Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5536-5544
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Consequences of
Functional Expression of the Plasma Membrane Ca
Pump
Isoform 1a (*)
(Received for publication, October 5,
1995; and in revised form, December 27, 1995)
Bei-Fang
Liu
(1), (§),
Xin
Xu(§)
(2),
Rafael
Fridman
(1),
Shmuel
Muallem
(2),
Tuan
H.
Kuo
(1)From the
(1)Department of Pathology, Wayne State
University School of Medicine, Detroit, Michigan 48201 and the
(2)Department of Physiology, University of Texas
Southwestern Medical Center, Dallas, Texas 76235
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
The plasma membrane Ca
-ATPase pump (PMCA) is
an integral component of the Ca signaling system
which participates in signal transduction during agonist stimulated
cell activation. To better understand the physiological function of the
pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial
cells using a stable transfection system under the control of a
cytomegalovirus promoter. The cell lines selected after transfection
with PMCA1a construct, expressed 3-4-fold increased pump protein
which was mostly targeted to the plasma membrane as indicated by
immunoperoxidase staining. Ca uptake assays in a
membrane preparation indicated a 3-4-fold increase in
Ca pumping activity in the transfected cells, and the
expressed PMCA1a showed typical dependence on Ca and
calmodulin for stimulation of activity. Measurement of
[Ca]
and
[Ca]
showed that expression
of PMCA1a had a profound effect on different aspects of the
Ca signal. The peak increase in
[Ca] evoked by ATP
and/or thapsigargin was lower but the plateau phase was similar in the
PMCA1a expressing cells. Accordingly, titration with ionomycin of
Ca content of internal stores, measurement of
Ca uptake into the thapsigargin- and
oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes,
Ca uptake into streptolysin O-permeabilized
cells, and analysis of SERCA mRNA and protein, showed that expression
and activity of the SERCA pump was down-regulated in cells expressing
PMCA1a pump. Expression of PMCA1a also down-regulated expression of the
inositol 1,4,5-trisphosphate (IP
)-activated Ca channel and the rate of IP-mediated Ca release in permeable cells, without affecting the affinity of the
channel for IP. On the other hand the rate of store
depletion-dependent Ca and Mn influx (Ca entry) into PMCA1a expressing cells
was increased by about 2.6-fold. These changes prevented estimating the
rate of pump-mediated Ca efflux from changes in
[Ca]. Measurement of
[Ca] showed that the rate of
Ca efflux in cells expressing PMCA1a was about
1.45-fold higher than Neo controls, despite the 4-fold increase in the
amount of functional pump protein. The overall study points to the
flexibility, interdependence, and adaptability of the different
components of the Ca signaling systems to regulate
the expression and activity of each component and maintain a nearly
constant Ca signal.
INTRODUCTION
The plasma membrane Ca pump (PMCA) (
)is the sole high affinity Ca extrusion
mechanism in the plasma membrane(1) . In non-muscle cells, the
pump plays a prominent role in Ca extrusion than
other mechanisms such as the Na
/Ca exchanger(2, 3, 4, 5, 6, 7) .
PMCA activity is believed to be activated during hormonal stimulation
of cells (2) and the increased Ca efflux is
concomitant with an enhanced phosphorylation of the Ca pump protein(8, 9) .
cDNA cloning indicates
the presence of at least four major isoforms for the PMCA, which are
encoded by distinct genes numbered ``1 to 4'' (1, 10, 11, 12) . The primary
transcripts of each gene may be alternatively spliced at their 3` ends
in a different way, further increasing the variety of pumps as denoted
by letters ``a to d''(1) . While these isozymes show
distinct tissue and cellular distribution, isoform 1b (PMCA1b) appears
to be the major isoform that is expressed in all tissues
examined(1, 13) . Recent attempts to over-express the
human gene 4 in COS cells have demonstrated the usefulness of transient
transfection in relating the structure of the pump to its biochemical
activity(14, 15, 16, 17) . However,
a transient expression system does not allow the study of intracellular
Ca signaling in a metabolically active cell type,
such as endothelial cells, due to the low transfection efficiency
(normally 15% or less of the cells were transfected by the transient
transfection system). (
)In this report we focus on the
stable transfection of the PMCA1a isoform of the pump in rat aortic
endothelial cells (RAECs), which endogenously contain a low level of
PMCA1b as the predominant species, while isoform 1a is either absent or
undetectable by reverse transcriptase-polymerase chain
reaction(13) . This over-expression system allowed us to study
the physiological function of PMCA during agonist stimulation. In
addition, since no information is available regarding how
over-expression of one calcium transporter may influence the expression
of other calcium transporters, we have examined the effect of PMCA1a
over-expression on the four major Ca transport
pathways responsible for cellular Ca homeostasis,
which includes intracellular pump (SERCA), the IP-activated
Ca channel, the plasma membrane calcium entry
pathway, and the PMCA(18, 19) . The overall findings
demonstrate the flexibility and coordinated regulation of all pathways,
possibly on the gene level, to maintain constant Ca signaling.
MATERIALS AND METHODS
Plasmid Construction
The cDNA encoding rat
plasma membrane Ca-ATPase isoform 1a (PMCA1a) was
excised from plasmid RB11-1(10) (a gift from Dr. Gary
Shull) with BclI and BamHI, cloned into the
eukaryotic expression vector pcDNAI/NEO (Invitrogen) in its BamHI site. The resulting construct, named PMCA construct and
the vector control (without the cDNA insert) were used to transfect rat
aortic endothelial cells separately.
Cell Culture and Stable Transfection
RAEC were
grown in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum, 2 mML-glutamine, and 25
µg/ml gentimycin as described previously(9) . To establish
cell lines that stably express rat PMCA1a, 6 µg/ml PMCA construct
or vector alone was transfected into RAEC by the Lipofectin method
(Life Technologies, Inc.). The neomycin-resistant colonies were
selected with 90 µg/ml G418 in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum. Colonies were picked
and grown as individual cell lines in the presence of 90 µg/ml
G418. Each cell line was assayed by Western blot to confirm PMCA
expression.
Northern Blot Analysis
Total RNA was isolated from
cells in logarithmic growth phase, and samples were used for Northern
analysis as described previously(9) . After electrophoresis and
transfer, the blots were hybridized with specific cDNA probes. The
SERCA cDNA probe was a 330-base pair EcoRI/PstI
fragment (nucleotides 892-1222) of the SERCA2 gene(20) . This
probe can recognize both SERCA isoforms 2 and 3 present in the RAEC. In
the case of Fig. 6, a full-length SERCA3 cDNA (21) was
used as a probe to detect SERCA3 mRNA.
Figure 6:
SERCA pump activity and mRNA levels. Panel A, ATP-mediated, oxalate-dependent Ca uptake was measured by incubating microsomes in 
Ca
uptake medium with and without Tg and in the presence of 7.5 mM oxalate. At the indicated times samples were removed to analyze Ca content in microsomes. The figure
shows the fraction of the Tg-sensitive Ca uptake. Panel B shows a Northern blot analysis of total
RNA prepared form Neo control and clone 8 cells. SERCA mRNA (4.4
kilobases) was identified by using a specific cDNA probe representing
SERCA 3, the major species in RAEC (T. H. Kuo, unpublished data). The
same blot was hybridized with an actin cDNA probe to show the equal
loading of RNA samples. Similar reductions in SERCA mRNA were obtained
in four additional experiments using clone 8 or 35
cells.
Western Blot Analysis
The protein samples (cell
lysate) were separated on 7% polyacrylamide gels that contained 0.1%
SDS and transferred to nitrocellulose membrane by electrophoretic
blotting as described previously(9) . A monoclonal antibody
5F10 (Affinity BioReagents) against the human erythrocyte PMCA was
described earlier (22) . This antibody recognizes several
plasma membrane calcium pump isoforms including PMCA1a in various
tissues(13) . The nitrocellulose membrane was treated with 5F10
and the expressed PMCA1a as well as endogenous pump detected by using
the alkaline phosphatase-conjugation method(9) .
Immunoperoxidase Staining
The cells were grown on
coverslips for immunostaining by avidin-biotin technique using a kit
from Vector Laboratory. Samples on coverslips were fixed in acetone and
then treated with monoclonal antibody 5F10 for 20 min at room
temperature. Subsequently, they were processed with biotinylated
anti-mouse IgG followed by treatment with a preformed avidin- and
biotinylated- horseradish peroxidase macromolecule complex according to
the manufacturer's protocol. This has been termed the ABC
technique. The coverslips were developed with substrate solution
containing 3-amino-9-ethylcarbazole and hydrogen peroxide and further
counterstained with hematoxylin for microscopy. The punctate staining
of the immunolabeled pumps was identified as reddish-brown spots in a
background of a blue hematoxylin. The two colors could be distinguished
very well.
Membrane Preparation
A crude membrane containing
both plasma membrane and microsomes was prepared according to the
procedure of Enyedi et al.(15) . The cells were
homogenized in a hypotonic solution of 10 mM Tris-HCl, pH 7.5,
1 mM MgCl, 0.1 mM phenylmethylsulfonyl
fluoride, 4 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 mM dithiothreitol. The homogenate was diluted with an equal volume of
0.5 M sucrose, 0.3 M KCl, 2 mM dithiothreitol, 10 mM Tris-HCl, pH 7.5, homogenized
again, and centrifuged at 5000 
g for 15 min. The
supernatant was made 0.6 M in KCl and 1.5 mM in EDTA
(to remove calmodulin). The suspension was centrifuged at 100,000
g for 40 min. The final pellet was resuspended in a
solution containing 0.25 M sucrose, 0.15 M KCl, 10
mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, and 20
µM CaCl.
CaUptake in
Microsomes
PMCA-mediated Ca uptake was
measured similar to the procedures of (15) . However, a major
difference being that under our conditions, it was not necessary to add
40 mM phosphate to improve the signal to noise ratio. Membrane
vesicles (6 µg of protein) were incubated at 37 °C in 140
µl of medium containing 100 mM KCl, 20 mM Hepes,
pH 7.4, 7 mM MgCl, 5 mM NaN,
4 µg/ml oligomycin, 0.5 mM ouabain, 100 µM CaCl (labeled with Ca), and sufficient
EGTA to obtain the desired free [Ca].
Thapsigargin (200 nM) was also included to inhibit the
activity of the SERCA pumps. The reaction was started by the addition
of 6 mM ATP. The incubation time was from 0 to 10 min.
Radioactive Ca accumulation at each time point was
determined by filtration(23) . The transport activity was
determined in the presence and absence of 240 nM calmodulin.
SERCA activity assay is based on the measurement of ATP-mediated,
oxalate-dependent calcium uptake into microsomes according to Lytton et al.(24) .
Measurements of Free Cytosolic
Ca
[Ca]
was measured by image acquisition and analysis using the Meridian
ACAS 570 or the PTI system described before(25) . RAEC grown or
attached to coverslips, were loaded with Indo-1 or Fura 2 by incubation
in loading buffer containing (in mM) 5.4 KCl, 137 NaCl, 0.44
KHPO
, 4.2 NaHCO, 0.34
NaHPO, 11.1 D-glucose, 5 Hepes, pH
7.3, 2 CaCl, 0.1% bovine serum albumin. After 60 min
incubation with Indo 1/AM or 30 min incubation with Fura 2/AM, the
cells were rinsed, the coverslips were placed in a holder and mounted
on the laser cytometer equipped with an inverted microscope (Indo 1) or
a stage of a Nikon inverted microscope (Fura 2). Indo 1 fluorescence
was measured at an excitation wavelength of 360 nm and emission
wavelengths of 405 and 485 nm. Fura 2 fluorescence was measured at
excitation wavelengths of 355 and 380 nm. For measurement of
[Ca] in cell suspension, Fura
2-loaded cells were suspended in a solution containing (in mM)
140 NaCl; 5 KCl; 1 MgCl; 10 Hepes (pH 7.4 with NaOH); 10
glucose (solution A) and with or without 1 mM CaCl. The cells were placed in a fluorimeter cuvette
maintained at 37 °C and the cells were continuously stirred.
Fluorescence was recorded at excitation wavelengths of 340 and 360 nm
and an emission wavelength of 500 nm. When appropriate, the signals
were calibrated as detailed before(26) .
Measurement of
[Ca]
Cells were
released from culture plates, washed twice in solution A, and kept on
ice until use. Before each measurement about 4 10
cells were washed once with Ca-free solution A
containing 0.2 mM MgCl and once with the same
solution that was treated with Chelex 100 (Sigma). The cells were
resuspended in 0.5 ml of a warm (37 °C) Chelex-treated solution
that contained 1 µM Fluo 3 and placed in a thermostated
fluorimeter cuvette. EGTA in increments of 0.2 µM was
added until medium [Ca] was reduced to
about 120 nM. The cells were then stimulated with a mixture of
(final concentrations) 100 µM ATP and 2 µM Tg. Fluo 3 fluorescence was measured at an excitation wavelength
of 480 nm and an emission wavelength of 530 nm. The fluorescence of the
dye present in the external medium was calibrated by addition of 1
mM CaCl to the incubation medium to obtain F
and then 10 mM EGTA and 20 mM NaOH to obtain F
.
[Ca] was calculated as described before
using a K
of 370 nM(2) .
Measurement of CaUptake and
Release in Permeable Cells
Between 5 
10 and 10
RAEC cells in solution A were washed twice
with a solution containing 145 mM KCl and 10 mM Hepes
(pH 7.4 with NaOH) and once with the same solution treated with Chelex
100. The cells were suspended in 0.5 ml of a warm, Chelex-treated
solution containing 1 µM Fluo 3, 3 mM ATP, 5
mM MgCl, 10 mM creatine phosphate, 5
units/ml creatine kinase, 10 µg/ml oligomycin, 10 µM antimycin A, and 3 mg/ml streptolysin O (SLO) reagent (Murex). The
cells were allowed to reduce medium [Ca]
using Ca uptake into the Tg-sensitive stores. After
stabilization of [Ca],
IP-mediated Ca release was measured by
addition of increasing concentrations of IP to the cuvette.
The Ca signal was calibrated as described
above(2) .
Calculation and Statistics
All measurements of
initial rates reported were derived from the maximal slope of a given
curve of changes in [Ca] or
[Ca]. When appropriate,
results are reported as mean ± S.E. of the indicated number of
experiments. Statistical analysis was by Student's t test. p < 0.05 and below was considered statistically
significant.
RESULTS
Over-expression of Rat PMCA Isoform 1a in
RAEC
Following G418 selection, 36 neomycin-resistant clones were
isolated from the culture transfected with the PMCA1a construct.
Screening by Western blot with the monoclonal antibody 5F10 (Fig. 1A) indicated varying degrees of PMCA1a
over-expression (from 1-4-fold increase over the level of the
native PMCA1b). Control cells that were transfected with the vector
alone (lane b) contained mainly the PMCA1b isoform and showed
low amounts of PMCA1a protein, similar to the nontransfected parental
RAEC (lane a). Fig. 1A shows only the
screening results of six clones. After complete screening of all 36
clones, three clones that expressed approximately 3-4-fold higher
PMCA1a protein, i.e. clones 8, 33 (Fig. 1A),
and 35 (not shown), were selected for further study. In a different
study using transient expression of PMCA1a construct in BSC-1 cells, we
have established that the over-expressed protein has a molecular mass
of 130 kDa (Fig. 1B, lower band) which matches the
theoretical size based on the cDNA sequence of isoform 1a(10) ,
while the major endogenous protein had a molecular mass of 135 kDa
corresponding to isoform 1b (Fig. 1B, upper band).
Over-expression of isoform 1a in RAEC was further confirmed by
detecting the corresponding mRNA by reverse transcriptase-polymerase
chain reaction, using a pair of primers previously described for
detecting alternatively spliced PMCA1 mRNA isoforms(13) .
Figure 1:
Western blot analysis of PMCA1a
over-expressing clones. Lysates from the different RAEC clones stably
over-expressing PMCA1a (A) and from BSC-1 cells transfected
with a vaccinia virus system that includes the PMCA1a construct (B) were prepared and separated by SDS-polyacrylamide gel
electrophoresis as described under ``Materials and Methods.''
The blots were probed with monoclonal antibody 5F10. Lanes a and b in panel A show membranes from control
nontransfected and control transfected with vector only, respectively. Lanes 2-7 show membrane from selected clones. Note the
higher PMCA1a levels in clones 8, 26, 33, and 34. Panel B illustrates the difference in molecular weight between PMCA1a and
PMCA1b. The left lane shows membranes from control BSC-1 cells
and the right lane shows membranes from BSC-1 cells
transfected with the PMCA1a construct.
The present study represents the first report that a PMCA isoform 1
cDNA was successfully over-expressed in mammalian cells. This is
different from an earlier report (14) describing the failed
attempt to express a full-length human PMCA isoform 1 cDNA. Presently,
the only other successful expression of the PMCA protein is with the
human isoform 4b(14, 17) . However, in one report
there was a problem in the correct targeting of the protein to the
plasma membrane of the COS cells(14) . Most recently isoform
PMCA4b was successfully expressed and targeted to the plasma
membrane(27) .
The Expressed PMCA1a Is Functional and Is Largely
Targeted to the Plasma Membrane
Fig. 2shows the
immunoperoxidase staining of the PMCA1a over-expressing clone as
compared to the control cells transfected with the vector alone. The
use of PMCA antibody 5F10 showed that higher levels of Ca pump were expressed in the PMCA-transfected cells (clone 8, Fig. 2A) as compared with control cells (Fig. 2B). A pattern of a punctate, peripheral
distribution of stained material was quite obvious in the clone 8
over-expressing PMCA (Fig. 2A) (observed as
reddish-brown spots in a blue hematoxylin background), suggesting that
at least a major portion of the over-expressed protein was associated
with the plasma membrane. An additional observation in Fig. 2A in support of plasma membrane localization is
the heaviest staining of the cytoplasmic perimeter of the cell where
the superficial and underlying plasma membrane come to join. Since the
distribution of the label on the cytoplasmic and/or nuclear surface is
evenly scattered, it is unlikely that they represent ER localization.
In control cells (Fig. 2B) minimal staining of
endogenous proteins are detected due to its low abundance in the plasma
membrane (9) . A similar pattern of staining was reported for
CHO cells over-expressing the PMCA4a(27) .
Figure 2:
Immunolocalization of PMCA1a. Clone 8
cells (panel A) and control cells transfected with the vector (panel B) were fixed and stained with monoclonal antibody 5F10
as described under ``Materials and Methods.'' Note the
punctate peripheral staining and absence of cytosolic, ER-like staining
in clone 8 cells over-expressing the PMCA1a pump. The amount of pump
protein in the control cells was below the detection limit at the
concentration of antibody used.
Additional
support for correct targeting of PMCA1a comes from the assay of PMCA
activity using isolated microsomes. It was reported previously (15) that over-expression of human PMCA4 led to an increased
Ca uptake in microsomes prepared from COS-1 cells,
and this activity is stimulated by oxalate. Stimulation by oxalate was
interpreted as an evidence that PMCA4 was retained in the
ER(15) . An assay of Ca uptake in microsomes
obtained from control and PMCA1a over-expressing RAECs indicated that
over-expression of PMCA1a led to an increase in the Ca uptake that is not stimulated by oxalate (see below). Thus the
combined evidence from immunoperoxidase staining and the properties of
microsomal Ca uptake suggest that the expressed
PMCA1a is not retained in the ER but rather localized to the plasma
membrane.
To determine whether the expressed protein was functional,
the properties of ATP-dependent Ca uptake were
measured. A 70-fold excess concentration of thapsigargin (Tg, 200
nM) was included in the uptake reaction to ensure complete
inhibition of SERCA pump activity, without affecting the
calmodulin-dependent Ca uptake mediated by the
PMCA(15) . As shown in Fig. 3A, the rate of
ATP-dependent Ca uptake in the membrane preparation
from the PMCA-transfected cells (clone 8) was about 3-fold higher than
the vector transfected controls (0.47 versus 0.16
nmol/mg/min). Similar results were obtained with microsomes prepared
from clone 35 (not shown). Thus the activity assay shows that the
over-expressed PMCA1a is functional in Ca transport.
Figure 3:
Ca uptake in isolated
microsomes. Isolated microsomes were incubated in uptake medium
containing Ca and 200 nM Tg. Uptake was initiated
by addition of ATP. Panel A shows the time course of
Ca uptake in microsomes prepared from Neo control
(
) or clone 8 (
) cells. Panels B and C show the dependence of Ca uptake on
[Ca] in the presence (
) or absence
() of 0.24 µM calmodulin in microsomes prepared
from clone 8 (B) or Neo control (C) cells. Pump
activity was evaluated from 10 min uptake and calculated as
nanomoles/mg of protein/10 min.
A critical parameter for the correct functioning of the PMCA is its
affinity for Ca and response to calmodulin.
Therefore, Ca transport activities of microsomes
isolated from the transfected cells were tested as a function of free
Ca concentration and in the presence or absence of
calmodulin. As shown in Fig. 3B, in membranes prepared
from the over-expressing cells (clone 8), the activity of PMCA1a was
dependent on Ca concentration. Addition of calmodulin
greatly enhanced the activity. Thus, the expressed isoform 1a shows a
characteristic dependence on Ca and calmodulin for
activation. Comparison of the PMCA over-expressing cells (Fig. 3B) with the vector-transfected cells (Fig. 3C) indicated a 3-4-fold increase in PMCA
activity at low and saturating Ca concentrations.
Over-expression of PMCA Modifies the
[Ca]i Signal of RAEC
Cells
To evaluate the role of PMCA1a in Ca homeostasis and in [Ca] signaling, we measured [Ca] in resting cells and cells treated with the purinergic agonist
ATP or the SERCA pump inhibitor thapsigargin (Tg). Fig. 4depicts examples of the
[Ca] signals of single control
and clone 8 cells stimulated with ATP or Tg. In both cell types ATP
caused a rapid increase in
[Ca], which then stabilized at
an elevated level, indicative of Ca release from
internal stores and subsequent activation of Ca entry(18) . Tg had a similar effect, although
Ca entry appeared more prominent. The variability of
the response of single cells did not allow firm conclusions as to the
differences between the two cell types, with the exception that the
initial [Ca] increase evoked by
ATP was usually lower in the PMCA1a over-expressing cells.
Figure 4:
Measurement of
[Ca] in single cells.
Neo control (traces a-d) or clone 8 (traces e-h)
cells grown on glass coverslips were loaded with Indo 1 (a, c, e, and g) or Fura 2 like (b, d, f, and h)
for measurements of
[Ca]. The cells were
continuously perfused with warm solution A. Where indicated 100
µM ATP or 2 µM Tg were included in the
perfusion medium. Similar results were obtained by imaging at least 10
cells in each coverslip in five separate experiments with Fura 2-loaded
cells.
To verify
this finding and obtain an averaged behavior of the cells we measured
[Ca] of a suspension of about
10 cells. Resting [Ca] was similar in control and clone 8 cells and averaged 77.5
± 7.5 (n = 51) and 75.8 ± 6.4 nM (n = 56), respectively. Hence, despite a 4-fold
over-expression of active PMCA1a in the plasma membrane, the cells were
able to maintain the same resting
[Ca]. Fig. 5shows that
when control cells were exposed to a mixture of ATP and Tg to discharge
most of the agonist-mobilizable Ca pool,
[Ca] increased to about 358
± 21 (n = 46) nM. ATP and Tg increased
[Ca] of clone 8 cells only to
266 ± 22 (n = 37) nM. The difference in
peak [Ca] was highly
significant (p < 0.01, Student's t test). To
exclude the possibility that modification of signal transduction was
responsible for the reduced response to agonist we estimated the size
of the intracellular Ca pool with the Ca ionophore ionomycin (Fig. 5, b and d).
Supermaximal concentration of ionomycin (10 µM) increased
[Ca] of Neo controls by 668
± 43 (n = 4) nM and that of clone 8
cells by 372 ± 19 (n = 4) nM.
Figure 5:
Measurement of
[Ca] in suspension of
cells. Fura 2-loaded Neo controls (traces a and b) or
clone 8 (traces c and d) cells were suspended in
solution A containing 1.5 mM CaCl (a and c) or Ca-free solution A containing 0.1
mM EGTA (b and d). Where indicated the cells
were exposed to a mixture of 100 µM ATP and 2 µM Tg and then 1.8 mM EGTA (a and c).
Cells in Ca-free medium were treated with 10
µM ionomycin to determine maximal Ca content of internal stores (b and d).
An
additional finding illustrated in Fig. 5, a and c, is that the second phase increase in
[Ca] appears to be somewhat
more prominent in clone 8 cells, suggesting a possible difference in
Ca influx rate. It is difficult to estimate the
difference in plasma membrane Ca pumping from the
rate of reduction in [Ca] after
ATP and Tg treatment, since the agonists increased
[Ca] to different levels in the
two cell types and Ca influx appears to be modified
by over-expression of the pump. Nonetheless, removal of external
Ca with EGTA caused faster reduction in
[Ca] in clone 8 relative to
control. Although not conclusive, this provides an indication that the
rate of Ca pumping across the plasma membrane of
clone 8 cells was higher than in controls.
Due to the apparent
multiple effects of PMCA1a over-expression on Ca signaling we proceeded to examine the expression and activity of
the different Ca transporting pathways involved in
regulating
[Ca](18, 19) .
Over-expression of PMCA1a Down-regulates SERCA
In
a previous successful over-expression of PMCA4b in CHO cells it was
shown that PMCA4b expression reduced the formation of a phosphorylated
intermediate by the SERCA pumps(27) , suggesting reduced SERCA
pump activity due to PMCA4b expression. We confirmed this finding by
showing similar reduction in phosphoenzyme formation by the SERCA pumps
of clone 8 and 35 cells (not shown). We proceeded to extend this
finding by testing Ca pumping activity, transcription
and translation of SERCA pumps in control and PMCA1a over-expressing
cells. Fig. 6A shows that SERCA-mediated Ca uptake was reduced by about 35% in microsomes isolated from
PMCA1a over-expressing cells. Further evidence is given in Fig. 8, A and B, below showing reduced
Ca pumping activity in permeabilized clone 8 cells.
Northern blot analysis of total RNA using a probe representing SERCA 3
showed more than 50% reduction in SERCA 3 mRNA levels in clone 8 cells (Fig. 6B). Finally, Western blot analysis using an
antibody that recognizes SERCA pumps showed a reduced level of SERCA
protein in clone 8 cells (Fig. 7, upper panel). Hence,
over-expression of PMCAa1 cDNA resulted in lower amount of SERCA mRNA
and protein to reduce Ca uptake into the ER.
Figure 8:
Ca uptake and
IP
-mediated Ca release in
SLO-permeabilized cells. Neo control (A and ) or clone 8
(B and 
) cells were washed and incubated in the SLO-containing
permeabilization medium. After stabilization of medium
[Ca] the cells were exposed to increasing
concentrations of IP as indicated and finally to 5
µM ionomycin. The resulting
[Ca] was plotted as a function of
[IP] (C) or the released Ca (Ca increase above resting) was calculated as a
% of the ionomycin-mobilizable pool (D). Two experiments with
each subpassage and four experiments from different subpassages showed
the same apparent affinity for IP in control and clone 8
cells.
Figure 7:
Western blot analysis of SERCA and
IPR proteins. Lysates prepared from Neo control and clone 8
cells were separated by SDS-polyacrylamide gel electrophoresis and
Western blotted. SERCA protein (100 kDa) and IPR (250 kDa)
were identified on separate blots by using specific antibody followed
by a chemiluminescence methods. SERCA antibody (monoclonal anti-SERCA2,
clone 2A7-A1 which cross-reacts with SERCA3) was purchased from
Affinity BioReagents and IPR (type 1) antibody was a gift
from Dr. Richard J. H. Wojcikiewicz, State University of New York at
Syracuse. The blots were hybridized with an actin antibody from Bio-Rad
to control for protein loading (not shown). Similar results were
obtained in one additional experiment.
Over-expression of PMCA1a Down-regulates
IPR
The next pathway examined was
Ca release from internal stores. As shown in Fig. 7(lower panel) over-expression of PMCA1a pump
down-regulated the abundance of the IPR protein. The
reduction in IPR protein level was comparable to that found
for the reduction in SERCA pump protein (Fig. 7).To evaluate
the impact of reduced levels of the IP-activated channel on
Ca release we used streptolysin O (SLO)-permeabilized
cells and measured Ca uptake and release in the
presence of mitochondrial inhibitors. Under these conditions
Ca uptake was completely dependent on the presence of
ATP in the permeabilization medium and was inhibited better than 96% by
0.1 µM Tg (not shown). Fig. 8, A and B, shows that the rate of Ca uptake by clone
8 cells was slower by about 38 ± 1.2% (n = 9)
compared to that measured in control cells. In addition, clone 8 cells
reduced medium [Ca] to 307 ± 26
nM (n = 9) as compared to control cells, which
reduced medium [Ca] to 203 ± 15
nM (n = 9). These results further support the
reduced SERCA pump activity in clone 8 cells.
After stabilization of
medium [Ca] the permeabilized cells were
used to measure the properties of IP-mediated
Ca release. Due to differences in SERCA pump activity
between the cells, the ability of IP to release
Ca was expressed as total Ca release (Fig. 8C) and as a percentage of
Ca mobilized by ionomycin (Fig. 8D).
It can be seen that in both cell types IP released about
50% of the ionomycin-mobilizable Ca pool with similar
apparent affinity. This would suggest that the IPR
remaining in clone 8 cells are not modified and that they are at
sufficiently high density to allow maximal Ca release.
The lower density of IPR in clone 8 cells
prompted testing the rate of IP-mediated Ca release. Fig. 9shows that Ca release in
clone 8 cells was slower than that of control cells. In four
determinations from two experiments the rate in clone 8 was reduced by
an average of 34 ± 4%.
Figure 9:
Rate
of IP-mediated Ca release. Experimental
protocol was as in Fig. 8except that after stabilization of
[Ca] the cells were exposed
to 2 µM IP. The integrated fluorescence signal
was recorded at a resolution of 3/s. Interruption in recording was
required for addition of IP.
Over-expression of PMCA1a Up-regulates CRAC
Activity
To study the influence of PMCA1a expression on
Ca release-activated Ca influx
(CRAC), cells in Ca-free medium were stimulated with
a mixture of Tg and ATP. After return of
[Ca] to resting levels
Ca influx was initiated by addition of 1.5 mM Ca to the medium. Fig. 10shows that the
initial rate of Ca influx into clone 8 cells was
faster than that into Neo control. In control cells the rate averaged
4.55 ± 0.36 (n = 4) nM/s, whereas in
clone 8 cells the rate averaged 10.52 ± 0.96 (n = 4) nM/s. Since changes in
[Ca] represent the sum of the
activity of all Ca transport pathways, the rate of
divalent cation influx was further evaluated by measuring the rate of
unidirectional Mn influx under the same conditions (Fig. 10, b and d). Mn influx in clone 8 cells was about 2.63 ± 0.13 (n = 4) fold faster than measured in Neo controls.
Figure 10:
Measurement of Ca and
Mn influx. Neo control (a and b) or
clone 8 (c and d) cells were suspended in
Ca-free solution A. Where indicated the cells were
stimulated with a mixture of 100 µM ATP and 2 µM Tg (b and d) and exposed to 1.5 mM CaCl and then 0.5 mM MnCl. To
determine maximal quench, at the end of each experiment the cells were
layered with 50 µM digitonin. The calcium signal is the
360/380 ratio and the Mn signal is the 360 nm portion
of the fluorescence signal.
Measurement of External
[Ca]
The multiple effect of
PMCA1a expression on the Ca transporting pathways,
the different levels of [Ca] increase caused by agonists in the two cell types and the fact
that [Ca] measurement reflects
the sum of the activity of all pathways made it impossible to evaluate
the rate of Ca pumping across the plasma membrane by
following [Ca]. To overcome
these difficulties we measured the rate of increase in external Ca concentration during cell stimulation. The
cells were stimulated with a mixture of ATP and Tg to release
Ca from the IP-sensitive pool and prevent
reuptake into the ER. Fig. 11, a and b, shows
the result of such experiments. In control cells, the agonist induced
Ca efflux at an initial rate of about 2.53 ±
0.24 (n = 6) nM/s. The same number of clone 8
cells and at the same external Ca buffering condition
extruded Ca at an initial rate of 3.52 ± 0.28 (n = 6) nM/s, which is 1.39-fold faster than
that measured in Neo controls.
Figure 11:
Unidirectional Ca efflux in control and clone 8 cells. Neo control (a and c) or clone 8 (b and d) cells were suspended
in Chelex-treated efflux medium and external
[Ca] was reduced to 115 nM with
0.4 µM EGTA. Within 1 min of EGTA titration the cells were
either stimulated with a mixture of 100 µM ATP and 2
µM Tg or exposed to 5 µM ionomycin.
In previous studies we showed that
agonist stimulation activates the plasma membrane Ca pump(2) . Thus, it was important to show that the faster
Ca pumping measured in clone 8 cells is independent
of cell stimulation. Fig. 11, c and d, shows
that when internal Ca was mobilized by ionomycin, the
initial rate of Ca extrusion by clone 8 cells was
about 1.35 ± 0.03 (n = 3) fold higher than that
of Neo controls. This was despite the lower increase in
[Ca] caused by ionomycin in
clone 8 cells (Fig. 5). This experiment also confirmed the
reduced size of the internal stores Ca pool in clone
8 cells.
DISCUSSION
Although the biochemical properties of the PMCA have been
extensively studied(1) , its functional role in resting and
stimulated cells is not well understood. In recent years it became
clear that the pump plays a pivotal role in the down-stroke phase of
the [Ca] signal evoked by
agonist stimulation (2, 18, 19) and during
[Ca] oscillations(7) .
Direct (2, 5) and indirect (4, 6) evidence suggests that the PMCA is activated by
agonists, and the activation is accompanied by phosphorylation of the
pump protein(8, 9) .
In an effort to better
understand the role and regulation of the PMCA in Ca signaling, we over-expressed the PMCA1a isoform and isolated
stable transfectant. A major difficulty in using expression systems to
study the role of PMCA in Ca transport in intact
cells was the correct targeting of the pump to the plasma
membrane(14) . The cell line, vector, transfection, and
selection procedures used in the present studies overcame this problem.
Two types of evidence suggest proper targeting of most of the PMCA1a to
the plasma membrane. Immunocytochemistry showed a typical punctate
staining in the plasma membrane of over-expressing cell lines with
minimal cytosolic staining. The high levels of PMCA1a colocalized with
markers of caveolae, ()similar to the localization of native
pump protein reported before(28, 29, 30) .
The increased Ca pumping activity in microsomes of
PMCA1a over-expressing cells was into an oxalate- and Tg-independent,
but calmodulin-sensitive compartment. These criteria firmly established
that in our cells a large fraction of the PMCA1a pump was correctly
targeted to the plasma membrane. While this work was in progress,
over-expression and successful targeting of the PMCA4b isoform to the
plasma membrane of CHO cells was reported(27) . Also in this
case the vector, transfection, and culture conditions were important in
the successful targeting of the pump(27) . The pattern of
immunostaining of PMCA4b over-expressing cells was similar to that
shown in Fig. 2A.
The successful over-expression of
high levels of PMCA1a in the plasma membrane revealed that pump
activity can be regulated on several levels. The first finding of note
was that despite the correct targeting and over-expression of the
PMCA1a, resting [Ca] was
identical in control cells and cells of the different clones
over-expressing the PMCA1a. Since resting
[Ca] is determined exclusively
by pump/leak turnover across the plasma membrane(18) , it was
clear that either the activity of the over-expressed pumps was
down-regulated in intact cells, Ca influx rate was
much higher in cells over-expressing the pump or a combination of both.
Measurement of Ca and Mn influx
into resting cells (such as those in Fig. 10, a and b) did not reveal major differences in Ca influx rate among the different cell types. Therefore, it was
likely that the over-expressed pumps were not active at resting
[Ca].
Following
[Ca] and
[Ca] to estimate the rate of
unidirectional Ca efflux revealed that the activity
of the over-expressed PMCA pumps was also regulated during agonist
stimulation and/or when [Ca] was high. Thus, Western blot analysis showed about 4-fold
increase in pump protein in the over-expressing clones used for the
present studies (clone 8 and 35). The over-expressed pumps were fully
functional as they increased Ca uptake in isolated
microsomes, had the expected apparent affinity for
Ca, and were regulated by calmodulin. Yet, the rate
of Ca pumping in intact clone 8 and clone 35 cells
was about 1.4-fold over that measured in control cells. Our
measurements of Ca pumping activity are not
influenced by the activity of other Ca transporting
pathways since we measured net Ca efflux by following
[Ca]. The lower than expected
increase in Ca pumping in intact cells is also not
because the lower increase in [Ca] evoked by agonist in these cells. When dCa/dt
for the two cell types is calculated at the same
[Ca] from experiments similar
to those in Fig. 10, b and d, and 11, the
increased rate of pumping due to PMCA1a over-expression is at best
1.67-fold over control. Our findings are further supported by the study
of Guerini et al.(27) . These authors reported a
20-fold increase in Ca pumping activity in microsomes
prepared from CHO cells over-expressing PMCA4b. Recalculation of the
data in their Fig. 5shows that such over-expression of active
pump protein in the plasma membrane caused only about a 1.7-fold
increase in Ca efflux rate(27) .
Considering the fact that agonist-induced Ca efflux is
influenced by reuptake of Ca into the ER and reflects
the sum of Ca extrusion and
Ca/Ca exchange through the CRAC
channel(18) , the 1.7-fold increased rate of Ca
efflux is an overestimation of the increase in Ca pump rate(27) .
The finding discussed above of the two
independent studies, using different cell type and different pump
isoforms, suggests that cells are capable of controlling Ca pumping rate independent of the number of the Ca pumps in the plasma membrane. Hence, a 4- (present studies) or a
20- ((27) ) fold increase in the number of Ca pumps increased Ca pumping rate in intact cells
to similar extent (1.4-1.7-fold). This suggests a set point for
the maximal possible Ca pumping rate which is
compatible with cell survival, since over-expression of PMCA strongly
affects the timing of the cell cycle(27) . How the cells
achieve such a set point is not clear at present. Two obvious
possibilities are down-regulation of pumping activity and/or shielding
the pumps from the increase in [Ca] by compartmentalizing the Ca pool and the areas
of Ca release (31, 32, 33, 34, 35, 36, 37) .
Discriminating between these and other possibilities requires further
studies. Using the PMCA over-expressing cells it should be now possible
to address such questions.
Another major and significant finding of
the present study was the adaptive regulation of all the major
Ca transporting pathways in response to
over-expression of the PMCA1a. The activity of the CRAC pathway in the
plasma membrane was up-regulated by as much as 2.6-fold, significantly
more than the net increase in Ca pumping activity.
The large increase in CRAC activity is likely to compensate for the
increase in net pumping rate and the reduced capacity of the
Ca stores (see below). It is interesting that
activation of CRAC in the PMCA1a over-expressing cells still required
Ca release from internal stores, indicating
maintained regulation of the CRAC pathway by Ca content of the ER(18, 38) . Since nothing is
known about the nature of the CRAC pathway (38) we could not
determine on what level CRAC activity was regulated in PMCA1a
over-expressing cells.
Over-expression of PMCA1a down-regulated the
contribution of the internal Ca pool to the
Ca signal by reducing the level and activity of both
the SERCA pumps and the IPR. Previous studies similarly
showed that over-expression of PMCA4b in the plasma membrane reduced
the phosphorylated intermediate formed during the turnover cycle of the
SERCA pump in CHO cells(27) . Here we show that the reduced
activity was largely due to a reduction in the number of the pumps.
Also in the case of IP-mediated Ca release, the reduced rate of release could be attributed to
reduced number of IPR, with normal apparent affinity for
IP. The reduction in the number of SERCA pumps resulted in
reduced rate of Ca pumping and Ca content in the ER. This was the case whether Ca content in the ER was measured in permeable or intact cells and
whether it was released by IP or ionomycin. The reduced
Ca content in the ER was reflected in a reduced
initial agonist-evoked [Ca] increase.
The regulation of the different Ca transporting pathways by over-expression of PMCA1a appears to be
on the gene level. In the case of the SERCA pump we were able to show
that over-expression of PMCA1a reduced SERCA gene expression by more
than 50%, which is likely to account for the reduced amount of SERCA
pump protein in these cells. The mechanism by which PMCA1a expression
regulates the SERCA gene(s) is not clear at present. Up-regulation of
SERCA2 gene by the hormone T and by the platelet-derived
growth factor have been reported in cardiac (39) and smooth
muscle cells(40) , respectively. Similarly, we have
demonstrated the stimulation of SERCA3 gene expression (the major
isoform) in RAEC by epidermal growth factor and angiotensin
II(41) . SERCA3 gene is also up-regulated in platelets from
hypertensive rats(42) . In fibroblasts, the expression of a
distinct Tg-resistant Ca pump is induced by
continuing exposure to Tg(43, 44) . These findings
together with the present studies, suggest that cells can regulate the
level of SERCA in response to external stimuli or a change in the level
or activity of the other Ca transport pathways.
In
summary, the present studies demonstrate that functional
over-expression of PMCA1a led to a down-regulation of SERCA gene
expression, a reduction in the number of IPR, and an
increase in the activity of the CRAC pathway. It is not clear at
present whether the regulation of all pathways was on the gene level.
However, if this is the case, it is possible that the genes of all the
major Ca transporting pathways are regulatorally
linked to concomitantly modulate the activities of all pathways. The
link can be provided by the [Ca] levels in resting cells. Considering the information encoded in
the [Ca] signal and the
involvement of Ca in numerous vital cell
functions(32, 38) , it is not surprising that its
concentration is regulated on several levels. Regulation on the level
of gene expression is likely to provide a constant
[Ca] signal, which is essential
for cell survival. This is emphasized in several recent studies showing
the modulation of the cell cycle (27, 43, 44) by over-expression of PMCA pumps
and/or down-regulation of SERCA pumps by Tg treatment.
FOOTNOTES
- *
- This work was supported by National Institutes of
Health Grant HL 39481 and a grant-in-aid from the American Heart
Association of Michigan (to T. H. K.) and by National Institutes of
Health Grants DK 38938 and DK 46591 (to S. M.). The costs of
publication of this article were defrayed in part by the payment of
page charges. This article must therefore by hereby marked
``advertisement'' in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
- §
- These authors contributed equally to this
study.
- ()
- The abbreviations used are: PMCA, plasma
membrane Ca-ATPase; RAEC, rat aortic endothelial
cell; SERCA, sarcoplasmic/endoplasmic reticulum
Ca-ATPase; ER, endoplasmic reticulum; IP,
inositol 1,4,5-trisphosphate; IPR, IP receptor;
CRAC, Ca release-activated Ca influx; Tg, thapsigargin;
[Ca], free
intracellular Ca concentration;
[Ca], free extracellular
Ca concentration; SLO, streptolysin O toxin; CHO,
Chinese hamster ovary.
- ()
- B.-F. Liu, X. Xu, R.
Fridman, S. Muallem, and T. H. Kuo, unpublished data.
- ()
- B.-F. Liu, X. Xu, R. Fridman, S. Muallem, and T.
H. Kuo, unpublished observation.
ACKNOWLEDGEMENTS
We thank Gary Shull for providing rat PMCA1a cDNA, P.
Tabacska and Ken Palmer for helping with the immunoperoxidase studies.
REFERENCES
- Carafoli, E. (1991) Physiol. Rev. 71, 129-153
[Free Full Text]
- Zhang, B. X., Zhao, H.,
Loessberg, P., and Muallem, S. (1992) J. Biol. Chem. 267, 15419-15425
[Abstract/Free Full Text]
- Williams, J. A. (1980) Cell Tissue Res. 210, 295-303
[Medline]
[Order article via Infotrieve]
- Rink, T. J., and Sage, S.
O. (1987) J. Physiol. 393, 513-524
[Abstract/Free Full Text]
- Duddy, S. K., Kass, G. E.
N., and Orrenius, S. K. (1989) J. Biol. Chem. 264, 20863-20866
[Abstract/Free Full Text]
- Randriamampita, C., and
Trautmann, A. (1990) J. Biol. Chem. 265, 18059-18062
[Abstract/Free Full Text]
- Teipikin, A. V.,
Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572
[Abstract/Free Full Text]
- Wang, K. K. W., Du, Y.
S., Diglio, C., Tsang, W., and Kuo, T. H. (1991) Arch.
Biochem. Biophys. 289, 103-108
[CrossRef][Medline]
[Order article via Infotrieve]
- Kuo, T. H., Wang, K. K.
W., Carlock, L., Diglio, C., and Tsang, W. (1991) J.
Biol. Chem. 266, 2520-2525
[Abstract/Free Full Text]
- Shull, G. E., and Greeb,
J. (1988) J. Biol. Chem. 263, 8646-8657
[Abstract/Free Full Text]
- Verma, A. K., Filoteo,
A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E.
E., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M.
A., James, P., Vorherr, T., Krebs, J., and Carafoli, E., (1988) J. Biol. Chem. 263, 14152-14159
[Abstract/Free Full Text]
- Gunteski-Hamblin, A.-M.,
Greeb, J., and Shull, G. E. (1988) J. Biol. Chem. 263, 15032-15040
[Abstract/Free Full Text]
- Sarkar, F. H., Ball, D.
E., Tsang, W., Li, Y.-W., and Kuo, T. H. (1993) DNA
Cell Biol. 12, 435-440
[Medline]
[Order article via Infotrieve]
- Adamo, H. P., Verma, A.
K., Sanders, M. A., Heim, R., Salisbury, J. L., Wieben, E. D., and
Penniston, J. T. (1992) Biochem. J. 285, 791-797
- Enyedi, A., Verma, A.
K., Filoteo, A. G., and Penniston, J. T. (1993) J.
Biol. Chem. 268, 10621-10626
[Abstract/Free Full Text]
- Enyedi, A., Verma, A.
K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and
Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43
[Abstract/Free Full Text]
- Zvaritch, E., Vellani,
F., Guerini, D., and Carafoli, E
