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

Abstract

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.

Full text

Identification of an Endogenous Inhibitor of the Cardiac Na
ⴙ
/Ca
2ⴙ
Exchanger, Phospholemman*
Received for publication, December 30, 2004, and in revised form, February 28, 2005
Published, JBC Papers in Press, March 17, 2005, DOI 10.1074/jbc.M414703200
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‡储‡‡
From the ‡Department of Cellular and Molecular Physiology and 储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
Rapid and precise control of Na
ⴙ
/Ca
2ⴙ
exchanger
(NCX1) activity is essential in the maintenance of beat-
to-beat Ca
2ⴙ
homeostasis in cardiac myocytes. Here, we
show that phospholemman (PLM), a 15-kDa integral sar-
colemmal phosphoprotein, is a novel endogenous pro-
tein inhibitor of cardiac NCX1. Using a heterologous
expression system that is devoid of both endogenous
PLM and NCX1, we first demonstrated by confocal im-
munofluorescence studies that both exogenous PLM
and NCX1 co-localized at the plasma membrane. Recip-
rocal 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 as-
says to assess Na
ⴙ
-dependent
45
Ca
2ⴙ
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
ⴙ
-K
ⴙ
-ATPase, also regulates Na
ⴙ
/Ca
2ⴙ
ex-
change in the heart.
Excitation-contraction coupling in cardiac myocytes depends
on the ability of regulatory proteins to maintain steady-state
Ca
2⫹
fluxes through each cycle of Ca
2⫹
influx, intracellular
Ca
2⫹
transient buffering, and Ca
2⫹
efflux (1). Among the many
transporters and ion channels involved in cardiac Ca
2⫹
fluxes,
the sarcolemmal Na
⫹
/Ca
2⫹
exchanger (NCX1)
1
is unique in
that it participates in all three major phases of Ca
2⫹
move-
ment. Depending on the thermodynamic driving force as deter-
mined by the membrane potential (E
m
) and the concentrations
of Na
⫹
and Ca
2⫹
ions sensed by the exchanger, NCX1 can
mediate both Ca
2⫹
influx (reverse mode) and Ca
2⫹
efflux (for-
ward mode). The forward mode of NCX1 is the major Ca
2⫹
efflux mechanism that extrudes the amount of extracellular
Ca
2⫹
that has entered the myocyte during a twitch, thereby
restoring cytosolic Ca
2⫹
concentration ([Ca
2⫹
]
i
) to resting lev-
els and maintaining steady-state Ca
2⫹
balance (1). NCX1 in-
volvement in Ca
2⫹
efflux and its additional assigned roles in
sarcoplasmic reticulum Ca
2⫹
loading (2) and release (3) are
likely to contribute alongside other proteins such as sarcoplas-
mic reticulum Ca
2⫹
ATPase, calmodulin, calbindin, and parval-
bumin as effective buffering mechanisms for short-term Ca
2⫹
transients. There have been significant advances made toward
understanding the intrinsic properties of NCX1 with regards to
the precise stoichiometry of the Na
⫹
/Ca
2⫹
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 Ca
2⫹
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 (Ni
2⫹
,La
3⫹
,Cd
2⫹
). Synthetic inhibitors in-
clude a 20-amino acid exchanger inhibitor peptide based on a
short sequence of the NCX1 intracellular loop, molluscan car-
dioexcitatory tetrapeptide (FMRFa) analogues, and an isothio-
urea 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 in-
hibitor of NCX1 called phospholemman (PLM). To unequivo-
cally demonstrate this, we have utilized a heterologous expres-
sion system of human embryonic kidney (HEK)293 cells that
* 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@psu.edu.
1
The abbreviations used are: NCX1, Na
⫹
/Ca
2⫹
exchanger; ANOVA,
analysis of variance; BSS, balanced salt solution; [Ca
2⫹
]
i
, cytosolic Ca
2⫹
concentration; [Ca
2⫹
]
o
, extracellular Ca
2⫹
concentration; C
m
, whole-cell
membrane capacitance; CMV, cytomegalovirus; E
m
, membrane poten-
tial; GFP, green fluorescent protein; HEK, human embryonic kidney;
HRP, horseradish peroxidase; I
NaCa
,Na
⫹
/Ca
2⫹
exchange current;
[Na
⫹
]
i
, cytosolic Na
⫹
concentration; [Na
⫹
]
o
, extracellular Na
⫹
concen-
tration; Ab, antibody; PBS, phosphate-buffered saline; PKA, protein
kinase A; PKC, protein kinase C; PLM, phospholemman.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 20, Issue of May 20, pp. 19875–19882, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 19875

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 dif-
ferent measures of NCX1 activity, Na
⫹
-dependent
45
Ca
2⫹
up-
take and NCX1 current (I
NaCa
). Co-expression of PLM with
NCX1 inhibited both forward and reverse I
NaCa
and decreased
Na
⫹
-dependent
45
Ca
2⫹
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-immunoprecipi-
tated with NCX1 in native adult cardiac myocyte membranes.
In summary, we have identified for the first time an endoge-
nous protein inhibitor of cardiac NCX1.
EXPERIMENTAL PROCEDURES
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 di-
rectly 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-di-
rected PLM mutant was authenticated by DNA sequencing and expres-
sion 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 al-
lowed us to identify successfully transfected HEK293 cells through a
separate cytomegalovirus (CMV) promoter present on the vector back-
bone that drives the expression of green fluorescent protein (GFP).
Transfection of HEK293 Cells—HEK293 cells (American Type Cul-
ture 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 ⫻10
6
cells/
100-mm dish. After 24 h, medium was changed and cells were trans-
fected with 25
␮
l of Lipofectamine (Invitrogen) and total of 3-
␮
g plasmid
DNA/dish (either pAdTrack-CMV alone (3
␮
g), pAdTrack-CMV-NCX1
(1.5
␮
g) ⫹pAdTrack-CMV (1.5
␮
g), pAdTrack-CMV-NCX1 (1.5
␮
g) ⫹
pAdTrack-CMV-PLM (1.5
␮
g), or pAdTrack-CMV-NCX1 (1.5
␮
g) ⫹
pAdTrack-CMV-PLMS68A (1.5
␮
g)) 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 for5hat37°C,5%CO
2
. 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 experi-
mentation. Cells for Western blot and co-immunoprecipitation applica-
tions 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
45
Ca
2⫹
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
2mMEGTA. 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 MNaCl, 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, Swit-
zerland) antibodies diluted in BLOTTO were added to the cells, incu-
bated 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 cover-
slip containing mounting solution (90% glycerol in PBS ⫹p-phe-
nylaminediamine) was applied. Images of HEK293 cells (green fluores-
cent 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
␮
l 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 ⫻15 s), 400
␮
l 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 ⫻g) for 10 min at 4 °C. The clarified
supernatant was then subjected to ultracentrifugation at 100,000 ⫻gat
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 pel-
lets 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 ad-
justed to 2 mg in 300
␮
l of Buffer III and combined with C
12
E
8
detergent
in 100
␮
l of Buffer III (at a detergent:protein ratio of 2:1) at room
temperature for 10 min. After the addition of another 400
␮
l of Buffer
III, samples were subjected to ultracentrifugation at 37,000 ⫻gusing
a Beckman TLA 100.3 rotor. The supernatant was transferred to a fresh
tube, and the protein content was determined. 400
␮
g 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 superna-
tants with 50
␮
l of protein A-agarose for1hat4°C.Precleared super-
natants were incubated with either 5
␮
g of preimmune rabbit IgG
(polyclonal Ab control), 5
␮
g of polyclonal PLM antibody (C2Ab), 5
␮
lof
polyclonal NCX1 Ab (
␲
11-13, Swant), 4
␮
l of monoclonal NCX1 Ab
(R3F1), or no Ab (monoclonal Ab control) overnight at 4 °C. The next
day, 40
␮
l (50% slurry) of washed suspended protein A-agarose beads
were added to each sample and incubated for a further2hat4°C.
Beads were pelleted, washed four times with 1.5 ml of Buffer III
containing 0.05% C
12
E
8
, and resuspended in 40
␮
lof2⫻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 sam-
ples were resolved on either 7.5 (NCX1) or 15% (PLM) SDS-PAGE in a
Tris-glycine electrode buffer. Proteins were transferred to polyvinyli-
dene difluoride membranes and blocked overnight in 5% nonfat milk/
Tris-buffered saline with 0.05% Tween 20. Primary antibody incubation
was performed for3hatroom 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. Sec-
ondary antibodies used were either 1:2000 donkey anti-rabbit IgG-
conjugated horseradish peroxidase (HRP*, Amersham Biosciences, Up-
psala, Sweden) or 1:2000 sheep anti-mouse IgG-HRP* (Amersham
Biosciences). Immunoreactivity was detected using an enhanced chemi-
luminescence kit, BioMax XAR film (Eastman Kodak Co., Rochester,
NY), and a developer.
Na
⫹
/Ca
2⫹
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
␮
m) with resistances
of 2.5–3.0 megohms when filled with standard internal solution were
used. Pipettes were filled with a buffered Ca
2⫹
solution containing the
following (in mM): 100 Cs
⫹
glutamate; 7.25 Na
⫹
HEPES; 1 MgCl
2
; 12.75
HEPES; 2.5 Na
2
ATP; 10 EGTA; and 6 CaCl
2
(pH 7.2). Free Ca
2⫹
in the
pipette solution was 205 nM, measured fluorimetrically with fura 2.
Inhibition of Na
⫹
/Ca
2⫹
Exchanger by Phospholemman
19876

Cells were bathed in an external solution containing the following (in
mM): 130 NaCl; 5 CsCl; 1.2 MgSO
4
; 1.2 NaH
2
PO
4
; 5 CaCl
2
; 10 HEPES;
10 Na
⫹
HEPES; and 10 glucose (pH 7.4). Verapamil (1
␮
M), ouabain (1
mM), and niflumic acid (30
␮
M) were used to block Ca
2⫹
,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), indi-
cating 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 [Ca
2⫹
]
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 mMCdCl
2
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 Cd
2⫹
(12). Currents were filtered at 1 kHz, and data were
acquired at 2 kHz. Whole-cell capacitance (C
m
) for each cell was meas-
ured 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 meas-
ured in current-clamp mode as previously described (13, 14, 16). Pipette
solution consisted of the following (in mM): 125 KCl; 4 MgCl
2
; 0.06
CaCl
2
; 10 HEPES; 5 K
⫹
EGTA; 3 Na
2
ATP; and 5 Na
2
-creatine phos-
phate (pH 7.2). External solution consisted of the following (in mM): 127
NaCl; 5.4 KCl; 1.8 CaCl
2
; 1.8 MgCl
2
; 0.6 NaH
2
PO
4
; 7.5 HEPES; 7.5 Na
⫹
HEPES; and 10 glucose (pH 7.4). To simulate Na
⫹
loading, 80 mMKCl
in pipette solution was replaced with NaCl.
Assay of Na
⫹
-dependent
45
Ca
2⫹
Uptake—Na
⫹
-dependent Ca
2⫹
up-
take 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) contain-
ing the following (in mM): 10 HEPES-Tris (pH 7.4); 146 NaCl; 4 KCl; 2
MgCl
2
; 0.1 CaCl
2
; 10 glucose; and 0.1% bovine serum albumin (BSA).
Ouabain (1 mM) and monensin (10
␮
M) were present during Na
⫹
load-
ing.
45
Ca
2⫹
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
45
CaCl
2
(1.5
␮
Ci/ml), [
3
H]mannitol (1
␮
Ci/ml), and 1 mMouabain. After 30 s,
45
Ca
2⫹
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 LaCl
3
. Preliminary studies showed
that
45
Ca
2⫹
uptake was linear during the first 30 s. Cells were solubi-
lized with 0.1 NNaOH and neutralized, and aliquots were taken for
determination of
45
Ca
2⫹
and
3
H radioactivity and protein. Extracellular
contamination as determined from [
3
H]mannitol counts was 0.134 ⫾
0.002
␮
l(n⫽96), and contamination by extracellular
45
Ca
2⫹
was
routinely subtracted before calculation of
45
Ca
2⫹
uptake. Na
⫹
-depend-
ent
45
Ca
2⫹
uptake was calculated by subtracting
45
Ca
2⫹
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 Mem-
branes—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
␮
l of Buffer III, and 1.2 mg of C
12
E
8
in 100
␮
l of Buffer III were
added. After mixing and incubation at room temperature for 10 min,
400
␮
l of Buffer III were added and the mixture was subjected to
ultracentrifugation at 37,000 ⫻g. The supernatant was precleared with
protein A-agarose and incubated with either IgG or 2
␮
l of polyclonal
PLM antibody, and co-immunoprecipitation experiments were per-
formed as described above for HEK293 cells. Crude membrane input
and immunoprecipitated samples were resolved on 12% 1.0 mM12-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
45
Ca
2⫹
uptake,
Student’s paired ttest 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.
RESULTS
Plasma Membrane Co-localization of PLM and NCX1 in
HEK293 Cells—Expression of PLM and of NCX1 in HEK293
was determined by confocal immunofluorescence microscopy.
As indicated by GFP expression driven via a separate CMV
promoter within both PLM and NCX1 pAdTrack expression
vectors, routine transfection efficiencies were between 30 and
50% (image not shown). Using both a monoclonal antibody
directed against the intracellular loop of NCX1 (R3F1; Fig. 1B)
and a polyclonal antibody directed against the cytoplasmic
domain of PLM (C2Ab; Fig. 1C) (11), we demonstrated that
both proteins were predominantly present at the plasma mem-
brane in permeabilized PLM ⫹NCX1-co-transfected cells. This
pattern is in contrast to GFP expression pattern, which was
cytoplasmic (Fig. 1A). These results suggest that both PLM-
and NCX1-transfected cDNAs underwent similar sorting
and/or processing of the translated protein as observed for
endogenous PLM and NCX1 in cardiac myocytes (14). There
was a high degree of co-localization of PLM and NCX1 at the
plasma membrane, as suggested by the merged image (Fig.
1D). In agreement with previous studies, neither NCX1 (8) nor
PLM (19) was detected in HEK293 cells transfected with only
the empty pAdTrack expression plasmid (image not shown;
Western blots shown in Fig. 2).
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 car-
diac 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
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 poly-
clonal 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)ofpanels A–C shows co-local-
ization of PLM and NCX1 but not GFP. Bar ⫽5
␮
m.
Inhibition of Na
⫹
/Ca
2⫹
Exchanger by Phospholemman 19877

pcDNA3-expressing PLM also demonstrated the minor bands
(19). The identities of these minor bands are at present unknown.
Association of NCX1 with PLM in HEK293 Cells—Co-local-
ization of NCX1 with PLM does not necessarily mean interac-
tion of these two proteins. To determine whether PLM associ-
ated with NCX1, we conducted co-immunoprecipitation
experiments in transfected HEK293 cells. Solubilized crude
membrane immunoprecipitates obtained using C2Ab to purify
PLM from cells that co-expressed PLM and NCX1 (Fig. 3,
bottom panel,lane 8) also contained NCX1 (Fig. 3, top panel,
lane 8). Recovery of NCX1 in immunoprecipitates obtained
using the PLM antibody C2Ab was dependent on PLM expres-
sion and was not observed in control experiments using cells
co-transfected with empty pAdTrack expression plasmid and/or
NCX1 (Fig. 3, lanes 5 and 7,top panel). Likewise, control
immunoprecipitates prepared from cells transfected with ei-
ther empty pAdTrack, PLM ⫹pAdTrack, NCX1 ⫹pAdTrack,
or PLM ⫹NCX1 using preimmume rabbit IgG did not contain
either PLM or NCX1 (Fig. 3, top and bottom panels,lanes 1– 4).
Equivalent levels of NCX1 and PLM protein were observed in
the starting material for immunoprecipitation whether ex-
pressed either alone or in combination in these cells (Fig. 3, top
and bottom panels,lanes 10 –12).
To provide further evidence for the specificity of this associ-
ation, the reciprocal experiment was performed in cells co-
expressing PLM and NCX1 in which anti-NCX1 immunopre-
cipitates 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 pre-
immume rabbit IgG did not contain PLM or NCX1 (Fig. 4, lanes
1and 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
⫹
/Ca
2⫹
exchange current (I
NaCa
) in HEK293 cells ex-
pressing NCX1. Fig. 5 shows the steady-state I
NaCa
measured
at various membrane potentials, at 30 °C and 5.0 mM[Ca
2⫹
]
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. Ex-
pression of NCX1 in HEK293 cells resulted in a large I
NaCa
compared with that measured in control HEK293 cells trans-
fected with empty vector (group effect, p⬍0.0001). In addition,
the differences in I
NaCa
between control cells and cells express-
ing 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 experi-
mental 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 impor-
tant to note that the decrease in I
NaCa
in PLM ⫹NCX1 cells
was not due to the reduction of NCX1 protein levels in cells
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
␮
g) from indicated cell cultures were sub-
jected 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.
FIG.3. Demonstration of association of NCX1 with PLM in
transfected HEK293 cells by immunoprecipitation. Top panel,
immunoblot of NCX1 immunoprecipitates from 400
␮
g of solubilized
crude membrane preparations using 5
␮
g of anti-PLM antibody (C2Ab)
or control rabbit IgG (Preimmune) probed with antibody to NCX1
(R3F1). Bottom panel, immunoblot of PLM immunoprecipitates from
400
␮
g of solubilized crude membrane preparations using 5
␮
gof
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 experi-
ment was performed three times with similar results.
FIG.4. Reciprocal co-purification of PLM with NCX1 in
HEK293 crude membrane extracts. Top panel, immunoblot of PLM
immunoprecipitates from 400
␮
g of solubilized crude membrane prep-
arations 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 molec-
ular mass markers (in kDa) are shown on the left. This experiment was
performed three times with similar results. WB, Western blot.
Inhibition of Na
⫹
/Ca
2⫹
Exchanger by Phospholemman
19878

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 in-
creased 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 pres-
ence 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 capac-
itance of 33 pF reported in HEK293 cells transfected with
NCX1 (8).
Effect of PLM on Na
⫹
-dependent
45
Ca
2⫹
Uptake in NCX1-
expressing Cells—Na
⫹
-dependent
45
Ca
2⫹
uptake in Na
⫹
-
loaded cells was measured during the initial 30 s since prelim-
inary experiments demonstrated that uptake was linear under
the conditions used. In Na
⫹
-loaded cells transfected with
empty control vector,
45
Ca
2⫹
uptake rates were very low and
similar in the presence and absence of Na
⫹
, consistent with
absent endogenous Na
⫹
/Ca
2⫹
exchange activity in HEK293
cells (Fig. 6, top panel). NCX1-expressing cells preloaded with
Na
⫹
exhibited a 16-fold greater
45
Ca
2⫹
uptake activity when
exposed to Na
⫹
-free uptake medium than that measured in
Na
⫹
-containing uptake medium (Fig. 6, top panel), consistent
with Na
⫹
/Ca
2⫹
exchange activity. Co-expression of PLM re-
sulted in a 15% reduction of
45
Ca
2⫹
uptake in Na
⫹
-loaded and
NCX1-expressing cells exposed to Na
⫹
-free uptake medium
(p⬍0.035). Na
⫹
-dependent
45
Ca
2⫹
uptake was calculated by
subtracting
45
Ca
2⫹
uptake in Na
⫹
-containing uptake medium
from that devoid of Na
⫹
. Co-expression of PLM significantly
inhibited Na
⫹
-dependent
45
Ca
2⫹
uptake in NCX1-expressing
cells (0.38 ⫾0.02 versus 0.33 ⫾0.02 nmol/mg/min; p⬍0.032)
(Fig. 6, bottom panel).
HEK293 Cell Resting Membrane Potential—Although both
measurements of I
NaCa
and Na
⫹
-dependent
45
Ca
2⫹
uptake in-
dicate 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
45
Ca
2⫹
uptake.
Because E
m
is an important factor in determining the thermo-
dynamic driving force of Na
⫹
/Ca
2⫹
exchange activity, we next
measured resting E
m
under conditions that simulated the in-
ternal 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
45
Ca
uptake. Using an identical Na
⫹
-loading protocol as used in the
present study, Iwamoto et al. (20) reported [Na
⫹
]
i
of 81 ⫾2mM
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 ⫺9mVfor
unloaded cells are similar to ⫺12 mV previously reported for
unloaded and non-transfected HEK293 cells (21).
Effect of a Single Amino Acid Substitution at Serine 68 in
PLM on I
NaCa
in NCX1-expressing Cells—One interpretation
of the effect of PLM on I
NaCa
in NCX1-expressing HEK293
cells is that the introduction of small integral membrane
proteins may exert nonspecific effects on cellular ion trans-
FIG.5.Inhibition of I
NaCa
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, I
NaCa
was measured at 5 mM
[Ca
2⫹
]
o
and 30 °C with a descending-ascending voltage ramp protocol
described under “Experimental Procedures.” Free [Ca
2⫹
] in the Ca
2⫹
-
buffered pipette solution was 205 nM. Holding potential was at the
calculated reversal potential of I
NaCa
(⫺73 mV) under our experimental
conditions. Ca
2⫹
,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.
FIG.6. Inhibition of Na
ⴙ
-dependent
45
Ca
2ⴙ
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 “Experi-
mental Procedures.”
45
Ca
2⫹
uptake into Na
⫹
-loaded cells was measured
during the initial 30 s in normal BSS (⫹[Na
⫹
]
o
)orNa
⫹
-free BSS
(⫺[Na
⫹
]
o
), both containing 1 mMouabain. *, p⬍0.035, NCX1 (⫺[Na
⫹
]
o
)
versus PLM/NCX1 (⫺[Na
⫹
]
o
). Bottom,Na
⫹
-dependent
45
Ca
2⫹
uptake
was calculated by subtracting
45
Ca
2⫹
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.
Inhibition of Na
⫹
/Ca
2⫹
Exchanger by Phospholemman 19879

port activity (22). To test this hypothesis, we co-transfected
HEK293 cells with NCX1 and a PLM mutant, PLMS68A, in
which a single amino acid at serine 68 was mutated to ala-
nine. Replacement of serine 68 by alanine abolished the effect
of wild-type PLM on I
NaCa
(Fig. 5, filled squares,n⫽7). This
is verified by two-way ANOVA of I
NaCa
between NCX1- and
PLMS68A ⫹NCX1-expressing cells: insignificant group (p⬍
0.36) and group x voltage interaction (p⬍0.99) effects. A lack
of PLMS68A mutant effect on I
NaCa
was not due to the lack of
expression or inability of the mutant to associate with NCX1
(Fig. 7). These results indicate that the effect of PLM on
NCX1 activity was specific.
Association of PLM with NCX1 in Native Cardiac Myocytes—
The results presented so far have been obtained in a heterolo-
gous expression system in which both PLM and NCX1 were
overexpressed. We next sought to evaluate whether PLM asso-
ciated with NCX1 in its native environment of cardiac mem-
branes. In both pig cardiac sarcolemmal vesicles and crude
membrane preparations from rat hearts, anti-PLM antibody
immunoprecipitated PLM and co-immunoprecipitated NCX1
(Fig. 8). Reciprocally, in rat cardiac membrane preparations,
anti-NCX1 antibody immunoprecipitated NCX1 and co-immu-
noprecipitated PLM (Fig. 8). The physical interaction between
PLM and NCX1 under native expression levels in cardiac myo-
cytes provided physiological relevance to our observations in
the heterologous expression system.
DISCUSSION
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 trans-
porter 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 phos-
phorylation 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 myo-
cytes, overexpression of PLM resulted in contraction and
[Ca
2⫹
]
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 infarc-
tion in the rat (30), an experimental model in which both NCX1
currents (15) and Na
⫹
-K
⫹
-ATPase activities (31) were de-
pressed. In addition, phenotypic changes associated with PLM
down-regulation (12) paralleled the contractile and [Ca
2⫹
]
i
transient changes observed after NCX1 overexpression in nor-
mal rat myocytes (10). In this study, we tested the hypothesis
that the effects of PLM on cardiac excitation-contraction cou-
pling 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 inter-
action of PLM with NCX1.
An advantage of the heterologous HEK293 expression sys-
tem is that high levels of NCX1 can be expressed in membranes
that do not possess endogenous NCX1, PLM, or a milieu of
other confounding ion transport pathways that are present in
cardiac cells. Under our experimental conditions in which K
⫹
,
Ca
2⫹
,Cl
⫺
, and Na
⫹
-K
⫹
-ATPase currents were blocked,
HEK293 cells displayed low background currents, resulting in
high signal-to-noise ratio for I
NaCa
. In addition, the relative
small size of HEK293 cells (C
m
⬃30 pF) compared with that of
adult rat cardiac myocytes (C
m
⬃180 pF) allowed stricter con-
trol of voltage clamp and intracellular ionic compositions. Our
precaution of holding the cell at the theoretical equilibrium
potential of Na
⫹
/Ca
2⫹
exchange (⫺73 mV under our experi-
FIG.7. PLMS68A mutant is expressed and associates with
NCX1 in transfected HEK293 cells. Top, immunoblot of NCX1 im-
munoprecipitates (Ip) from 400
␮
g of solubilized crude membrane prep-
arations 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
␮
g 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).
FIG.8.Association of PLM with NCX1 in native cardiac mem-
branes. Panel A, immunoprecipitates (IP) from 600
␮
g of solubilized
pig sarcolemmal (SL) vesicles using 2
␮
l of anti-PLM antibody or
control IgG were obtained. NCX1 and PLM were identified by immu-
noblotting with R3F1 and anti-PLM antibodies, respectively. Solubi-
lized sarcolemmal vesicles (Mem input) were derived from pig hearts
expressing native levels of PLM and NCX1. Panel B, immunoprecipi-
tates from 2 mg of solubilized crude membrane preparations (Mem
Input) from rat hearts using 5
␮
g 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, immuno-
precipitates from 2 mg of solubilized crude membrane preparations
(Mem Input) from rat hearts using 5
␮
l 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, West-
ern blotting.
Inhibition of Na
⫹
/Ca
2⫹
Exchanger by Phospholemman
19880

mental conditions) for at least 5 min before application of the
voltage ramp minimized ion fluxes via the exchanger and al-
lowed [Na
⫹
]
i
and [Ca
2⫹
]
i
to equilibrate with those present in
the pipette solution. Finally, there were little-to-no differences
in currents measured between the descending and ascending
voltage ramps, indicating that [Na
⫹
]
i
and [Ca
2⫹
]
i
sensed by the
Na
⫹
/Ca
2⫹
exchanger were not appreciably changed by NCX1
fluxes during the brief (880 ms) voltage ramp.
Previous studies suggested an overlap in the expression pat-
tern 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-contrac-
tion coupling are known to be concentrated at the t-tubules,
thereby ensuring spatial and temporal control of Ca
2⫹
move-
ment throughout the cell (33). Although immunohistochemical
co-localization of PLM and NCX1 to the same membrane re-
gions 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 thermody-
namic considerations alone, increased [Na
⫹
]
i
due to inhibition
of Na
⫹
-K
⫹
-ATPase by PLM should lead to reductions in for-
ward Na
⫹
/Ca
2⫹
exchange (Ca
2⫹
efflux) but increases in reverse
Na
⫹
/Ca
2⫹
exchange (Ca
2⫹
influx). This prediction is not con-
sistent 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 signifi-
cantly 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 quali-
tative inhibition of NCX1 activity by PLM. However, quantita-
tive 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
45
Ca
2⫹
uptake was measured at
E
m
of ⫺5to⫺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, meas-
uring Na
⫹
/Ca
2⫹
exchange activity at different membrane po-
tentials may partly account for the differences in the absolute
magnitudes of PLM inhibition on Na
⫹
/Ca
2⫹
exchange as as-
sessed 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 [Ca
2⫹
]
o
of 12 and 5 mM, respectively, whereas Na
⫹
-
dependent
45
Ca
2⫹
uptake was measured at [Na
⫹
]
i
and [Ca
2⫹
]
o
of 80 and 0.1 mM, respectively. At the high [Na
⫹
]
i
(80 mM) used
in Na
⫹
-dependent
45
Ca
2⫹
uptake experiments, Na
⫹
-dependent
inactivation of the Na
⫹
/Ca
2⫹
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
45
Ca
2⫹
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
[Ca
2⫹
]
i
of 205 nMbut Na
⫹
-dependent
45
Ca
2⫹
uptake was meas-
ured at resting [Ca
2⫹
]
i
of HEK 293 cells, which had been
reported to be 97 ⫾5n
M(35). Due to allosteric regulation of
NCX1 by cytosolic Ca
2⫹
, a substantial portion of NCX1 may be
in a deactivated state at resting [Ca
2⫹
]
i
(⬃100 nM) (36). Finally,
it should be noted that the usual degree of inhibition or acti-
vation observed with NCX1 modulators in
45
Ca
2⫹
flux assays
was generally in the order of 20 –30% (18, 20).
The purpose in performing the PLM serine 68 mutant exper-
iments was 2-fold. The first was that previous studies con-
ducted 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 speci-
ficity of wild-type PLM on NCX1 activity. The second reason
was that PLM contains two PKC phosphorylation sites at ser-
ine 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 substi-
tution 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 re-
sults with the PLMS68A mutant are consistent with the hy-
pothesis that phosphorylation of PLM at serine 68 by PKA
and/or PKC has an important effect upon NCX1 activity. How-
ever, 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
⫹
/Ca
2⫹
exchange
activity. Phospholemman co-localized and associated with Na
⫹
/
Ca
2⫹
exchanger in transfected HEK293 cells. Phospholemman
co-immunoprecipitated with Na
⫹
/Ca
2⫹
exchanger in native
adult cardiac membranes. Using two independent techniques
to assay Na
⫹
/Ca
2⫹
exchange activity, we demonstrated that
phospholemman inhibited Na
⫹
/Ca
2⫹
exchange activity. We hy-
pothesized that phosphorylation of serine 68 in phospholem-
man may be an important mechanism by which it regulates
Na
⫹
/Ca
2⫹
exchange activity.
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19882