurements.

For the determination of effects of PKA stimulation on the apparent K⁺ and Na⁺ affinities of Na,K-ATPase, oocytes were injected with Na,K-ATPase cRNA alone or together with wild type or mutant PLM plus β2 adrenergic receptor cRNA, and the β2 adrenergic receptor signaling pathway was activated by incubation of oocytes for 2 min with 0.1 mM epinephrine before electrophysiological measurements. Epinephrine was present during the duration of electrophysiological measurements (e.g. for about 3 min for K⁺ affinity measurements and about 1 h for Na⁺ affinity measurements).

For the determination of effects of PKC stimulation on the apparent external K⁺ affinity of Na,K-ATPase, oocytes were incubated for 3 min in modified Barth's solution containing 100 nM PDBu before measurements.

Maximal Na,K-ATPase Current Measurements—Measurements of maximal Na,K-pump currents (I_max) were performed by the two-electrode voltage clamp technique 2 days after oocyte injection with human Na,K-ATPase 1 or 2 cRNAs and β1 cRNA in the presence or absence of wild type or mutant PLM cRNAs. Oocytes were loaded with Na⁺ by overnight incubation in a K⁺-free medium (30). The membrane potential was set at -50 mV, and the Na,K-pump current was measured at room temperature as the outward current induced by the addition of 10 mM K⁺.

For the determination of effects of PKA stimulation on I_max of Na,K-ATPase, oocytes were treated for 4 h with 10 µM forskolin and 1 mM IBMX before measurements. I_max measurements were performed within less than 1 min. Moreover, the effect of PKA activation on I_max of Na,K-ATPase 1/β1 isozymes was determined by the following protocol. I_max was determined on oocytes by the addition of 10 mM K⁺ until plateau values were reached. K⁺ was removed, 0.1 mM epinephrine was added, and K⁺-induced currents were measured after 2, 5, 10, and 15 min in the continuous presence of epinephrine.

For the determination of effects of PKC stimulation on I_max of Na,K-ATPase, oocytes were treated for 3 or 10 min with 100 nM PDBu before measurements or in the continuous presence of PDBu over a 15-min time period. In separate experiments, it was verified that the K⁺-induced currents corresponded to ouabain-inhibitable Na,K-pump currents (data not shown).

[³H]Ouabain Binding on Intact Oocytes—Two days after injection of oocytes with human 1 or 2 cRNAs and β1 cRNA in the presence or absence of wild type or mutant PLM cRNAs, the total number of Na,K-ATPase isozymes expressed at the cell surface was determined by [³H]ouabain binding essentially as described (26). Briefly, oocytes were loaded with Na⁺ in a K⁺-free solution containing 90 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM Hepes, pH 7.4, for 2 h at 19 °C. Oocytes were then incubated at room temperature in a K⁺-free solution containing 0.3 µM [21,22-³H]ouabain (Amersham Biosciences; specific activity 15 Ci/mmol) and 0.7 µM of cold ouabain for 30 min. Oocytes were extensively washed with a buffer containing 90 mM NaCl, 30 mM imidazole, pH 7.4, individually transferred to scintillation tubes, and solubilized with 100 ml of 5% SDS. Solubilized oocytes were counted after the addition of 2 ml of Scintillator 299 (Packard). Ouabain binding measured in noninjected oocytes was subtracted from values obtained in cRNA-injected oocytes. Previous experiments have shown that non-specific binding determined in the presence of a 1000-fold excess of cold ouabain amounts to 3-7% of total [³H]ouabain binding (26). For the determination of effects of PKA stimulation on cell surface expression of Na,K-ATPase, oocytes were treated for 4 h with 10 µM forskolin and 1 mM IBMX before incubation with [³H]ouabain. For the determination of effects of PKC stimulation on cell surface expression of Na,K-ATPase, oocytes were treated for 3 or 10 min before incubation with [³H]ouabain.

Turnover Number of Na,K-ATPase—The turnover number of Na,K-ATPase 1/β1 and 2/β1 was calculated as the ratio between the maximal Na,K-pump current and the number of ouabain binding sites.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To study the role of PLM phosphorylation on Na,K-ATPase expressed in Xenopus oocytes, we have produced the following PLM mutants: 1) a S68A mutant in which the PKA phosphorylation site (17) was abolished; 2) a S63A/S68A mutant in which the two PKC phosphorylation sites (17) were abolished; and 3) a S63D/S68D mutant that mimics constitutive phosphorylation of PLM. To test whether these mutants associate with Na,K-ATPase, they were expressed in Xenopus oocytes together with Na,K-ATPase and metabolically labeled. Oocyte microsomes were then subjected to immunoprecipitation with a Na,K-ATPase subunit antibody under nondenaturing immunoprecipitation. Similar to wild type PLM (Fig. 1, lanes 4-6), all three mutants could be co-immunoprecipitated with a Na,K-ATPase subunit antibody over prolonged chase periods (lanes 7-15), indicating that they can stably associate with Na,K-ATPase.

View larger version (72K): [in this window] [in a new window]   FIGURE 1. Association of wild type and mutant PLM with Na,K-ATPase. Ten ng of rat Na,K-ATPase 1 subunit cRNA plus 1 ng of Na,K-ATPase β1 subunit cRNA were injected in Xenopus oocytes in the absence (lanes 1-3) or presence of 3 ng of wild type (lanes 4-6) or mutant PLM cRNAs (lanes 7-15). Xenopus oocytes were metabolically labeled with [35S]methionine for 6 h, followed by 24- and 48-h chase periods. Microsomes were prepared and subjected to immunoprecipitation under nondenaturing conditions with antibodies against the Na,K-ATPase subunit. Complexes were separated by SDS-PAGE and revealed by fluorography. cg, coreglycosylated β subunit; fg, fully glycosylated β subunit. One of two similar experiments is shown.

PKA Phosphorylation of PLM Does Not Influence the Apparent K⁺ Affinity of Na,K-ATPase 1/β1 and 2/β1 Isozymes—Based on the observation that the effect of PKA and PKC phosphorylation of PLM on Na,K-ATPase activity in myocytes was additive, despite shared phosphorylation sites, it was suggested that PKA and PKC regulation may target different Na,K-ATPase isozymes (11). We tested this hypothesis and determined the effect of PKA phosphorylation of PLM on the transport properties of Na,K-ATPase 1/β1 and 2/β1 isozymes present in the human heart.

We first tested whether PKA phosphorylation of PLM affects the effect of PLM on the K⁺ affinity of rat Na,K-ATPase isozymes. As shown in Fig. 2B, forskolin treatment of oocytes expressing Na,K-ATPase 1/β1 isozymes without PLM had no effect on the apparent K⁺ affinity of Na,K-ATPase. As previously described (7), co-expression of Na,K-ATPase 1/β1 isozymes with PLM slightly decreased its apparent K⁺ affinity over a large range of membrane potentials. This effect on the K⁺ affinity was not altered by forskolin treatment (Fig. 2B). Similar results were obtained when oocytes, expressing β-adrenergic receptors together with PLM and/or Na,K-ATPase, were treated with epinephrine (Fig. 2C). Consistent with these results, the phosphorylation site mutant S63A/S68A and the constitutive phosphorylation mutant S63D/S68D mutant did not influence the effect of PLM on the K⁺ affinity of Na,K-ATPase 1/β1 isozymes (Fig. 2D). Parallel analysis of the phosphorylation state of PLM showed that epinephrine treatment reduced the amount of the wild type PLM population not phosphorylated at Ser⁶⁸ by about 60% but had no effect on the S63A/S68A and the S63D/S68D mutants (Fig. 2, E and F).

Similar experiments performed in oocytes expressing Na,K-ATPase 2/β1 isozymes with PLM showed that phosphorylation of PLM by PKA also did not influence the effect of PLM on the K⁺ affinity of 2/β1 isozymes (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Functional effects of PKA and PKC phosphorylation of PLM on Na,K-ATPase 1/β1 and 2/β1 isozymes

Similar experiments performed in oocytes expressing Na,K-ATPase 2/β1 isozymes with PLM showed that phosphorylation of PLM by PKA also abolished the effect of PLM on the Na⁺ affinity of 2/β1 isozymes (Table 1).

PKA Phosphorylation of PLM Does Not Influence the Maximal Na,K-ATPase Activity—Contradictory results have been reported concerning the mechanism by which PKA phosphorylation of PLM increases Na,K-ATPase activity in cardiac myocytes. Increased Na,K-ATPase activity has either been attributed to an increase in its apparent Na⁺ affinity (13) or to an increase in V_max (12). To test whether PLM phosphorylation by PKA increases not only the apparent Na⁺ affinity of Na,K-ATPase, as confirmed in this study, but also its V_max, we investigated whether PLM phosphorylation by PKA influences the maximal Na,K-pump currents (I_max) in Xenopus oocytes. Forskolin treatment of oocytes had no effect on I_max of human Na,K-ATPase 1/β1 isozymes expressed alone or together with wild type PLM or the S63A/S68A mutant (Fig. 4A). Analysis of the phosphorylation state confirmed that wild type PLM but not the S63A/S68A mutant was phosphorylated after the electrophysiological measurements (Fig. 4D). The pool of nonphosphorylated, wild type PLM decreased by about 88% after forskolin treatment (Fig. 4E). Since I_max is determined either by the cell surface expression or by the turnover number of Na,K-pumps, we performed [³H]ouabain binding on intact oocytes to measure the number of Na,K-ATPase isozymes at the cell surface. Forskolin treatment did not influence the cell surface expression of Na,K-ATPase expressed alone or together with wild type PLM or the S63A/S68A mutant (Fig. 4B). Thus, PLM phosphorylation by PKA has no effect on the cell surface expression or the turnover number (Fig. 4C) of Na,K-pumps. The lack of an effect of PKA phosphorylation of PLM on I_max of Na,K-ATPase was confirmed by continuous incubation of oocytes with epinephrine over a 15-min time period (Fig. 4F) during which phosphorylation of PLM was maintained (Fig. 4G).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. PLM phosphorylation by PKA has no effect on the apparent K+ affinity of Na,K-ATPase 1/β1 isozymes. A, phosphorylated PLM is associated with Na,K-ATPase. Two days after injection of rat Na,K-ATPase 1 plus β1 subunit cRNAs together with 3 ng of wild type PLM cRNA, oocytes were incubated for 4 h with forskolin. Microsomal proteins were either directly loaded on a gel (50µg) (lanes 1 and 2) or immunoprecipitated (IP) (100µg) under nondenaturing conditions with an antibody against Na,K-ATPase subunit (lanes 3 and 4). Proteins were transferred onto nitrocellulose membranes and immunodetected with an antibody recognizing PLM phosphorylated on Ser68 (CP68). After stripping of nitrocellulose membranes, PLM was revealed by an antibody recognizing PLM not phosphorylated at Ser68 (non-phosph PLM). B, 2 days after injection of Na,K-ATPase 1 plus β1 subunit cRNAs alone or together with 3 ng of wild type PLM cRNA, oocytes were incubated or not for 4 h with forskolin. The apparent K+ affinity of Na,K-ATPase, KK+ was determined by two-electrode voltage clamp techniques over a range of membrane potentials. C and D, 2 days after injection of Na,K-ATPase 1 plus β1 subunit cRNAs and of β2 adrenergic receptor (5 ng) cRNA, alone or together with 3 ng of wild type (C) or mutant (D) PLM cRNA, the apparent K+ affinity of Na,K-ATPase (KK) was determined after pretreatment or not of oocytes for 2 min with 0.1 mM epinephrine and in the continuous presence or absence of the activator, respectively. D, only the KK values at a membrane potential of -50 mV are represented. For all measurements, the endogenous oocyte Na,K-ATPase was inhibited by the presence of 1 µM ouabain. Closed diamonds, nonactivated oocytes expressing Na,K-ATPase (B and C); open diamonds, forskolin-activated (B) or epinephrine-activated (C) oocytes expressing Na,K-ATPase; closed squares, nonactivated oocytes expressing Na,K-ATPase plus PLM (B and C); open squares, forskolin-activated (B) or epinephrine-activated (C) oocytes expressing Na,K-ATPase plus PLM. Shown are means ± S.E. of 15 oocytes from three different batches. E, after electrophysiological measurements, oocyte microsomes were prepared, 50 µg of proteins were loaded on a gel and transferred onto nitrocellulose membranes, and PLM was immunodetected with an antibody recognizing PLM not phosphorylated at Ser68. After stripping, the nitrocellulose membrane was probed with a Na,K-ATPase subunit antibody. F, quantification of the amount of PLM not phosphorylated at Ser68 remaining after epinephrine treatment and electrophysiological measurements. Shown are arbitrary units obtained by densitometric scanning of data shown in E normalized by the amount of Na,K-ATPase subunit. Shown are means ± S.E. of four independent experiments.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. PLM phosphorylation by PKA increases the apparent Na+ affinity of Na,K-ATPase 1/β1 isozymes. A, one day after injection of rat Na,K-ATPase 1 and β1 subunit cRNAs plus β2 adrenergic receptor cRNA, alone or together with wild type or mutant PLM cRNAs, the epithelial Na+ channel subunit cRNAs (0.5 ng of , β, and subunit cRNAs) were injected. The following day, the apparent Na+ affinity of Na,K-ATPase (KNa+) was determined at -50 mV in the presence or absence of 0.1 µM epinephrine. Shown are means ± S.E. of 10 oocytes from five different batches. p < 0.01, 1/β1, and PLM without epinephrine versus plus epinephrine and versus 1/β1 and S63D/S68D without and plus epinephrine. B, microsomes were prepared from oocytes treated with epinephrine for the duration of the electrophysiological measurements and 50 µg of proteins were loaded on a gel, transferred onto nitrocellulose membranes, and probed with an antibody recognizing PLM not phosphorylated at Ser68 (bottom). After stripping of nitrocellulose membrane, the amount of Na,K-ATPase subunit was revealed with a Na,K-ATPase subunit antibody (top). C, quantification of PLM not phosphorylated at Ser68 after epinephrine stimulation and electrophysiological measurements as shown in B and normalized by the amount of Na,K-ATPase subunit. Shown are means ± S.E. of three independent experiments.

PKC Phosphorylation of PLM Increases the Maximal Activity of Na,K-ATPase 2/β1 Isozymes—We next tested the effect of PKA and PKC phosphorylation of PLM on I_max of human Na,K-ATPase 2/β1 isozymes. Neither forskolin nor phorbol ester treatment of oocytes had an effect on I_max of Na,K-ATPase expressed without PLM (Fig. 6A, bars 1-4). Forskolin also did not affect I_max of Na,K-ATPase 2/β1 isozymes expressed with PLM (bars 5 and 6). Interestingly, however, phorbol ester treatment for 3 min significantly increased I_max of 2/β1 isozymes expressed with PLM by about 30% (bar 7), in parallel with PLM phosphorylation (Fig. 7, A and B). After 10 min of phorbol ester treatment, the effect on I_max was lost (Fig. 6A, bar 8), coinciding with a decrease in phosphorylation of PLM (Fig. 7C, lane 10). Consistent with an effect of PLM phosphorylation on I_max of 2/β1 isozymes, the constitutive phosphorylation mutant S63D/S68D produced a similar increase in I_max of 2/β1 isozymes in the absence and in the presence of phorbol ester for 3 min (Fig. 6A, bars 9 and 10). A slight decrease of I_max of 2/β1 isoforms was observed after exposure of oocytes to phorbol esters for 10 min (bar 11), suggesting that compensatory mechanisms, independent of PLM phosphorylation, might operate during prolonged phorbol ester treatment. In any case, the increase in I_max of 2/β1 isozymes after phorbol ester treatment for 3 min is entirely due to PLM phosphorylation, since the double S63A/S68A (bars 12 and 13) as well as the single S68A (bars 15 and 16) mutant did not produce an effect on I_max of 2/β1 isozymes. This was also confirmed in an experiment of continuous exposure to PDBu, which showed that maximal activation of I_max of 2/β1 isozymes associated with wild type PLM was reached after about 5 min, whereas that of 2/β1 isozymes associated with the S68A mutant was not affected. Moreover, I_max of 2/β1 isozymes associated with the S63D/S68D mutant was already increased in the absence of PDBu to the level of I_max of 2/β1 isozymes associated with wild type PLM treated for 5 min with PDBu (Fig. 6D).

View larger version (31K): [in this window] [in a new window]   FIGURE 4. PLM phosphorylation by PKA has no effect on the turnover number of Na,K-ATPase 1/β1 isozymes. Two days after injection of human Na,K-ATPase 1 (10 ng) and β1 (1 ng), subunit cRNAs alone or together with 3 ng of wild type or mutant PLM cRNA, oocytes were incubated or not for 4 h with forskolin. A, maximal Na,K-pump currents (Imax) at a membrane potential of -50 mV. The pump currents activated by 10 mM K+ were measured by the two-electrode voltage-clamp technique in Na+-loaded oocytes. Na,K-ATPase currents measured in noninjected oocytes (35-55 nA) were subtracted. Data are means ± S.E. of 20 oocytes from four different batches. B,[3H]ouabain binding to intact oocytes. Values obtained in noninjected oocytes (without forskolin, 150-203 dpm; with forskolin, 154-227 dpm) were subtracted. Data are means ± S.E. of 30 oocytes from four different batches. C, turnover number of Na,K-ATPase. The turnover number (charges transported/s/molecule) was calculated as the ratio between the maximal Na,K-pump current and the number of ouabain binding sites. D, after electrophysiological measurements, oocyte microsomes were prepared, 50 µg of proteins were loaded on a gel and transferred onto nitrocellulose membranes, and PLM was immunodetected with an antibody recognizing PLM not phosphorylated at Ser68. After stripping, the nitrocellulose membrane was probed with a Na,K-ATPase subunit antibody. E, quantification of the amount of PLM not phosphorylated at Ser68 remaining after forskolin treatment and electrophysiological measurements. Shown are arbitrary units obtained by densitometric scanning of data shown in D normalized by the amount of Na,K-ATPase subunit. Shown are means ± S.E. of two independent experiments. F, 2 days after injection of human 1 and β1 cRNAs in the absence (closed diamonds) or presence of wild type PLM (closed squares) cRNAs, Imax of Na,K-ATPase was measured by the addition of 10 mM K+ at various time points in the continuous presence of epinephrine as described under "Experimental Procedures." Data are means ± S.E. of 10 oocytes of two different batches. G, after electrophysiological measurements, oocyte microsomes were prepared, 10 µg of proteins were loaded on a gel and transferred onto nitrocellulose membranes, and PLM was immunodetected with an antibody recognizing PLM not phosphorylated at Ser68. After stripping, the nitrocellulose membrane was probed with an Na,K-ATPase subunit antibody.

View larger version (39K): [in this window] [in a new window]   FIGURE 5. PLM phosphorylation by PKC has no effect on the turnover number of Na,K-ATPase 1/β1 isozymes. Two days after injection of human Na,K-ATPase 1 and β1 subunit cRNAs alone or together with wild type or mutated PLM cRNAs, oocytes were incubated or not with PDBu. A, microsomal proteins from Xenopus oocyte not treated (lanes 1, 3, 5, and 7) or treated for 3 min (lanes 2, 4, 6, and 8), 5 min (lane 9), or 10 min (lane 10) with PDBu were either directly loaded on a gel (50 µg) (lanes 1-4 and 7-10) or immunoprecipitated (IP) (100 µg) under nondenaturing conditions with a Na,K-ATPase subunit antibody (lanes 5 and 6). Proteins were transferred onto nitrocellulose membranes and probed with an antibody recognizing PLM phosphorylated at Ser68 (CP68), with an antibody recognizing PLM phosphorylated at Ser68, or with a Na,K-ATPase subunit antibody. B, maximal Na,K-ATPase currents (Imax) at a membrane potential of -50 mV of oocytes treated for 3 min with PDBu. C,[3H]ouabain binding to intact oocytes treated for 3 min with PDBu. Values obtained in noninjected oocytes (without PDBu, 167-251 dpm; with PDBu, 155-246 dpm) were subtracted. D, turnover number of Na,K-ATPase 1/β1 isozymes. Data are means ± S.E. of 15 oocytes from four different batches.

Phosphorylation analysis confirmed that wild type PLM was phosphorylated at Ser⁶⁸ and that the S63A/S68A, the S68A and the S63D/S68D mutants were not phosphorylated at the point of functional measurements (Fig. 7, A and B). Moreover, Fig. 7C shows that both nonphosphorylated and PKC-phosphorylated PLM are associated with Na,K-ATPase 2/β1 isozymes.

Since, due to the transient phosphorylation of PLM by PKC, it was not possible to measure the apparent Na⁺ affinity of 2/β1-PLM complexes exposed to phorbol ester, we cannot entirely exclude the possibility that the increased I_max may be due to an increased apparent Na⁺ affinity rather than to an effect on the turnover number of Na,K-ATPase 2/β1 isozymes. This is, however, unlikely, since intracellular Na⁺ concentrations in Na⁺-loaded oocytes used for I_max measurements reach values up to 80 mM (32). Under these conditions, it is improbable that an increase in the apparent Na⁺ affinity would be translated into an increased I_max, considering a K Na⁺ of about 12 mM for human Na,K-ATPase 2/β1 isozymes (23).

View larger version (46K): [in this window] [in a new window]   FIGURE 6. PLM phosphorylation by PKC increases the turnover number of Na,K-ATPase 2/β1 isozymes. A-C, 2 days after injection of human Na,K-ATPase 2 and β1 subunit cRNAs alone or together with wild type or mutant PLM cRNA, oocytes were incubated for 3 min (bars 3, 7, 10, 13, and 16) or 10 min (bars 4, 8, 11, 14, and 17) with PDBu or for 4 h with forskolin (bars 2 and 6). A, maximal Na,K-ATPase currents (Imax) at a membrane potential of -50 mV. B,[3H]ouabain binding to intact oocytes. Values obtained in noninjected oocytes (without activator, 192-380 dpm; with forskolin, 177-352 dpm; with PDBu, 193-368 dpm) were subtracted. C, turnover number of Na,K-ATPase. Data are means ± S.E. of 25 oocytes from five different batches. D, 2 days after injection of human 2 and β1 cRNAs in the absence (closed diamonds) or presence of wild type PLM (closed squares), S68A mutant (open diamonds), or S63D/S68D mutant (open squares) cRNAs; Imax of Na,K-ATPase was measured by the addition of 10 mM K+ at various time points in the continuous presence of PDBu. Shown are means ± S.E. of 10 oocytes from three different batches. *, p < 0.01, Imax or turnover number of Na,K-ATPase 2/β1 isozymes associated with wild type PLM or the S63A/S68A mutant in the presence of PDBu versus Imax in the absence of PDBu (bar 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Using the Xenopus oocyte as an expression system, our studies on the functional effects of PLM phosphorylation on Na,K-ATPase show that PKA phosphorylation of PLM increases the apparent Na⁺ affinity of Na,K-ATPase 1/β as well as 2/β isozymes but has no effect on the maximal Na,K-pump activity. Moreover, we provide evidence that PKC phosphorylation of PLM produces a distinct functional effect on Na,K-ATPase 2/β isozymes.

Regulation of Na,K-ATPase by PLM and its phosphorylation has extensively been studied in cardiac myocytes of wild type and PLM-deficient mice (8, 9, 11-13, 33) as well as in some expression systems (7, 10) and on a shark phospholemman-like protein (19). These studies, in particular those performed in myocytes, have provided some controversial results and have left several open questions with respect to the mechanism by which PKA and PKC phosphorylation of PLM might influence Na,K-ATPase activity. In the present study, we have used the Xenopus oocyte expression system to address certain unanswered questions, which are difficult to assess in cardiac myocytes. The Xenopus oocyte expression system, even if it may not be as physiologically relevant as cardiac myocytes, permits dissection of the intrinsic effects of PLM phosphorylation on Na,K-ATPase activity, independently of the complex physiological conditions existing in cardiac myocytes and the presence of different Na,K-ATPase isozymes, which may determine the outcome of the overall response to PLM phosphorylation. In this respect, it has been proposed that the contradictory results obtained in different laboratories on PLM action may be due to comparison of myocytes of knock-out mice with myocytes of wild type mice with different proportions of phosphorylated and nonphosphorylated PLM determined by the adrenergic state of the heart upon preparation (35).

Effects of PKA and PKC Phosphorylation of PLM on Association with Na,K-ATPase—Co-immunoprecipitation experiments performed on Xenopus oocyte extracts suggest that PLM remains associated with Na,K-ATPase after PKA and PKC phosphorylation. This result is in line with observations made in cardiac myocytes (8, 11, 33). Moreover, recent experimental evidence shows that PKA and PKC phosphorylation of PLM reduces but does not abolish fluorescence resonance energy transfer between Na,K-ATPase and PLM (36). Together, these data favor the hypothesis that PLM regulates Na,K-ATPase in a manner, which is similar to regulation of sarcoendoplasmic reticulum Ca²⁺-ATPase by phospholamban (37). PKA- and PKC-mediated phosphorylation of PLM may change interaction with Na,K-ATPase but does not result in a complete dissociation.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. Phosphorylation and association of wild type and mutant PLM with Na,K-ATPase 2 and β1 isozymes. A, 2 days after injection of human Na,K-ATPase 2 (12 ng) and β1 (1 ng) subunit cRNAs alone (lanes 1 and 2) or together with 3 ng of wild type (lanes 3 and 4) or mutant (lanes 5-10) PLM cRNAs, oocytes were incubated for 3 min with PDBu. Fifty µg of proteins from Xenopus oocyte microsomes were directly loaded on a gel, transferred onto nitro-cellulose membranes, and probed with an antibody recognizing PLM not phosphorylated at Ser68 or with a Na,K-ATPase subunit antibody. B, quantification of the amount of PLM not phosphorylated at Ser68 remaining after PDBu stimulation normalized for Na,K-ATPase subunit expression. Shown are means ± S.E. of three independent experiments. C, rat Na,K-ATPase 2 subunit and β1 subunit cRNAs were injected in Xenopus oocytes in the absence (lanes 1 and 2) or presence of wild type (lanes 3-10) PLM cRNA. Two days after injection, oocytes were incubated for 3 min (lanes 2, 4, 6, and 8), 5 min (lane 9), or 10 min (lane 10) with PDBu. Microsomal proteins were either directly loaded on a gel (lanes 1-4 and 7-10) or immunoprecipitated (IP) under nondenaturing conditions with a Na,K-ATPase subunit antibody (lanes 5 and 6). Proteins were transferred onto nitrocellulose membranes and probed with an antibody recognizing PLM phosphorylated at Ser68 or an antibody recognizing PLM not phosphorylated at Ser68.

View larger version (14K): [in this window] [in a new window]   FIGURE 8. PLM phosphorylation by PKC has no effect on the apparent K+ affinity of Na,K-ATPase 1/β1 and 2/β1 isozymes. Two days after injection of rat Na,K-ATPase 1 (A) or ouabain-resistant 2 (B) and β1 subunit cRNAs alone or together with wild type PLM cRNA, oocytes were incubated or not for 3 min with PDBu, and the apparent K+ affinity of Na,K-ATPase (KK+) was determined. Closed diamonds, nonactivated oocytes expressing Na,K-ATPase; open diamonds, PDBu-activated oocytes expressing Na,K-ATPase; closed squares, nonactivated oocytes expressing Na,K-ATPase plus PLM; open squares, PDBu-activated oocytes expressing Na,K-ATPase plus PLM.

Effects of PKA Phosphorylation of PLM on the Apparent Na⁺ Affinity of Na,K-ATPase Isozymes—As we have previously shown, PLM not only has an effect on the K⁺ affinity of Na,K-ATPase but also decreases the apparent Na⁺ affinity of 1/β1 and 2/β1 isozymes (7). The effect of PLM on the Na⁺ affinity was later confirmed in cardiac myocytes by showing that the K_m(Na) of Na,K-ATPase was lower in myocytes from PLM knock-out mice than in myocytes from wild type mice (13). It remains unexplained why Zhang et al. (14) observed inhibition of Na,K-ATPase after overexpression of PLM in cardiac myocytes but no effect on its apparent Na⁺ affinity. In the present study, we confirm the conclusions drawn by Despa et al. (13) from comparative studies in cardiac myocytes of wild type and knock-out mice that PKA phosphorylation of PLM increases the apparent Na⁺ affinity of Na,K-ATPase and thus abolishes the inhibitory effect of PLM. Notably, our results show that PLM phosphorylation by PKA influences the effect of PLM on the Na⁺ affinity of both Na,K-ATPase 1/β1 and 2/β1 isozymes. Consistently, our results show that PLM mutated in its phosphorylation site Ser⁶⁸ has no effect on the apparent Na⁺ affinity of Na,K-ATPase upon PKA activation, whereas a constitutive phosphorylation mutant produces an effect even in the absence of PKA activation (Fig. 3A). These latter results, together with the observation that PKA activation has no effect on the apparent Na⁺ affinity of Na,K-ATPase expressed without PLM, also indicate that PKA activation produces its functional effect on PLM-associated Na,K-ATPase by phosphorylation of PLM rather than by phosphorylation of the Na,K-ATPase subunit. This conclusion is in line with results obtained in cardiac myocytes (13) and with studies showing that forskolin treatment of cardiac myocytes leads to phosphorylation of PLM at Ser⁶⁸ but not of the PKA consensus site in Na,K-ATPase 1 subunits (9).

Effects of PKA Phosphorylation of PLM on the Maximal Activity of Na,K-ATPase Isozymes—It remains controversial whether PKA phosphorylation of PLM influences the apparent Na⁺ affinity or the maximal Na,K-ATPase activity or both. In agreement with studies by Despa et al. (13) in cardiac myocytes, we find an effect of PKA phosphorylation of PLM on the apparent Na⁺ affinity but not on V_max of both Na,K-ATPase 1/β1 and 2/β1 isozymes. An increase in the apparent Na⁺ affinity without an effect on V_max was also correlated with PLM phosphorylation of Na,K-ATPase coexpressed in P. pastoris (10). This contrasts with studies that describe an increase in V_max of Na,K-ATPase after PKA phosphorylation of PLM (9, 12). The reasons for these discrepancies are not clear, but it is possible that experimental conditions (such as temperature), variations in the proportion of phosphorylated and unphosphorylated PLM in different cell types, or other undefined variables may be the cause for different results.

Effects of PKC Phosphorylation of PLM on Na,K-ATPase Activity—Recent studies by Han et al. (11) show that PKC phosphorylation of PLM does not influence the apparent Na⁺ affinity of Na,K-ATPase but increases its V_max. Moreover, this study provides evidence that successive PKA and PKC activation leads to additive effects on PLM-associated Na,K-ATPase despite common phosphorylation sites on PLM. In the present study, we tested the interesting hypothesis that PKA and PKC may target different pools of PLM associated with different Na,K-ATPase isozymes. Our results indeed show that PKC phosphorylation of PLM has no effect on I_max of Na,K-ATPase 1/β1 isozymes but significantly increases I_max of 2/β1 isozymes.

The study by Han et al. (11) has not assessed the question of whether the increase in V_max of Na,K-ATPase upon PKC phosphorylation of PLM is due to an increase in the cell surface expression or an increase in the turnover number of Na,K-ATPase. We show in this study that the increase in I_max of 2/β isozymes observed after PKC phosphorylation of PLM cannot be attributed to an increase in cell surface expression but most likely to an increase in their turnover number. Since it was not possible to measure the apparent Na⁺ affinity of Na,K-ATPase after PKC treatment of Xenopus oocytes, we cannot entirely exclude the possibility that the increase in I_max of 2/β isozymes is due to an effect of PKC phosphorylation of PLM on the apparent Na⁺ affinity or that PKC phosphorylation of PLM may have an additional effect on the apparent Na⁺ affinity of either Na,K-ATPase 1/β or 2/β isozymes. However, since no effect on the apparent Na⁺ affinity of Na,K-ATPase has been observed in cardiac myocytes after PKC activation (11), this is unlikely and would probably be physiologically irrelevant.

Overall, our studies suggest that PKA can target PLM associated with Na,K-ATPase 1/β and 2/β isozymes to increase their apparent Na⁺ affinity. On the other hand, PKC can phosphorylate PLM associated either with 1/β or 2/β isozymes but produces a functional effect, namely an increased turnover number, only on 2/β isozymes. If we consider that PKA phosphorylates Ser⁶⁸ and PKC phosphorylates Ser⁶³ and Ser⁶⁸ in PLM, and taking into account that mutation of Ser⁶⁸ abolishes the effect of PKA phosphorylation of PLM on the apparent Na⁺ affinity as well as the effect of PKC phosphorylation of PLM on the turnover number of Na,K-ATPase, we may speculate that the effect of PKA-phosphorylated PLM on the Na⁺ affinity of 1/β and 2/β isozymes might be mediated by phosphorylation of Ser⁶⁸, whereas the V_max effect of PKC phosphorylation of PLM associated with 2/β isozymes might necessitate phosphorylation of both Ser⁶³ and Ser⁶⁸.

Since both 1/β and 2/β isozymes are present in cardiac myocytes (40-42), the molecular basis that permit the differential effects of PKC-mediated phosphorylation of PLM on 1/β and 2/β isozymes remains to be determined. 2/β isozymes may only make up about 11% of the total Na,K-ATPase (43), but they may play a more important and different role in cardiac contractility than 1/β isozymes (43, 44) due to their preferential localization in T-tubules and their co-localization with the Na/Ca exchanger (34, 43).

Physiological Relevance—Our results support the hypothesis that PLM phosphorylation by PKA and PKC play differential roles in the regulation of different Na,K-ATPase isozymes. This result raises the interesting possibility that the additive effects of PKA and PKC phosphorylation of PLM on Na,K-ATPase, which are observed in cardiac myocytes, might reflect that 1) PKA activated by β-adrenergic receptor stimulation can target PLM associated with Na,K-ATPase 1/β and 2/β isozymes to increase their apparent sodium affinity, and 2) PKC activated by -adrenergic receptor stimulation targets, in addition, PLM associated with 2/β isozymes to increase their turnover number. According to our results, PKC phosphorylation of PLM associated with Na,K-ATPase 1/β isozymes is possible but does not lead to an increase in their turnover number. Physiologically, the overall response to concomitant PKA and PKC activation would be an additive increase in sodium extrusion, which may limit sodium load during increased heart activity and hence may limit positive inotropy during sympathetic stimulation of the heart by favoring calcium extrusion through the sodium/calcium exchanger.

	FOOTNOTES

 
^* This work was supported by Swiss National Science Foundation Grant 3100A0-107513 (to K. G.). 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 To whom correspondence should be addressed. Tel.: 41-21-692-54-10; Fax: 41-21-692-53-55; E-mail: Kaethi.Geering{at}unil.ch.

² The abbreviations used are: PLM, phospholemman; PKA, protein kinase A; PKC, protein kinase C; IBMX, isobutylmethylxanthine; PDBu, 4-β-phorbol-12,13-dibutyrate.

	ACKNOWLEDGMENTS

 
We thank H. Garty, S. Karlish, and D. Bers for PLM antibodies.

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