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(Received for publication, May 17, 1995; and in revised form, August 30, 1995) From the
Sorcin is a 22-kDa calcium-binding protein initially identified
in multidrug-resistant cells; however, its patterns of expression and
function in normal tissues are unknown. Here we demonstrate that sorcin
is widely distributed in rodent tissues, including the heart, where it
was localized by immunoelectron microscopy to the sarcoplasmic
reticulum. A >500-kDa protein band immunoprecipitated from cardiac
myocytes by sorcin antiserum was indistinguishable in size on gels from
the 565-kDa ryanodine receptor/calcium release channel recognized by
ryanodine receptor-specific antibody. Association of sorcin with a
ryanodine receptor complex was confirmed by complementary
co-immunoprecipitations of sorcin with the receptor antibody. Forced
expression of sorcin in ryanodine receptor-negative Chinese hamster
lung fibroblasts resulted in accumulation of the predicted 22-kDa
protein as well as the unexpected appearance of ryanodine receptor
protein. In contrast to the parental host fibroblasts, sorcin
transfectants displayed a rapid and transient rise in intracellular
calcium in response to caffeine, suggesting organization of the
accumulated ryanodine receptor protein into functional calcium release
channels. These data demonstrate an interaction between sorcin and the
ryanodine receptor and suggest a role for sorcin in modulation of
calcium release channel activity, perhaps by stabilizing the channel
protein.
Sorcin was initially identified as a 22-kDa protein in cultured
cells selected for resistance to natural product cancer drugs, such as
vincristine, adriamycin, and actinomycin D, i.e. multidrug-resistant
cells(1, 2, 3, 4, 5) . One
of the major mechanisms of resistance in these cells is mediated by
overexpression of the membrane-bound drug transporter,
P-glycoprotein(6) . Molecular cloning studies demonstrated that
the sorcin and P-glycoprotein genes are tightly linked and that both
may be amplified during the acquisition of multidrug resistance.
However, while P-glycoprotein overexpression correlates with resistance
development, increased sorcin expression is not obligatory, and its
abundance does not correlate with the degree of
resistance(4, 5, 6, 7, 8, 9) .
Complementary DNA for sorcin has been isolated from hamster (2) and human (10) multidrug-resistant cells, which
amplify the sorcin gene. The highly conserved sequence, with 95%
homology between hamster and human sorcin, predicts a 22-kDa protein
with four putative Ca In the
present study, we began to characterize the expression of sorcin in
normal tissues and gain some insight into its function. We found that
sorcin is widely expressed in mammalian tissues, including the heart,
where it was localized to cardiac sarcoplasmic reticulum (SR). (
Metabolic labeling with 100 µCi/ml
[
DC-3F, DC-3F/VCRd-5L, transfected
DC-3F cells and other controls were grown on two-chamber glass slides
(Lab-Tek Chamber Slide System, VWR Scientific, Rochester, NY) or glass
coverslips. The cells were rinsed and fixed as described above for
heart tissue. Cells were incubated for 12-18 h in 0.1 M Tris-buffered saline containing 0.1% bovine serum albumin and
either a 1:1000 dilution of the rabbit antiserum against sorcin C
terminus peptide or a 1:500 dilution of GP561, a polyclonal antiserum
raised in guinea pig against purified rabbit brain ryanodine receptor.
The cells were then processed with the ABC method described above, and
the bound peroxidase was visualized by light microscopy.
Figure 1:
Distribution of sorcin
among mouse tissues. Western blot analysis of homogenates of liver (lane 1), small intestine (lane 2), heart (lane
3), brain (lane 4), lung (lane 5), skeletal
muscle (lane 6), spleen (lane 7), kidney (lane
8), and DC-3F/VCRd-5L cells (lane 9) with antiserum
raised against the sorcin C terminus peptide is shown. Samples
containing 60 µg of protein from tissues or 2 µg of
DC-3F/VCRd-5L cell protein were heated at 100 °C for 2 min before
electrophoresis on 13% acrylamide gels.
Figure 2:
Specificity of antiserum to sorcin C
terminus peptide. Western blot analysis of 60 µg of mouse heart
homogenate (lanes 1 and 3) and 2 µg of
DC-3F/VCRd-5L cell protein (lanes 2 and 4) per lane
with peptide antisera (lanes 1 and 2) or with
antisera preadsorbed with C terminus peptide (lanes 3 and 4). Samples were treated as described in Fig. 1. The
minor band at 35 kDa was present in some tissue (see Fig. 1, lane 6) and cultured cell samples.
Figure 3:
Northern blot analysis of rodent tissue
RNA with sorcin cp6 cDNA probe. Lanes 1, 4, and 5 contain 10 µg of total RNA from rat heart, rat spleen, and
mouse heart, respectively. Lanes 2 and 3 contain 2
µg of poly (A)
Figure 4:
Immunofluorescent labeling of rat cardiac
myocytes with sorcin antiserum. Cells were labeled with rabbit antibody
raised against sorcin C terminus peptide with rhodamine-conjugated goat
anti-rabbit secondary antiserum (right panel) and
double-labeled with mouse monoclonal antibody directed against
sarcomeric myosin heavy chain with fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antiserum (left
panel). Labeling with sorcin N terminus peptide antibody was
identical in distribution to that seen in the right panel (not
shown).
Figure 5:
Localization of sorcin to rat ventricular
SR by immunoelectron microscopy. Sections were labeled with unadsorbed
or preadsorbed antibody directed against the sorcin N terminus peptide. A, section shows intense labeling of SR (arrow) near
transverse tubule (*) and mitochondrion (m) with unadsorbed
antibody. B, section labeled as in panel A shows
intense peroxidase reaction product in the SR (arrow) near the
plasmalemma of the myocyte and nearby mitochondrion (m).
Plasmalemma (arrowheads) and mitochondrial membranes are also
peroxidase-positive near points of apposition with the labeled SR.
Other portions of the plasmalemma (small arrows) appear
markedly less electron dense. C, section collected from tissue
processed as in panels A and B but at depths in the
tissue having limited access to immunoreagents. No peroxidase product
is associated with the SR (arrow) near the transverse tubule
(*) or mitochondrion (m), as was the case when preadsorbed
antibody was used for labeling. Bars, 0.5
µm.
Figure 6:
Immunoprecipitation of
[
To address the question of
whether the >500-kDa band represented aggregated material or a true
association between sorcin and the RyR, additional immunoprecipitation
experiments were carried out with the use of RyR-specific antibody.
Immunoprecipitates of proteins from rat cardiac myocytes were
solubilized in Laemmli buffer containing 2-mercaptoethanol and examined
by Western blot analysis with sorcin antiserum, resulting in display of
a prominent 22-kDa protein (Fig. 7, lane 1).
Interestingly, no sorcin was detected when the myocytes were initially
lysed in the presence of a reducing agent (Fig. 7, lane
2), suggesting a disulfide-linkage between sorcin and the RyR
complex. RyR antibody did not recognize sorcin, nor did sorcin
antibodies recognize RyR, by Western blot analysis.
Figure 7:
Immunoprecipitation/immunoblot analysis of
sorcin in unlabeled rat cardiac myocytes. Aliquots of cell homogenates
containing 100 µg of protein were immunoprecipitated by RyR
antibody (C3-33) (lane 1), RyR antibody (with myocytes
lysed in the presence of 2-mercaptoethanol) (lane 2), sorcin C
terminus peptide antiserum (lane 3), and preimmune sorcin
antiserum (lane 4). Antigen-antibody complexes were
solubilized by heating in Laemmli buffer containing 2-mercaptoethanol (21) at 100 °C for 2 min, and products were subjected to
gel electrophoresis on 13% acrylamide gels before analysis by Western
blot with antiserum raised against sorcin C terminus peptide. The arrow indicates 22-kDa sorcin. Sorcin was also detected if
myocyte proteins were cross-linked before immunoprecipitation. The
45-50-kDa proteins in the figure are observed in all lanes, including the preimmune sorcin antiserum samples (lane 4), suggesting lack of
specificity.
Figure 8:
Immunocytochemical labeling of Chinese
hamster cells with sorcin antiserum or GP561 RyR antibody. Panels A and B show DC-3F/VCRd-5L cells, panels C and D show DC-3F cells, and panels E and F show
DC-3F/sor.3 sorcin transfectants. Panels A, C, and E show labeling with sorcin C terminus peptide antiserum, and panels B, D, and F show labeling with RyR
antibody. Bar, 100 µm.
Figure 9:
Effect of 10 mM caffeine on
intracellular Ca
The time
frame of effects of caffeine on intracellular Ca
Figure 10:
Time course of the effect of 10 mM caffeine on free intracellular Ca
Sorcin was initially identified as a protein differentially
expressed during the acquisition of multidrug
resistance(1, 2, 3, 4, 5) .
Although the mechanism underlying sorcin's enhanced expression in
these cells has been determined, its pattern of expression and function
in normal tissues have not previously been characterized. With the use
of immunological reagents, we now demonstrate that 22-kDa sorcin is
widely expressed in mammalian tissues and is highly conserved among
mammalian species. Antibodies raised against hamster sorcin peptide
sequences recognized the 22-kDa protein in a number of other species,
including mouse, rat (this paper), and human(4, 5) .
Higher molecular weight immunoreactive bands are observed in some
tissue and cultured cell samples. Their identity has not been
determined; however, they may represent different forms of sorcin or
sorcin dimers. A related 28-kDa protein, grancalcin, has been shown to
exist in dimeric form, possibly through covalent linkage stable to
thiol reagents(33) . The primary sequence of sorcin gives
some clues as to its potential function. The two EF hand
Ca Biochemical studies presented here
suggest that sorcin interacts directly with the cardiac RyR. Sorcin
antisera immunoprecipitated a >500-kDa species from cardiac myocytes
that co-migrated on gels with the protein immunoprecipitated by
antibody raised against the 565-kDa RyR receptor. This finding suggests
that sorcin is part of a complex, and an association between sorcin and
the cardiac RyR/SR Ca Sorcin
transfectants, generated to study a function for the
Ca Increase
in RyR immunoreactivity in sorcin transfectants and in DC-3F/VCRd-5L
cells suggested that sorcin influences the abundance of RyR protein.
Given the demonstrated biochemical association between the two
proteins, it is conceivable that sorcin directly stabilizes the RyR
protein and retards its degradation, a mechanism that seems more
plausible than a transcriptional effect on RyR gene expression.
Clearly, sorcin alone is insufficient to promote the accumulation of
the RyR in all cell types, since the former protein is much more widely
expressed than the latter. However, in the appropriate cellular
environment, such as sarcomeric muscle, the levels of sorcin may
influence the abundance of co-expressed RyR. Thus, it will be of
interest to compare the profiles of sorcin and RyR expression during
myogenic differentiation, both in vivo and in cell culture
model systems, such as the C2C12 mouse myoblast cell line(34) ,
where our preliminary studies suggest a marked accumulation of sorcin
during the process of differentiation into myotubes. It should be
pointed out, with regard to a possible role for sorcin in
multidrug-resistant cells, that forced expression of sorcin in Chinese
hamster lung cells did not, on its own, confer the multidrug resistance
phenotype. Both DC-3F/VCRd-5L and DC-3F/sor.3 cells, with increased
levels of sorcin and RyR protein, demonstrated a characteristic
property of the RyR/Ca Although
these data suggest an important interaction between sorcin and the RyR,
they do not indicate whether interaction between sorcin and RyR is
direct or through a third or intermediary protein. The data also do not
address whether sorcin modulates any gating parameters of the
Ca
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26411-26418
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-binding domains, two with
strong homology to calmodulin ``EF hand'' motifs(2) .
In a classification of Ca
-binding proteins based on
sequence, sorcin was placed among members of the closely related
calpain and sarcoplasmic Ca
-binding protein
subfamilies (11) . Direct
Ca
binding to sorcin has been demonstrated by in vitro assays(3) , and Ca
affinity in the
µM range has been determined by fluorescence
spectroscopy(12) . Sorcin also contains two putative
recognition sites for protein kinase A, and phosphorylation of sorcin
in drug-resistant cell-free extracts or of purified sorcin in the
presence of the catalytic subunit of protein kinase A has been
observed(4, 5, 13) . Although initially
characterized as a soluble protein, our recent studies have shown that
sorcin undergoes Ca
-mediated translocation from
soluble to cellular membrane sites(12) . Despite these
biochemical data suggesting a role for sorcin in Ca
handling, a function for sorcin in multidrug resistance or in
P-glycoprotein activity remains
speculative(2, 3, 4, 5) .
)Co-immunoprecipitation studies revealed an association
between sorcin and the ryanodine receptor (RyR), the calcium release
channel located in muscle SR at the junction of transverse tubules and
SR terminal cisternae(14) . Sorcin transfectants in DC-3F
Chinese hamster lung fibroblasts were generated to study a possible
role for sorcin in intracellular calcium transport. These cells were
characterized immunocytochemically for sorcin and ryanodine receptor
expression as well as functionally, by digital
Ca-imaging for their response to caffeine, a
potentiator of Ca
release from the SR through
RyR/Ca
release channels(14, 15) .
Antibodies
A mouse monoclonal antibody raised
against a peptide from the sorcin N terminus (amino acids 21-56) (2) (Berkeley Antibody Company, Richmond, CA) and rabbit
polyclonal antiserum raised against a peptide from the sorcin C
terminus (amino acids 178-198) (2) (Pocono Rabbit Farm
and Laboratory, Inc., Canadensis, PA) were developed for these studies.
Both antibodies recognized a 22-kDa protein as the predominant
immunoreactive species in immunoblot analysis of multidrug-resistant
cells, bacterially expressed recombinant protein(12) , and in vitro translation products. In some cases minor higher
molecular weight bands were also observed. Detection of all
immunoreactive bands was abrogated when antibodies were preadsorbed
with appropriate antigenic peptide for 15 min at 4 °C. Guinea pig
antiserum recognizing cardiac and brain RyR (GP561) was a generous gift
from Dr. Kevin Campbell of the Howard Hughes Medical Institute
(University of Iowa) (16) and a mouse monoclonal antibody
raised against canine cardiac RyR (C3-33) was a generous gift
from Dr. Gerhard Meissner, University of North Carolina(17) .Cell Lines
The spontaneously transformed Chinese
hamster lung cell line, DC-3F, vincristine-resistant subline,
DC-3F/VCRd-5L, and actinomycin D-resistant line, DC-3F/AD X, have been
described and characterized in previous studies(1) . The cells
were maintained in 1:1 Eagle's minimum essential medium and
Ham's F12 supplemented with 5% fetal bovine serum, and, for the
multidrug-resistant cells, 50 µg/ml of vincristine (generously
supplied by Eli Lilly and Co., Indianapolis, IN) or 10 µg/ml
actinomycin D (Sigma). DC-3F/VCRd-5L cells express 10-15 times
more sorcin than DC-3F (as a result of amplification of the sorcin
gene), unlike DC-3F/AD X cells, which do not overexpress sorcin and do
not contain amplified genes for sorcin(18) . The two resistant
lines express equivalent, high levels of P-glycoprotein (19) .Preparation of Tissue and Cell Samples
Tissue
samples from adult BALB/c mice were minced and sonicated in 50 mM Hepes buffer (pH 7.4) containing 1% Triton X-100, 10% glycerin, 1
mM EDTA, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride for study of tissue distribution of
sorcin. Cardiac myocytes were enzymatically dissociated from neonatal
Sprague-Dawley rat heart as described previously (20) and
plated in 60-mm tissue culture dishes for short term culture. In some
experiments cardiac myocytes were separated from
fibroblasts(20) .S]methionine (DuPont NEN) in methionine-free
medium was accomplished by overnight incubation of monolayer cells at
37 °C. Labeled or unlabeled cardiac myocytes or cultured Chinese
hamster cells and sublines were lysed in 20 mM sodium
phosphate buffer (pH 7.4) containing 0.15 M NaCl, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride by needle
extrusion. Where noted for some experiments lysis buffers also
contained 1% 2-mercaptoethanol. Cross-linking of proteins was carried
out by treating cell lysates with 1 mM
dithiobis(succinimidylpropionate) (Pierce) in dimethyl sulfoxide for 30
min at room temperature, followed by quenching with 1 M Tris-HCl (pH 7.4), as suggested by the manufacturer. All
procedures involving use of animal tissues were undertaken in
accordance with institutional guidelines.
Immunochemical Analysis
Aliquots of tissue or cell
homogenates were subjected to electrophoresis and transferred to
nitrocellulose according to published
procedures(3, 21, 22) . For Western blot
analysis with sorcin peptide antiserum, nitrocellulose papers were
incubated in 5% dry milk in 0.1 M Tris buffer (pH 7.4) for 1 h
and then in primary antibody diluted in 0.1 M Tris buffer (pH
7.4), containing 0.15 M NaCl and 0.05% Tween 20 (Sigma) or in
primary antibody preincubated with the corresponding antigenic peptide
for 15 min at 4 °C. The papers were washed with diluting buffer and
finally incubated with peroxidase-conjugated Protein A (Bio-Rad
Laboratories, Hercules, CA) before analysis by chemiluminescence (ECL
Western blotting System, Amersham Corp.). For immunoprecipitations,
aliquots of cell homogenates containing 100-200 µg of protein
were incubated for 1 h at room temperature with 1:500 dilutions of
antibodies to sorcin or to RyR (C3-33) (17) in buffer
containing 10 mM Tris (pH 7.4), 0.15 M NaCl, 1%
sodium deoxycholate, 1% Triton X-100, and 0.1% sodium dodecyl sulfate.
Antibody-antigen complexes were precipitated with immobilized Protein A
(for antiserum to sorcin C terminus peptide) or G (for N terminus
peptide antibody or RyR antibody) (Sigma). Antigens were solubilized by
heating the washed complexes in Laemmli buffer (21) at 100
°C for 2 min (for sorcin detection) or by treating the complexes
with 2% sodium dodecyl sulfate in 6.25 mM Tris-HCl and 10%
glycerol for 10 min at room temperature (for RyR detection) before
examination by gel electrophoresis (21) and autoradiography
(for labeled samples) by exposure of dried gels to Eastman Kodak X-AR
film for 30 h or Western blot analysis with sorcin peptide antibody as
described above.Northern Blot Analysis
Total RNA samples were
isolated from rat and mouse heart and spleen with the use of TRIzoL
reagent (Life Technologies, Inc.) according to the manufacturer's
procedures, and poly(A) RNA was prepared with Dyna
beads mRNA Direct kit (Dynal A.S., Oslo, Norway). RNA samples were
electrophoresed on 0.8% agarose/formaldehyde gels and capillary-blotted
onto Nytran membranes (Schleicher and Schuell). Membranes were
prehybridized at 42 °C in 5
SSC, 50 mM sodium
phosphate, pH 7.4, 1 Denhardt's solution, 2% sodium
dodecyl sulfate with 100 µg/ml denatured salmon sperm DNA and then
hybridized in that buffer with random prime-labeled sorcin cp6 probe (2, 18) for 16 h at 42 °C. The cp6 probe contains
the 1.0-kilobase sorcin coding region isolated from
hamster(2) . Blots were washed with 0.2 SSC, 0.2% SDS
at 60 °C and exposed to Kodak X-AR film (Kodak, Rochester, NY) for
20 h.Immunofluorescent Staining of Cardiac
Myocytes
Cardiac myocytes were isolated as described above,
plated on coverslips, and washed with phosphate-buffered saline before
fixation in 3% formaldehyde and permeabilization in acetone at
-20 °C. After extensive washing with phosphate-buffered
saline, cells were incubated for 16 h at 4 °C with mouse monoclonal
antibody F59 directed against sarcomeric myosin heavy chain (23) together with the rabbit antiserum raised against the
sorcin C terminus peptide or with antibody raised against the N
terminus peptide alone. Cells were then incubated with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG and/or
rhodamine-conjugated goat anti-rabbit IgG antiserum (Sigma) and
visualized by immunofluorescence microscopy.Electron Microscopic Immunocytochemistry
Adult
male Sprague-Dawley rats (200-300 g) were deeply anesthetized
with sodium pentabarbital (50 mg/kg intraperitoneally). The thoracic
cavity was opened, and the excised heart ventricle was removed and
placed in a solution containing 3.7% acrolein and 2% paraformaldehyde
in 0.1 M phosphate buffer at pH 7.4. Sections (30-50
µm in thickness) were cut with the use of a Lancer Vibratome and
collected in phosphate buffer. Tissue sections were rinsed in 0.1 M Tris buffer (pH 7.4) and then placed in 1% sodium borohydride
solution in the same buffer for 30 min to neutralize aldehydes prior to
immunocytochemical labeling(24) . Tissue sections were
incubated for 24 h at room temperature with antibody raised against the
sorcin N terminus or C terminus peptide or preadsorbed control antibody
at dilutions ranging from 1:100 to 1:1000 in 0.1 M Tris-buffered saline containing 1% bovine serum albumin and 0.035%
Triton X-100. Preadsorbed control antiserum was prepared by incubating
1 ml of the working dilution of antibodies with excess sorcin peptide.
After primary antibody incubations, sections were washed, placed for 30
min each in biotinylated goat anti-mouse or anti-rabbit IgG (1:400) and
peroxidase-avidin complex (ABC, Elite Kit, Vector Laboratories,
Burlingame, CA)(25) , and then treated with
3,3`-diaminobenzidine (40 mg/100 ml) and 0.01% hydrogen peroxide for 6
min. All incubations and washes between each step were carried out in
0.1 M Tris-buffered saline. Immunolabeled sections were then
processed for electron microscopy using conventional
methods(24) . Ultrathin sections from outer and inner portions
of the tissue sections were examined with a Phillips CM-10 electron
microscope. Selective peroxidase immunoreactivity was recognized by
detection of a granular precipitate that was seen only near the
accessible surface of tissue sections incubated with the nonadsorbed
antibody.Sorcin Expression Vector, Transfection, and
Immunocytochemistry
Stable transfectants that overexpressed
sorcin were generated in DC-3F host cells. Transfections were carried
out by the calcium phosphate technique (26) with a sorcin
expression vector, pFRCMVSOR, in which transcription was directed by
the human CMV promoter and enhancer (a generous gift from Dr. Piet
Borst, Director of the Netherlands Cancer Institute)(27) . In
brief, cells were transfected with 15 µg of pFRCMVSOR and 5 µg
of p308 plasmid (American Type Culture Collection, Rockville, MD,
number 37613) containing the neo
gene(28) . Cells were selected in 100 µg/ml G418
(Sigma) for approximately 2 weeks until distinct colonies were visible.
Clonal populations of G418-resistant cells were obtained by serial
dilution. Control DC-3F cells transfected with the p308 neo gene alone were generated in parallel.
Integration of the sorcin expression gene was confirmed by Southern
blot analysis (29) of genomic DNA from individual
transfectants. The sorcin transfectants were analyzed for response to
vincristine, actinomycin D, and adriamycin (drugs to which cells become
multidrug-resistant). The clones were not resistant to those drugs;
doses for 50% cell kill were identical in transfectants and DC-3F host
cells. Increase in P-glycoprotein expression in sorcin transfectants
was not observed (data not shown).Digital Ca
Cells were plated on cover glasses (25-mm
circle 1, VWR Scientific) in 35-mm tissue culture dishes. The cells
were maintained and experiments were performed in a Hepes-buffered (pH
7.4) Krebs-Henseleit solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaClImaging
, 2 mM
MgCl, 10 mM glucose, 10 mM Hepes, and
0.1% bovine serum albumin with or without 10 mM caffeine
(Sigma) and loaded with 5 µM fura-2/AM (Molecular Probes,
Inc., Eugene, OR) for 30-40 min at 37 °C to initiate the
experiments. The cells were then transferred to Hepes-buffered solution
without fura-2/AM and incubated for 1 h, and the cover glasses were
placed in a tissue chamber mounted on the stage of a Nikon microscope
as described previously(30) . Sequential excitation at 340 and
380 nm was used, and the emitted fluorescent images were acquired at
500 nm by a silicon-intensified target camera (Dage MTI 65, Michigan
City, IN). After determination of base-line Ca levels, cells were perfused with medium containing 10 mM
caffeine until termination of the experiment. Fluorescent ratio images
were calculated off-line on a pixel-by-pixel basis by dividing the
340-nm image by the 380-nm image after background subtraction.
Intracellular Ca
levels, correlated with the
instrument-derived false color spectrum, were determined by in
vitro calibration(31) . Quantitation of the effect of
caffeine on intracellular Ca
levels in the cells was
based on values obtained for an average of 10 determinations with at
least 50 cells/microscope field.
Sorcin Distribution among Normal Tissues
Western
blot analysis of mouse tissue homogenates with the sorcin C terminus
peptide antiserum revealed that the 22-kDa protein was present in a
wide variety of tissues, including heart, spleen, lung, skeletal
muscle, liver, and brain (Fig. 1). The slightly faster migrating
band in kidney samples was consistently observed and may represent a
degradation product. Antibody specificity was demonstrated by
immunoblot analysis of DC-3F/VCRd-5L and heart proteins with
preadsorbed antiserum (Fig. 2). The predominant 22 kDa species
as well as a minor 35 kDa band observed in some tissue and cultured
cell samples, were not detected by preadsorbed antiserum. Quantitative
Western blotting showed that the abundance of sorcin in lysates of
heart is 50-70-fold less than that found in the DC-3F/VCRd-5L
cells (not shown).
Sorcin mRNA in Heart and Spleen
Two major sorcin
transcripts of 1.0 and 2.5 kilobases were detected in normal mouse
heart and spleen poly(A)
samples, using a hamster cDNA
probe (Fig. 3). These sizes were identical to sorcin transcripts
expressed in DC-3F/VCRd-5L cells(18) , where they have been
shown to represent the use of alternative polyadenylation
sites(2) . The transcripts were present at markedly lower
levels in the normal tissue samples, in parallel with the protein
quantitation comparison, and required examination of purified mRNA
samples for adequate detection. The relative abundance of sorcin mRNA
in normal tissues, i.e. heart and spleen, paralleled the
amount of encoded protein detected immunologically (Fig. 1).
RNA from mouse spleen and heart,
respectively. Sizes of the transcripts are identical to those detected
in DC-3F/VCRd-5L cells(18) .
Neonatal Rat Heart Cells Are Sorcin-positive by
Immunofluorescent Labeling
Preparations enriched for cardiac
myocytes were double-labeled with antibody to sarcomeric myosin heavy
chain (Fig. 4, left panel) and to the antibody raised
against the sorcin C terminus (Fig. 4, right panel).
Myocytes were identified by their characteristic filamentous labeling
pattern(20, 23) . These cells were strongly reactive
with the sorcin antiserum; immunolabeling was mainly extranuclear and
was distributed throughout the cytoplasm, although some punctate
nuclear labeling was observed. Antibody raised against the N terminus
sorcin peptide produced an identical labeling pattern (data not shown).
Preferential expression of sorcin in myocytes was observed; the
occasional non-myocyte was not positive for myosin heavy chain and had
reduced or nondetectable sorcin fluorescence.
Cardiac SR Is Selectively Labeled with Sorcin
Antibody
The intracellular localization of sorcin within cardiac
cells was investigated with immunoelectron microscopy. Intense
immunoperoxidase labeling of SR tubules in rat ventricular
cardiomyocytes was observed with either the C or N terminus peptide
antibodies. N terminus peptide antibody labeling is shown in Fig. 5; use of C terminus peptide antiserum produced the same
pattern. Punctate labeling of the lateral saccules of the reticulum was
seen near the transverse tubules (Fig. 5A).
Mitochondrial membranes were not immunolabeled except at points
directly in contact with or in close proximity to the SR near the
transverse tubules (Fig. 5B). Plasma membranes also had
no detectable sorcin except near points of contact with SR. Control
sections immunolabeled with adsorbed antibody and specifically labeled
sections collected at depths within the tissue having limited access to
primary antibody showed no immunolabeling (Fig. 5C).
Cross-immunoprecipitation with Antibodies to RyR and
Sorcin Indicate an Association between the Two
Proteins
Antibodies to peptides representing N and C termini of
sorcin immunoprecipitated a >500-kDa protein from rat or mouse
cardiac myocytes metabolically labeled with
[S]methionine (Fig. 6, lanes 1 and 2), which co-migrated with immunoprecipitated RyR (Fig. 6, lane 3; arrow at left indicates RyR) and with RyR detected in isolated myocyte SR by
Western blot with RyR antibodies (not shown). Neither the sorcin nor
RyR antibody immunoprecipitated high molecular weight proteins from
cardiac fibroblasts (Fig. 6, lane 7) or DC-3F/VCRd-5L
cells (Fig. 6, lanes 4-6). However, both RyR
antibody (Fig. 6, lane 4) and sorcin antiserum (Fig. 6, lanes 5 and 6) immunoprecipitated an
110-kDa protein from the drug-resistant cells, similar in size to
a 106-kDa SR protein shown to bind ryanodine and have Ca
release channel properties when incorporated into planar lipid
bilayers(14, 32) .
S]methionine-labeled proteins with sorcin and
RyR antibodies. Mouse (lane 1) and rat (lane 2)
cardiac myocyte proteins were immunoprecipitated with antibody raised
against sorcin N terminus peptide; rat cardiac myocyte proteins were
immunoprecipitated with antibody to RyR (C3-33) (lane 3) (arrow at left indicates 565-kDa RyR protein);
DC-3F/VCRd-5L cell proteins were immunoprecipitated with RyR antibody (lane 4), antibody to sorcin N terminus peptide (lane
5), and antiserum to sorcin C terminus peptide (lane 6);
and cardiac fibroblasts were immunoprecipitated with RyR antibody
(C3-33) (lane 7). Aliquots of homogenates containing 200
µg of protein (at equivalent specific radioactivities) were
analyzed by immunoprecipitation on 5% acrylamide gels. The >500-kDa
band present in lanes 1 and 2 was also detected when
myocyte lysates were treated with dithiobis(succinimidylpropionate)
cross-linking agent before
immunoprecipitation.
Sorcin Transfectants Express both Sorcin and the
RyR
To identify a functional role for sorcin, cell lines that
stably overexpressed the protein were generated, and 10 clones of DC-3F
cells co-transfected with pFRCMVSOR and p308 and five clones
transfected with p308 alone were analyzed for sorcin expression by
Western blot. Nine sorcin transfectant clones overexpressed sorcin, and
one of those, DC-3F/sor.3, was selected for further analysis by
immunocytochemistry. Surprisingly, in addition to the expected
accumulation of sorcin in the transfected cell line, RyR
immunoreactivity was now detected (Fig. 8, panels E (sorcin) and F (RyR)). In contrast, the DC-3F parental
line (Fig. 8, panels C and D) was negative for
both sorcin and RyR. Sham transfectants (DC-3F/neo), as well as the
non-sorcin-overproducing drug-resistant DC-3F/AD X cells, were also
negative for these proteins (not shown). To further establish a
relationship between sorcin and the RyR, DC-3F/VCRd-5L drug-resistant
cells, which constitutively overexpress sorcin as a result of gene
amplification, were assessed. In these cells, like the DC-3F/sor.3
transfectants, both sorcin and RyR protein were detected (Fig. 8, panels A (sorcin) and B (RyR)),
suggesting that the increase in sorcin was associated with the
coordinate accumulation of the RyR protein.
RyR-positive Sorcin Transfectants Exhibit
Caffeine-sensitive Ca
To examine whether increased expression of sorcin and RyR
protein was associated with functional Ca Release from Intracellular
Stores
channel
release activity, the effects of caffeine on intracellular
Ca
in the transfectant clone (DC-3F/sor.3) and the
various control lines were carried out with the use of digital
fluorescent Ca
imaging. In DC-3F/sor.3 cells, 10
mM caffeine stimulated a transient increase in intracellular
Ca
from 103 ± 6 nM to 240 ± 20
nM (Fig. 9, C and D) followed by
gradual decay to near basal levels. More than 90% of cells in every
field studied were responsive. No change in free intracellular
Ca
was observed in caffeine-treated DC-3F/neo cells (Fig. 9, A and B). A similar rapid increase
and gradual decay in intracellular Ca
levels was
observed in DC-3F/VCRd-5L cells treated with 10 mM caffeine (Fig. 9, A`-D`). Caffeine did not elicit
increase in intracellular Ca
in parental DC-3F cells
or in low sorcin multidrug-resistant DC-3F/AD X cells.
by digital fluorescent
Ca
imaging with fura-2/AM indicator. False color
images show levels of intracellular Ca
in
sham-transfected DC-3F cells (DC-3F/neo) of about 100 nM in
the absence (A) or presence (B) of 10 mM caffeine. Caffeine stimulates an increase in intracellular
Ca
in DC-3F/sor.3 sorcin-transfected cells from about
100 nM (C) to 240 nM (D).
Sorcin-overproducing DC-3F/VCRd-5L cells also respond to 10 mM
caffeine with a rise in intracellular Ca
from about
100 nM (A`), to about 250 nM (B`),
followed by a gradual decay toward base line in 4-8 min (C`, D`). More than 90% of sorcin-transfected or
drug-resistant cells in each field were caffeine-responsive. The rainbow spectrum on the right correlates with
Ca
concentration as calculated from in vitro calibration methods described under ``Materials and
Methods.'' Addition of EGTA to extracellular solutions during
caffeine perfusion did not affect these
results.
levels is shown in Fig. 10. Responsive cells showed peak
Ca
levels within 1 min after application of caffeine,
and levels gradually returned to base line over the course of 2-8
min. Removal of extracellular Ca
by the addition of 5
mM EGTA to the Hepes-buffered Krebs-Henseleit medium did not
affect the response to caffeine, confirming that caffeine induced
cytosolic Ca
increase by releasing Ca
from intracellular stores and not by Ca
influx
from the extracellular medium.
. Change in
intracellular Ca
levels with time in DC-3F/sor.3 (solid circles) and in DC-3F or sham-transfected DC-3F
(DC-3F/neo) (open circles) and low level sorcin DC-3F/AD X
cells (open triangles) is shown. No change in intracellular
free Ca
was measured for DC-3F, DC-3F/neo, or
DC-3F/AD X cells over the course of the experiments. Values represent
the average of 10 determinations with ±S.E. represented by error bars; there were at least 50 cells/microscope
field.
-binding domains are homologous with those in a
number of Ca
-binding proteins, including calpain and
calmodulin(2) , and sorcin has been shown to bind
Ca
by in vitro assays(3, 12) . Immunoelectron microscopy
directly localized sorcin to the SR, at or near transverse tubules,
suggesting that sorcin might participate in SR-mediated intracellular
Ca
regulation.
release channel was confirmed
by co-immunoprecipitation of 22-kDa sorcin from cardiac myocytes with
RyR-specific antibody. Immunoblot analysis of isolated SR protein with
antibody to RyR and to sorcin revealed the presence of the 565-kDa RyR
and of 22-kDa sorcin in a location protected from proteolysis by
proteinase K (not shown), supporting the possibility of an
intramembrane interaction between sorcin and the RyR complex.
-binding protein, were unexpectedly found to be
RyR-positive. DC-3F/VCRd-5L cells, which overproduce sorcin by an
entirely different mechanism, also displayed RyR immunoreactivity. The
species accounting for the immunoreactivity in the drug-resistant cells
may be the
110-kDa protein immunoprecipitated by RyR antibody (Fig. 6, lane 4) (also identified by Western blot with
RyR antibody in other experiments), although the apparent absence of
the 565-kDa RyR as an immunoprecipitated protein in those cells could
be associated with differences between C3-33 and GP561 RyR
antibodies, assay sensitivity, or low expression of RyRs.
release channel, i.e. caffeine-induced intracellular Ca
release. The
temporal profile of Ca
movement exhibited by the
sorcin-transfected fibroblasts in response to caffeine paralleled that
observed in similarly treated cardiac myocytes. These results suggest
that nonexcitable cells may serve as a useful model to study some
aspects of intracellular Ca
movement normally
associated with excitable tissues, analogous to the recent use of such
cells for the study of contractile protein function(35) . The
ultimate goal of such analyses is to produce new information about
cardiac or muscle cell function and diagnose dysfunction.
release channel. Detailed functional studies using
expression systems in which sorcin and/or RyR abundance can be
manipulated and Ca
movement determined will be
required to investigate the latter possibility.
)
We gratefully acknowledge the gift of the pFRCMVSOR
construct from Drs. Piet Borst and Frank Baas (The Netherlands Cancer
Institute), the GP561 antiserum against ryanodine receptor from Dr.
Kevin Campbell (an Investigator at the Howard Hughes Medical
Institute), and the C3-33 ryanodine receptor antibody from Dr.
Gerhard Meissner (University of North Carolina). We thank Ms. Aileen
Heras, June Chan, Kathy Clark, and Chan Hee Song for valuable technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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