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J. Biol. Chem., Vol. 280, Issue 20, 19875-19882, May 20, 2005

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Identification of an Endogenous Inhibitor of the Cardiac Na⁺/Ca²⁺ Exchanger, Phospholemman^*

Belinda A. Ahlers, Xue-Qian Zhang, J. Randall Moorman¶, Lawrence I. Rothblum, Lois L. Carl, Jianliang Song, JuFang Wang, Lisa M. Geddis¶, Amy L. Tucker¶, J. Paul Mounsey¶, and Joseph Y. Cheung||

From the Department of Cellular and Molecular Physiology and ^||Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania 17822, and ^¶Department of Internal Medicine (Cardiovascular Division), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

Received for publication, December 30, 2004 , and in revised form, February 28, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Rapid and precise control of Na⁺/Ca²⁺ exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca²⁺ homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na⁺-dependent ⁴⁵Ca²⁺ uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na⁺-K⁺-ATPase, also regulates Na⁺/Ca²⁺ exchange in the heart.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Excitation-contraction coupling in cardiac myocytes depends on the ability of regulatory proteins to maintain steady-state Ca²⁺ fluxes through each cycle of Ca²⁺ influx, intracellular Ca²⁺ transient buffering, and Ca²⁺ efflux (1). Among the many transporters and ion channels involved in cardiac Ca²⁺ fluxes, the sarcolemmal Na⁺/Ca²⁺ exchanger (NCX1)¹ is unique in that it participates in all three major phases of Ca²⁺ movement. Depending on the thermodynamic driving force as determined by the membrane potential (E_m) and the concentrations of Na⁺ and Ca²⁺ ions sensed by the exchanger, NCX1 can mediate both Ca²⁺ influx (reverse mode) and Ca²⁺ efflux (forward mode). The forward mode of NCX1 is the major Ca²⁺ efflux mechanism that extrudes the amount of extracellular Ca²⁺ that has entered the myocyte during a twitch, thereby restoring cytosolic Ca²⁺ concentration ([Ca²⁺]_i) to resting levels and maintaining steady-state Ca²⁺ balance (1). NCX1 involvement in Ca²⁺ efflux and its additional assigned roles in sarcoplasmic reticulum Ca²⁺ loading (2) and release (3) are likely to contribute alongside other proteins such as sarcoplasmic reticulum Ca²⁺ATPase, calmodulin, calbindin, and parvalbumin as effective buffering mechanisms for short-term Ca²⁺ transients. There have been significant advances made toward understanding the intrinsic properties of NCX1 with regards to the precise stoichiometry of the Na⁺/Ca²⁺ exchange ratio (4, 5) and the direction of ion fluxes during an action potential in excitable cells where NCX1 function has interdependency on the activities of various Na⁺ and K⁺ channels and the ATP-driven Na⁺-K⁺-ATPase (1, 6).

Not surprisingly, there is a current focus upon identifying both synthetic and endogenous factors that regulate NCX1 function. Known activators of NCX1 in the cardiac muscle include Ca²⁺ ions, phosphatidylinositol 4,5-bisphosphate, phospholipase C-activating agonists, protein kinase C (PKC) activators, non-exchanged monovalent cations (Na⁺, K⁺, Li⁺), redox reagents, and mild proteolysis of the internal surface of NCX1. Inhibitors include high [Na⁺]_i in the absence of ATP or at low pH_i (Na⁺-dependent inactivation), H⁺, and divalent and trivalent cations (Ni²⁺, La³⁺, Cd²⁺). Synthetic inhibitors include a 20-amino acid exchanger inhibitor peptide based on a short sequence of the NCX1 intracellular loop, molluscan cardioexcitatory tetrapeptide (FMRFa) analogues, and an isothiourea derivative (KB-R7943) (7). Of note is, to date, there has been no endogenous protein regulator of NCX1 described.

Here we describe a newly identified endogenous protein inhibitor of NCX1 called phospholemman (PLM). To unequivocally demonstrate this, we have utilized a heterologous expression system of human embryonic kidney (HEK)293 cells that are devoid of PLM and NCX1 and are electrically silent (8). Confocal immunofluorescence and co-immunoprecipitation studies of transiently transfected HEK293 cells clearly showed co-localization and association of both exogenous PLM and NCX1 at the plasma membrane. The functional consequence of this association was investigated using two fundamentally different measures of NCX1 activity, Na⁺-dependent ⁴⁵Ca²⁺ uptake and NCX1 current (I_NaCa). Co-expression of PLM with NCX1 inhibited both forward and reverse I_NaCa and decreased Na⁺-dependent ⁴⁵Ca²⁺ uptake rate. Inhibition of NCX1 by PLM was specific, because ablation of a dual protein kinase A (PKA) and PKC phosphorylation site (serine 68) in PLM by alanine replacement led to loss-of-function and abolished its inhibitory effect on NCX1 activity. PLM co-immunoprecipitated with NCX1 in native adult cardiac myocyte membranes. In summary, we have identified for the first time an endogenous protein inhibitor of cardiac NCX1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Construction of Rat PLM and NCX1 Clones—A portion of the left ventricle from Sprague-Dawley rat heart was frozen in liquid nitrogen at the time of euthanasia and stored at -80 °C. Rat cDNA library was constructed from left ventricular poly(A)⁺-selected RNA (mRNA) using Superscript double-stranded cDNA synthesis kit (Invitrogen). A PCR-based cloning strategy utilizing pfu polymerase was carried out in order to obtain a rat PLM clone containing the complete coding region (279 bp). The forward primer (carrying HindIII and BglII restriction sites as underlined), 5'-AAG CTT AGA TCT ATG GCA TCT CCC GGC CAC ATC CTG-3', and reverse primer (carrying HindIII and XhoI restriction sites as underlined), 5'-AAG CTT CTC GAG TTA CCG CCT GCG GGT GGA CAG ACG-3', were used for the PCR reaction and also separately for subsequent DNA sequence verification. Rat PLM was inserted directly into the mammalian expression vector pAdTrack-CMV (9) using the restriction endonucleases BglII and XhoI. PLM serine-to-alanine substitution at amino acid position 68 on the mature protein (PLMS68A) was constructed with PLM in pAlter-1 using Altered Sites II in vitro mutagenesis system (Promega, Madison, WI). The site-directed PLM mutant was authenticated by DNA sequencing and expression analysis. Rat cardiac NCX1 clone in pcDNA3.1(+) was a generous gift from Dr. J. Lytton and subcloned into pAdTrack-CMV as previously described (10). We chose the pAdTrack shuttle vector, because it allowed us to identify successfully transfected HEK293 cells through a separate cytomegalovirus (CMV) promoter present on the vector backbone that drives the expression of green fluorescent protein (GFP).

Transfection of HEK293 Cells—HEK293 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium /Ham's F-12 (Cellgro, Herndon, VA) containing 10% heat-inactivated fetal bovine serum at a density of 1.2 x 10⁶ cells/100-mm dish. After 24 h, medium was changed and cells were transfected with 25 µl of Lipofectamine (Invitrogen) and total of 3-µg plasmid DNA/dish (either pAdTrack-CMV alone (3 µg), pAdTrack-CMV-NCX1 (1.5 µg) + pAdTrack-CMV (1.5 µg), pAdTrack-CMV-NCX1 (1.5 µg) + pAdTrack-CMV-PLM (1.5 µg), or pAdTrack-CMV-NCX1 (1.5 µg) + pAdTrack-CMV-PLMS68A (1.5 µg)) according to the manufacturer's instructions. Levels of DNA and lipid were optimized in transfection assays to ensure minimal toxicity to cells. The lipid-DNA complex was left on cells for 5 h at 37 °C, 5% CO₂. Medium was then replaced with DMEM/Ham's F12 + 10% fetal bovine serum, and cells were cultured for an additional 48 h before experiments. For confocal and patch clamp applications, cells were trypsinized at 24 h post-transfection using trypsin-EDTA (Invitrogen), transferred to 35-mm dishes containing sterile glass coverslips, and incubated a further 24 h prior to experimentation. Cells for Western blot and co-immunoprecipitation applications were left in 100-mm dishes until 48 h post-transfection. Cell-seeding density, lipid, and DNA amounts were scaled down according to surface area for 24-well plate transfections that were used to measure Na⁺-dependent ⁴⁵Ca²⁺ uptake. Transfections according to this protocol routinely yielded 30–50% transfection efficiency.

Confocal Microscopy—HEK293 cells transiently transfected for 24 h with either pAdTrack alone or PLM/NCX1 were plated on laminin-coated glass slide chambers (Nunc, Lab-Tek Division, Naperville, IL) and cultured for an additional 24 h. Adherent cells were washed three times with phosphate-buffered saline (PBS, Sigma) containing 2 mM EGTA and then fixed for 30 min in 3% paraformaldehyde in PBS with 2 mM EGTA. After two rinses with PBS, cells were permeabilized for 2 min in 0.05% Triton X-100 followed by two additional rinses with PBS and once with BLOTTO (5% nonfat dry milk, 0.1 M NaCl, and 50 mM Tris-HCl (pH 7.4)). Primary polyclonal PLM (1:250 dilution, C2Ab) (11) and monoclonal NCX1 (1:250 dilution, R3F1, Swant, Bellinzona, Switzerland) antibodies diluted in BLOTTO were added to the cells, incubated in room temperature in the dark for 60 min, and rinsed three times with BLOTTO. Secondary antibodies diluted in BLOTTO were added to cells and incubated in the dark for 30 min followed by three PBS rinses. Secondary antibodies were Alexa Fluor 546-labeled goat anti-mouse IgG (1:50, Molecular Probes, Eugene, OR) for R3F1 Ab and Alexa Fluor 647-labeled goat anti-rabbit IgG (1:50, Molecular Probes) for C2Ab. The slide was then removed from the chamber, and a coverslip containing mounting solution (90% glycerol in PBS + p-phenylaminediamine) was applied. Images of HEK293 cells (green fluorescent protein, excitation 488, emission 515 nm; R3F1 Ab, excitation 546, emission 570 nm; and C2Ab, excitation 633, emission 674 nm) were acquired with a Leica TCS SP2 confocal microscope and processed with LCS software.

Crude Membrane Preparation—HEK293 cells were washed three times with ice-cold Hanks' balanced salt solution and scraped into 400 µl of ice-cold Buffer I containing the following (in mM): 10 Tris (pH 7.5); 1 sodium vanadate; 1 phenylmethylsulfonyl fluoride; 100 NaF; 1 EGTA; and a combination of complete protease inhibitor (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor cocktails (catalog numbers P-2850 and P-5726, Sigma). After sonication (3 x 15 s), 400 µl of ice-cold Buffer II containing the following (in mM): 10 Tris (pH 7.5); 300 KCl; 1 Na⁺ vanadate; 1 phenylmethylsulfonyl fluoride; 100 NaF; 1 EGTA; 20% sucrose; and complete protease inhibitor mixture were added. Cell sonicates were centrifuged (10,000 x g) for 10 min at 4 °C. The clarified supernatant was then subjected to ultracentrifugation at 100,000 x g at 4 °C for 1 h. The resultant pellet (crude membrane fraction) was washed with Hanks' balanced salt solution and stored at -80 °C until use.

Co-immunoprecipitation of PLM and NCX1—Crude membrane pellets were resuspended in a minimal volume of Buffer III (in mM: 140 NaCl; 25 imidazole; 1 EDTA; and a combination of complete protease inhibitor and phosphatase inhibitor cocktails (pH 7.4)) and then adjusted to 2 mg in 300 µl of Buffer III and combined with C₁₂E₈ detergent in 100 µl of Buffer III (at a detergent:protein ratio of 2:1) at room temperature for 10 min. After the addition of another 400 µl of Buffer III, samples were subjected to ultracentrifugation at 37,000 x g using a Beckman TLA 100.3 rotor. The supernatant was transferred to a fresh tube, and the protein content was determined. 400 µg of solubilized crude membrane preparation was used in both preimmune control and antibody immunoprecipitation experiments. Samples were precleared before the addition of relevant antibodies by preincubation of supernatants with 50 µl of protein A-agarose for 1 h at 4 °C. Precleared supernatants were incubated with either 5 µg of preimmune rabbit IgG (polyclonal Ab control), 5 µg of polyclonal PLM antibody (C2Ab), 5 µl of polyclonal NCX1 Ab (11-13, Swant), 4 µl of monoclonal NCX1 Ab (R3F1), or no Ab (monoclonal Ab control) overnight at 4 °C. The next day, 40 µl (50% slurry) of washed suspended protein A-agarose beads were added to each sample and incubated for a further 2 h at 4 °C. Beads were pelleted, washed four times with 1.5 ml of Buffer III containing 0.05% C₁₂E₈, and resuspended in 40 µl of 2x Laemmli sample buffer (+dithiothreitol). Beads were boiled for 5 min at 95 °C and stored until further use for immunoblotting.

Western Blot—Crude membrane input and immunoprecipitated samples were resolved on either 7.5 (NCX1) or 15% (PLM) SDS-PAGE in a Tris-glycine electrode buffer. Proteins were transferred to polyvinylidene difluoride membranes and blocked overnight in 5% nonfat milk/Tris-buffered saline with 0.05% Tween 20. Primary antibody incubation was performed for 3 h at room temperature (PLM) or overnight at 4 °C (NCX) in 5% nonfat milk/Tris-buffered saline with 0.05% Tween 20 containing 1:1000 R3F1, 1:5000 11-13, or 1:5000 C2 antibodies. Secondary antibodies used were either 1:2000 donkey anti-rabbit IgG-conjugated horseradish peroxidase (HRP^*, Amersham Biosciences, Uppsala, Sweden) or 1:2000 sheep anti-mouse IgG-HRP^* (Amersham Biosciences). Immunoreactivity was detected using an enhanced chemiluminescence kit, BioMax XAR film (Eastman Kodak Co., Rochester, NY), and a developer.

Na⁺/Ca²⁺ Exchange Current (I_NaCa) Measurements—Whole-cell patch clamp recordings were performed at 30 °C as described previously (12–15). Fire-polished pipettes (tip-diameter 2–3 µm) with resistances of 2.5–3.0 megohms when filled with standard internal solution were used. Pipettes were filled with a buffered Ca²⁺ solution containing the following (in mM): 100 Cs⁺ glutamate; 7.25 Na⁺ HEPES; 1 MgCl₂; 12.75 HEPES; 2.5 Na₂ATP; 10 EGTA; and 6 CaCl₂ (pH 7.2). Free Ca²⁺ in the pipette solution was 205 nM, measured fluorimetrically with fura 2. Cells were bathed in an external solution containing the following (in mM): 130 NaCl; 5 CsCl; 1.2 MgSO₄; 1.2 NaH₂PO₄; 5 CaCl₂; 10 HEPES; 10 Na⁺ HEPES; and 10 glucose (pH 7.4). Verapamil (1 µM), ouabain (1 mM), and niflumic acid (30 µM) were used to block Ca²⁺, Na⁺-K⁺-ATPase, and Cl^- currents, respectively. K⁺ currents were minimized by Cs⁺ substitution for K⁺ in both pipette and external solutions. Only cells that fluoresced green (excitation 380 nm, emission 510 nm), indicating successful pAdTrack transfection, were selected for current measurements. For current measurements, cell capacitance and series resistance were compensated with the analog circuitry of the patch clamp amplifier. Membrane potential (E_m) was held at the calculated reversal potential of I_NaCa (-73 mV) for 5 min before stimulation. This precaution minimized fluxes through NCX1 before the voltage ramp and thus allowed [Na⁺]_i and [Ca²⁺]_i to equilibrate with those present in pipette solution. A descending voltage ramp (from +100 to -120 mV; 500 mV/s) was immediately followed by an ascending voltage ramp (from -120 to +100 mV; 500 mV/s). The voltage ramp was repeated after the addition of 1 mM CdCl₂ to the external solution. Currents were derived from measurements during the descending voltage ramp. I_NaCa was defined as the difference current measured in the absence and presence of Cd²⁺ (12). Currents were filtered at 1 kHz, and data were acquired at 2 kHz. Whole-cell capacitance (C_m) for each cell was measured by applying a small hyperpolarizing pulse (-10 mV, 16 ms) and integrating the resulting current change (digitized at 50 kHz, 0.5-kHz filter) over time. To facilitate comparison of NCX1 currents, I_NaCa of each cell was divided by C_m to account for variations in cell sizes.

Resting Membrane Potential Determinations—Resting E_m was measured in current-clamp mode as previously described (13, 14, 16). Pipette solution consisted of the following (in mM): 125 KCl; 4 MgCl₂; 0.06 CaCl₂; 10 HEPES; 5 K⁺ EGTA; 3 Na₂ATP; and 5 Na₂-creatine phosphate (pH 7.2). External solution consisted of the following (in mM): 127 NaCl; 5.4 KCl; 1.8 CaCl₂; 1.8 MgCl₂; 0.6 NaH₂PO₄; 7.5 HEPES; 7.5 Na⁺ HEPES; and 10 glucose (pH 7.4). To simulate Na⁺ loading, 80 mM KCl in pipette solution was replaced with NaCl.

Assay of Na⁺-dependent ⁴⁵Ca²⁺ Uptake—Na⁺-dependent Ca²⁺ uptake measurements were performed essentially as previously described (17, 18). Transfected HEK293 cells in 24-well plates were preloaded with Na⁺ at 37 °C for 20 min in a balanced salt solution (BSS) containing the following (in mM): 10 HEPES-Tris (pH 7.4); 146 NaCl; 4 KCl; 2 MgCl₂; 0.1 CaCl₂; 10 glucose; and 0.1% bovine serum albumin (BSA). Ouabain (1 mM) and monensin (10 µM) were present during Na⁺ loading. ⁴⁵Ca²⁺ uptake was initiated by replacing the loading medium with 0.5 ml of either Na⁺-free BSS (NaCl replaced with equimolar methyl-D-glucosamine) or normal BSS, both of which contained 0.1 mM ⁴⁵CaCl₂ (1.5 µCi/ml), [³H]mannitol (1 µCi/ml), and 1 mM ouabain. After 30 s, ⁴⁵Ca²⁺ uptake was terminated by washing cells four times with an ice-cold solution containing the following (in mM): 10 HEPES-Tris (pH 7.4); 120 choline chloride; and 10 LaCl₃. Preliminary studies showed that ⁴⁵Ca²⁺ uptake was linear during the first 30 s. Cells were solubilized with 0.1 N NaOH and neutralized, and aliquots were taken for determination of ⁴⁵Ca²⁺ and ³H radioactivity and protein. Extracellular contamination as determined from [³H]mannitol counts was 0.134 ± 0.002 µl (n = 96), and contamination by extracellular ⁴⁵Ca²⁺ was routinely subtracted before calculation of ⁴⁵Ca²⁺ uptake. Na⁺-dependent ⁴⁵Ca²⁺ uptake was calculated by subtracting ⁴⁵Ca²⁺ uptake values in the presence of extracellular Na⁺ from those obtained in the absence of Na⁺ and normalized to milligram cell protein.

Co-immunoprecipitation of NCX1 and PLM in Native Cardiac Membranes—Sarcolemmal vesicles were prepared from the left ventricles of pig hearts essentially according to the method of Larry R. Jones (24, 25). Washed membrane pellets (adjusted to 2 mg/ml) were resuspended in 300 µl of Buffer III, and 1.2 mg of C₁₂E₈ in 100 µl of Buffer III were added. After mixing and incubation at room temperature for 10 min, 400 µl of Buffer III were added and the mixture was subjected to ultracentrifugation at 37,000 x g. The supernatant was precleared with protein A-agarose and incubated with either IgG or 2 µl of polyclonal PLM antibody, and co-immunoprecipitation experiments were performed as described above for HEK293 cells. Crude membrane input and immunoprecipitated samples were resolved on 12% 1.0 mM 12-well bis-tris gels (Invitrogen). The primary antibody used to detect NCX1 was R3F1.

In a second series of experiments, left ventricles were excised from adult male Sprague-Dawley rat hearts (300 g body weight). Crude membrane preparations were prepared with the protocol described above for HEK293 cells. Crude membrane preparations (2 mg) were used in co-immunoprecipitation experiments as described above for HEK293 cells.

Statistical Analysis—All of the results are expressed as means ± S.E. For the analysis of a parameter (e.g. I_NaCa) as a function of group (e.g. NCX1 versus NCX1 + PLM) and voltage, two-way ANOVA was used to determine statistical significance. For the analysis of C_m, one-way ANOVA was used. For the analysis of Na⁺-dependent ⁴⁵Ca²⁺ uptake, Student's paired t test was used. A commercial software package (JMP, version 4.0.5, SAS Institute; Cary, NC) was used. In all of the analyses, p < 0.05 was taken to be statistically significant.

View larger version (47K): [in this window] [in a new window]   FIG. 1. Co-localization of PLM with cardiac NCX1 by confocal immunofluorescence microscopy in transfected HEK293 cells. Cells were transiently transfected with plasmid DNA encoding both PLM + NCX1. After 48 h, cells were fixed, permeabilized, and doubly labeled with monoclonal antibody to NCX1 (R3F1; panel B) and polyclonal antibody to PLM (C2Ab; panel C). GFP expressed under the control of a second CMV promoter is localized to the cytosol (panel A). Primary antibodies were visualized with Alexa Fluor 546-labeled goat anti-mouse IgG (red, panel B) and Alexa Fluor 647 goat anti-rabbit IgG (blue, panel C). Merged image (panel D) of panels A–C shows co-localization of PLM and NCX1 but not GFP. Bar = 5 µm.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Western blotting experiments on crude membrane fractions further confirmed membrane localization of PLM and NCX1 (Fig. 2). Importantly, the expression levels of NCX1 were similar in the absence or presence of co-transfected PLM. Three bands were detected after SDS-PAGE for PLM with minor bands detected at 8 and 18 kDa and a major band at 14 kDa, respectively. The major band corresponds to the size previously found in rat cardiac myocytes where PLM (with a predicted size of 7.2 kDa) is known to display aberrant migration on SDS-PAGE (11, 25). Previous studies on HEK293 cells transfected with pcDNA3-expressing PLM also demonstrated the minor bands (19). The identities of these minor bands are at present unknown.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Exogenous expression of PLM and cardiac NCX1 in HEK293 cells. Equal amounts of total plasmid DNA were transiently transfected in HEK293 cells with pAdTrack alone (pAdT), pAdT + PLM (PLM), pAdT + NCX1 (NCX1), or PLM + NCX1 (PLM/NCX1). Crude membrane preparations (6 µg) from indicated cell cultures were subjected to immunoblot analysis at 48 h post-transfection using either monoclonal anti-NCX1 (R3F1, top panel) or polyclonal anti-PLM (C2Ab, bottom panel) antibodies. The antibodies used are indicated on the right and molecular mass markers (in kDa) are shown on the left.

To provide further evidence for the specificity of this association, the reciprocal experiment was performed in cells co-expressing PLM and NCX1 in which anti-NCX1 immunoprecipitates from solubilized crude membrane preparations were obtained using either monoclonal R3F1 or polyclonal 11-13 NCX1 antibodies. Both types of NCX1 antibodies efficiently purified NCX1 and co-purified PLM (Fig. 4, lanes 2 and 3). By contrast, control immunoprecipitates using beads alone or preimmume rabbit IgG did not contain PLM or NCX1 (Fig. 4, lanes 1 and 4, respectively).

Effect of PLM on I_NaCa in NCX1-expressing Cells—Having confirmed co-localization and direct association between PLM and NCX1, we next examined whether PLM had any effect on the Na⁺/Ca²⁺ exchange current (I_NaCa) in HEK293 cells expressing NCX1. Fig. 5 shows the steady-state I_NaCa measured at various membrane potentials, at 30 °C and 5.0 mM [Ca²⁺]_o, in empty vector control (open squares; n = 3), NCX1 (open circles; n = 11), and PLM + NCX1 (filled circles; n = 8) transfected cells. Consistent with previous observations (8), no endogenous I_NaCa was detectable in control HEK293 cells. Expression of NCX1 in HEK293 cells resulted in a large I_NaCa compared with that measured in control HEK293 cells transfected with empty vector (group effect, p < 0.0001). In addition, the differences in I_NaCa between control cells and cells expressing NCX1 were amplified at more positive membrane voltages (group x voltage interaction effect, p < 0.0001). The reversal potential of I_NaCa was approximately -60 mV, close to the theoretical equilibrium potential of -73 mV under our experimental conditions. Co-expression of PLM with NCX1 resulted in a significant decrease in I_NaCa when compared with cells expressing NCX1 alone (group effect, p < 0.0001). It is important to note that the decrease in I_NaCa in PLM + NCX1 cells was not due to the reduction of NCX1 protein levels in cells co-expressing PLM and NCX1 (Figs. 2 and 3). The differences in I_NaCa between NCX1 and NCX1 + PLM-expressing cells are amplified at more positive voltages (group x voltage interaction effect, p < 0.0001). In both NCX1 and PLM + NCX1-expressing cells, depolarization to more positive membrane potentials increased the absolute magnitude of I_NaCa (voltage effect, p < 0.0001). Comparing I_NaCa between PLM + NCX1 cells and HEK293 cells transfected with empty vector showed significant group effect (p < 0.002), indicating residual I_NaCa in the presence of PLM. There were no significant differences in the C_m among control, NCX1, and NCX1 + PLM-expressing cells (33.9 ± 3.1, 29.6 ± 2.3, and 25.8 ± 2.2 pF, respectively; p < 0.22). Our C_m values are in agreement with the average capacitance of 33 pF reported in HEK293 cells transfected with NCX1 (8).

View larger version (41K): [in this window] [in a new window]   FIG. 3. Demonstration of association of NCX1 with PLM in transfected HEK293 cells by immunoprecipitation. Top panel, immunoblot of NCX1 immunoprecipitates from 400 µg of solubilized crude membrane preparations using 5 µg of anti-PLM antibody (C2Ab) or control rabbit IgG (Preimmune) probed with antibody to NCX1 (R3F1). Bottom panel, immunoblot of PLM immunoprecipitates from 400 µg of solubilized crude membrane preparations using 5 µg of anti-PLM antibody (C2Ab) or control rabbit IgG (Preimmune) probed with antibody to PLM (C2Ab). Crude membrane preparations (Mem Input) were derived from HEK293 cells transiently transfected (48 h) with pAdTrack alone (pAdT), pAdTrack + PLM (PLM), pAdTrack + NCX1 (NCX1), or PLM + NCX1 (PLM/NCX1) encoding plasmid DNA. The antibodies used for immunoblots are indicated on the right, and molecular mass markers (in kDa) are shown on the left. This experiment was performed three times with similar results.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Reciprocal co-purification of PLM with NCX1 in HEK293 crude membrane extracts. Top panel, immunoblot of PLM immunoprecipitates from 400 µg of solubilized crude membrane preparations obtained using either NCX1 monoclonal antibody (R3F1) or polyclonal antibody (11-13) and probed with antibody to PLM (PLM C2Ab). Controls for monoclonal and polyclonal immunoprecipitations (IP) were either beads alone (Beads) or preimmune rabbit IgG (IgG). Immunoblots of corresponding NCX1 immunoprecipitates probed with either polyclonal NCX1 antibody (11-13, middle panel) or monoclonal NCX1 antibody (R3F1, bottom panel) are also shown. Crude membrane preparations (Mem Input) were derived from HEK293 cells transiently transfected for 48 h with PLM + NCX1 encoding plasmid DNA. The antibodies used for immunoblots are indicated on the right, and molecular mass markers (in kDa) are shown on the left. This experiment was performed three times with similar results. WB, Western blot.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Inhibition of INaCa by PLM but not PLMS68A mutant in transfected HEK293 cells. HEK293 cells were transfected with pAdTrack (open squares, n = 3), pAdTrack + NCX1 (open circles, n = 11), PLM + NCX1 (filled circles, n = 8), and PLMS68A + NCX1 (filled squares, n = 7). At 48 h post-transfection, INaCa was measured at 5 mM [Ca2+]o and 30 °C with a descending-ascending voltage ramp protocol described under "Experimental Procedures." Free [Ca2+] in the Ca2+-buffered pipette solution was 205 nM. Holding potential was at the calculated reversal potential of INaCa (-73 mV) under our experimental conditions. Ca2+, Na+-K+-ATPase, Cl-, and K+ currents were blocked by appropriate inhibitors. Error bars are not shown if they fall within boundaries of the symbols.

HEK293 Cell Resting Membrane Potential—Although both measurements of I_NaCa and Na⁺-dependent ⁴⁵Ca²⁺ uptake indicate that PLM inhibited NCX1 activity, the magnitude of PLM inhibition of I_NaCa was much more impressive than the magnitude of PLM inhibition of Na⁺-dependent ⁴⁵Ca²⁺ uptake. Because E_m is an important factor in determining the thermodynamic driving force of Na⁺/Ca²⁺ exchange activity, we next measured resting E_m under conditions that simulated the internal ionic composition of the cell. E_m was also recorded with internal ionic compositions that simulated those of the Na⁺-loaded cells used in the measurement of Na⁺-dependent ⁴⁵Ca uptake. Using an identical Na⁺-loading protocol as used in the present study, Iwamoto et al. (20) reported [Na⁺]_i of 81 ± 2 mM after 20 min of Na⁺-loading in non-excitable mammalian cells. At rest ([Na⁺]_i = 16 mM), E_m in HEK293 cells was -8.8 ± 1.1 mV (n = 4). Under conditions that simulated Na⁺-loading ([Na⁺]_i = 80 mM), E_m was -4.7 ± 1.4 mV (n = 4) and slightly less negative than that measured under basal conditions (p < 0.05). Our values of -5 mV for Na⁺-loaded cells and -9 mV for unloaded cells are similar to -12 mV previously reported for unloaded and non-transfected HEK293 cells (21).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Inhibition of Na+-dependent 45Ca2+ uptake by PLM in transfected HEK293 cells. Top, HEK293 cells transfected for 48 h with either pAdTrack alone (pAdT), pAdT + NCX1 (NCX1), or NCX1 + PLM (PLM/NCX1) were loaded with Na+ as described under "Experimental Procedures." 45Ca2+ uptake into Na+-loaded cells was measured during the initial 30 s in normal BSS (+[Na+]o) or Na+-free BSS (-[Na+]o), both containing 1 mM ouabain. *, p < 0.035, NCX1 (-[Na+]o) versus PLM/NCX1 (-[Na+]o). Bottom, Na+-dependent 45Ca2+ uptake was calculated by subtracting 45Ca2+ uptake values obtained in the presence of Na+ from those obtained in the absence of Na+. *, p < 0.032, NCX1 versus PLM/NCX1. Data represent the mean ± S.E. of four independent experiments.

View larger version (42K): [in this window] [in a new window]   FIG. 7. PLMS68A mutant is expressed and associates with NCX1 in transfected HEK293 cells. Top, immunoblot of NCX1 immunoprecipitates (Ip) from 400 µg of solubilized crude membrane preparations using anti-PLM antibody and probed with antibody to NCX1. Crude membrane preparations (Mem input) were derived from HEK293 cells transiently transfected (48 h) with pAdTrack alone (pAdT), pAdT + NCX1 (NCX1), NCX1 + PLM (NCX1/PLM), or NCX1 + PLMS68A mutant (NCX1/S68A) plasmid DNA. Bottom, immunoblot of PLM immunoprecipitates from 400 µg of solubilized crude membrane preparations using anti-PLM antibody and probed with antibody to PLM. Crude membrane preparations (Mem input) were identical to those described for the top panel. The antibodies used for immunoblots are indicated on the right, and molecular mass markers (in kDa) are shown on the left. This experiment was performed three times with similar results. Beads alone were used as a negative control for the immunoprecipitation (results not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
PLM or FXYD1, an integral membrane protein composed of 72 amino acids, contains only a single transmembrane domain and is a member of the FXYD gene family of small ion transporter regulators (23). PLM is expressed in a tissue-specific manner in heart and skeletal muscle and contains a C-terminal multi-site phosphorylation motif that is absent from all of the other FXYD family members. It is a major substrate for cAMP-dependent PKA and PKC (24, 25). Furthermore, rapid phosphorylation of PLM following - and -adrenergic stimulation paralleled positive inotropic response of the heart (24–26).

Early studies suggested that PLM acted as channels or as ion channel modulators (27, 28). More recently, PLM and other FXYD family members have been demonstrated to be tissue-specific modulators of Na⁺-K⁺-ATPase (29). In rat cardiac myocytes, overexpression of PLM resulted in contraction and [Ca²⁺]_i transient abnormalities (11) similar to those observed in myocytes in which NCX1 was down-regulated (13), leading us to suggest that another function of PLM was to regulate NCX1 activity. Other circumstantial evidence also supports the hypothesis that PLM inhibits NCX1. For example, expression of PLM was significantly elevated following myocardial infarction in the rat (30), an experimental model in which both NCX1 currents (15) and Na⁺-K⁺-ATPase activities (31) were depressed. In addition, phenotypic changes associated with PLM down-regulation (12) paralleled the contractile and [Ca²⁺]_i transient changes observed after NCX1 overexpression in normal rat myocytes (10). In this study, we tested the hypothesis that the effects of PLM on cardiac excitation-contraction coupling are not simply due to changes in the local Na⁺ ion gradient resulting from PLM inhibition of Na⁺-K⁺-ATPase (32), but rather are mediated via a direct protein-protein interaction of PLM with NCX1.

View larger version (46K): [in this window] [in a new window]   FIG. 8. Association of PLM with NCX1 in native cardiac membranes. Panel A, immunoprecipitates (IP) from 600 µg of solubilized pig sarcolemmal (SL) vesicles using 2 µl of anti-PLM antibody or control IgG were obtained. NCX1 and PLM were identified by immunoblotting with R3F1 and anti-PLM antibodies, respectively. Solubilized sarcolemmal vesicles (Mem input) were derived from pig hearts expressing native levels of PLM and NCX1. Panel B, immunoprecipitates from 2 mg of solubilized crude membrane preparations (Mem Input) from rat hearts using 5 µg of anti-PLM antibody or control IgG were obtained. NCX1 and PLM were identified by immunoblotting with R3F1 and anti-PLM antibodies, respectively. Panel C, immunoprecipitates from 2 mg of solubilized crude membrane preparations (Mem Input) from rat hearts using 5 µl of anti-NCX1 antibody (R3F1) were obtained. Control was using beads alone. NCX1 and PLM were identified by immunoblotting with R3F1 and anti-PLM antibodies, respectively. The antibodies used for immunoblots are indicated on the right, and molecular mass markers (in kDa) are shown on the left. This experiment was performed twice with similar results. WB, Western blotting.

Previous studies suggested an overlap in the expression pattern of NCX1 (7) and PLM (14) on the sarcolemma, intercalated disks, and t-tubules of guinea pig, rat, and rabbit ventricular myocytes. This overlap has important functional implications for PLM, because major proteins involved in excitation-contraction coupling are known to be concentrated at the t-tubules, thereby ensuring spatial and temporal control of Ca²⁺ movement throughout the cell (33). Although immunohistochemical co-localization of PLM and NCX1 to the same membrane regions of the myocyte provides anatomic support for a functional interaction, it does not at all prove a direct interaction. Thus the first major finding of our present study is that, using isolated and detergent-solubilized membrane proteins from transiently transfected HEK293 cells, we showed that this co-localization was due to a direct and specific interaction of PLM with NCX1.

Our second major finding that NCX1 activity was inhibited by PLM is not a secondary effect due to interaction of PLM with the -subunit of Na⁺-K⁺-ATPase. On the basis of thermodynamic considerations alone, increased [Na⁺]_i due to inhibition of Na⁺-K⁺-ATPase by PLM should lead to reductions in forward Na⁺/Ca²⁺ exchange (Ca²⁺ efflux) but increases in reverse Na⁺/Ca²⁺ exchange (Ca²⁺ influx). This prediction is not consistent with our data in which both forward and reverse NCX1 currents were inhibited by PLM (Fig. 5). Additionally, under whole-cell patch clamp conditions in which [Na⁺]_i in the well dialyzed HEK293 cell was likely to be "clamped" at pipette [Na⁺] and in which Na⁺-K⁺-ATPase activity would be minimal due to the presence of ouabain and the absence of K⁺ in both external and pipette-filling solutions, I_NaCa was still significantly lower in NCX1 + PLM-expressing cells when compared with NCX1-expressing cells.

Two independent measures of exchanger activity employed in the current study have consistently demonstrated a qualitative inhibition of NCX1 activity by PLM. However, quantitative differences in the magnitude of NCX1 inhibition by PLM were apparent. Some of the quantitative differences between the two techniques may be due to differences in experimental design. For example, I_NaCa was measured over a wide range of E_m, whereas Na⁺-dependent ⁴⁵Ca²⁺ uptake was measured at E_m of -5 to -9 mV. The magnitude of I_NaCa measured at -10 mV was 2.37 ± 0.50 pA/pF for NCX1 cells and 0.50 ± 0.27 pA/pF for cells co-expressing PLM and NCX1 (Fig. 5). These I_NaCa values are substantially less than those measured at +100 mV (8.81 ± 1.50 and 2.58 ± 0.77 pA/pF for NCX1- and NCX1 + PLM-expressing cells, respectively). Therefore, measuring Na⁺/Ca²⁺ exchange activity at different membrane potentials may partly account for the differences in the absolute magnitudes of PLM inhibition on Na⁺/Ca²⁺ exchange as assessed by electrophysiology versus radioactive tracer uptake techniques. A second difference in experimental design is the ionic compositions used. For example, I_NaCa was measured at [Na⁺]_i and [Ca²⁺]_o of 12 and 5 mM, respectively, whereas Na⁺-dependent ⁴⁵Ca²⁺ uptake was measured at [Na⁺]_i and [Ca²⁺]_o of 80 and 0.1 mM, respectively. At the high [Na⁺]_i (80 mM) used in Na⁺-dependent ⁴⁵Ca²⁺ uptake experiments, Na⁺-dependent inactivation of the Na⁺/Ca²⁺ exchanger is likely to occur and thus may result in lower exchanger activity. A third difference is that I_NaCa was measured at 30 °C, whereas Na⁺-dependent ⁴⁵Ca²⁺ uptake was measured at room temperature of 20–22 °C. It is well known that NCX1 activity is very temperature-sensitive (34). A fourth difference is that I_NaCa was measured at [Ca²⁺]_i of 205 nM but Na⁺-dependent ⁴⁵Ca²⁺ uptake was measured at resting [Ca²⁺]_i of HEK 293 cells, which had been reported to be 97 ± 5 nM (35). Due to allosteric regulation of NCX1 by cytosolic Ca²⁺, a substantial portion of NCX1 may be in a deactivated state at resting [Ca²⁺]_i (100 nM) (36). Finally, it should be noted that the usual degree of inhibition or activation observed with NCX1 modulators in ⁴⁵Ca²⁺ flux assays was generally in the order of 20–30% (18, 20).

The purpose in performing the PLM serine 68 mutant experiments was 2-fold. The first was that previous studies conducted in Xenopus oocytes demonstrated nonspecific effects on membrane currents as a direct result of simply overexpressing integral membrane proteins (22). Thus our negative results obtained with PLMS68A mutant (Fig. 5) confirmed the specificity of wild-type PLM on NCX1 activity. The second reason was that PLM contains two PKC phosphorylation sites at serine 63 and serine 68 and a PKA phosphorylation site at serine 68 (37). Because PLM is a major target for both PKA and PKC in the heart (24, 25), we specifically wanted to test the effects of dual PKA/PKC phosphorylation site ablation at serine 68 on NCX1 activity. Most strikingly, this single amino acid substitution completely negated the capability of PLM to inhibit NCX1 activity (Fig. 5). The loss of function was not due to decreased expression of the PLMS68A mutant at the plasma membrane or loss of association with NCX1 (Fig. 7). Our results with the PLMS68A mutant are consistent with the hypothesis that phosphorylation of PLM at serine 68 by PKA and/or PKC has an important effect upon NCX1 activity. However, our current studies were not designed to explore in-depth the specific regulatory role of PLM phosphorylation on NCX1 activity.

In summary, we have identified phospholemman as the first endogenous protein regulator of cardiac Na⁺/Ca²⁺ exchange activity. Phospholemman co-localized and associated with Na⁺/Ca²⁺ exchanger in transfected HEK293 cells. Phospholemman co-immunoprecipitated with Na⁺/Ca²⁺ exchanger in native adult cardiac membranes. Using two independent techniques to assay Na⁺/Ca²⁺ exchange activity, we demonstrated that phospholemman inhibited Na⁺/Ca²⁺ exchange activity. We hypothesized that phosphorylation of serine 68 in phospholemman may be an important mechanism by which it regulates Na⁺/Ca²⁺ exchange activity.

	FOOTNOTES

 
^* This work was supported in part by the National Institutes of Health Grants HL-58672 (to J. Y. C.), DK-46678 (to J. Y. C.), HL-70548 and GM-64640 (to J. R. M.), HL-69074 (to A. L. T.), American Heart Association Pennsylvania Affiliate Grants-in-aid for scientific research 0265426U (to X.-Q. Z.) and 0355744U (to J. Y. C.), American Heart Association Pennsylvania Affiliate Post-Doctoral Fellowship 0425319U (to B. A. A.), and by grants from the Geisinger Foundation (to J. Y. C. and L. I. R.). 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.

To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, MC-H166, Hershey, PA 17033. Tel.: 717-531-5748; Fax: 717-531-7667; E-mail: jyc1{at}psu.edu.

¹ The abbreviations used are: NCX1, Na⁺/Ca²⁺ exchanger; ANOVA, analysis of variance; BSS, balanced salt solution; [Ca²⁺]_i, cytosolic Ca²⁺ concentration; [Ca²⁺]_o, extracellular Ca²⁺ concentration; C_m, whole-cell membrane capacitance; CMV, cytomegalovirus; E_m, membrane potential; GFP, green fluorescent protein; HEK, human embryonic kidney; HRP, horseradish peroxidase; I_NaCa, Na⁺/Ca²⁺ exchange current; [Na⁺_i]_i, cytosolic Na⁺ concentration; [Na⁺]_o, extracellular Na⁺ concentration; Ab, antibody; PBS, phosphate-buffered saline; PKA, protein kinase A; PKC, protein kinase C; PLM, phospholemman.

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