From the
Triadin is a major membrane protein that is specifically
localized in the junctional sarcoplasmic reticulum of skeletal muscle
and is thought to play an important role in muscle
excitation-contraction coupling. In order to identify the proteins in
the skeletal muscle that interact with triadin, the cytoplasmic and
luminal domains of triadin were expressed as glutathione
S-transferase fusion proteins and immobilized to
glutathione-Sepharose to form affinity columns. Using these affinity
columns, we find that triadin binds specifically to the ryanodine
receptor/Ca
In muscle cells, depolarization of the transverse tubules
(T-tubules)(
Here, we
report that triadin associates with both the ryanodine
receptor/Ca
The reassociated calsequestrin
was calculated by subtracting the residual calsequestrin in the
junctional face membrane from the total amount of calsequestrin in the
reassociated calsequestrin/junctional face membrane complex. The
reassociated calsequestrin in the absence of triadin fusion protein was
designated as 100%.
To identify the proteins that interact with triadin, domains
of triadin that are localized in the cytoplasm and the lumen of the
sarcoplasmic reticulum (C-triadin and L-triadin, respectively) were
expressed as glutathione S-transferase (GST) fusion proteins
and immobilized on glutathione-Sepharose to form affinity columns.
Homogenates were prepared from rabbit skeletal muscle and solubilized
with 1.5% CHAPS. Detergent-solubilized material was diluted 10-fold and
applied to the column, washed, and eluted with a buffer containing 0.8
M NaCl and 20 m
M EDTA. Two proteins with molecular
masses
Calsequestrin is a soluble protein that remains associated
with the luminal side of the junctional membrane of the sarcoplasmic
reticulum through its interaction with a previously unidentified
membrane protein
(10) . Calsequestrin can be extracted from the
junctional face membrane by treatment with EDTA or high concentrations
of NaCl
(11, 29) , indicating that the interaction
between calsequestrin and its membrane anchoring protein can be
inhibited by EDTA and is disruptable by high ionic strength. Notably,
the association between calsequestrin and triadin-GST fusion protein is
also inhibited by EDTA, and high salt concentration can elute
calsequestrin from L-triadin-Sepharose. These binding properties
resemble those of the interaction of calsequestrin with its anchoring
protein in the junctional face membrane. In addition, like
calsequestrin, triadin is an abundant protein that is specifically
localized in the junctional region, with the bulk of this protein in
the lumen of the sarcoplasmic reticulum
(15, 16) .
Together, these characteristics strongly suggest that triadin is the
physiological anchoring protein for calsequestrin at the junctional
face of sarcoplasmic reticulum. To further test this hypothesis, we
examined whether the triadin luminal domain-GST fusion protein was
capable of inhibiting the reassociation of calsequestrin with the
junctional face membrane. As shown in Fig. 4, most of the calsequestrin
was removed from the junctional face membrane by treating triads
vesicles twice with a buffer containing 20 m
M Tris-HCl, pH
7.4, 0.1% Triton X-100, 0.75
M NaCl (Fig. 4 A,
lane 1). When calsequestrin was incubated with the
junctional face membrane in a buffer containing 150 m
M NaCl
and 1 m
M CaCl
We thank D. Witcher, V. Scott, and S. Kahl for helpful
discussion. We also thank Drs. B. Adams, A. Jorgensen, G. Koretzky, and
M. Welsh for helpful comments on the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
release channel and the
Ca
-binding protein calsequestrin from CHAPS
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid)-solubilized skeletal muscle homogenates. The luminal but not the
cytoplasmic domain of triadin-glutathione S-transferase fusion
protein binds [
H]ryanodine receptor, whereas
neither the cytoplasmic nor the luminal portion of triadin binds
[
H]PN-200-100-labeled dihydropyridine receptor.
In addition, the luminal domain of triadin interacts with calsequestrin
in a Ca
-dependent manner and is capable of inhibiting
the reassociation of calsequestrin to the junctional face membrane.
These results suggest that triadin is the previously unidentified
transmembrane protein that anchors calsequestrin to the junctional
region of the sarcoplasmic reticulum, and is involved in the functional
coupling between calsequestrin and the ryanodine
receptor/Ca
release channel.
)
results in Ca
release from the sarcoplasmic reticulum and the elevation of the
cytoplasmic Ca
leads to muscle contraction.
Considerable research has been focused on identifying and
characterizing the protein components that are important in the
regulation of calcium storage and release from the sarcoplasmic
reticulum. Several proteins that are specifically localized to the
triad junction play essential roles in excitation-contraction coupling.
The T-tubular dihydropyridine receptor senses the voltage across the
membrane, and activation of this receptor leads to the release of
Ca
from the sarcoplasmic reticulum
(1, 2, 3, 4) . The ryanodine
receptor/Ca
release channel is localized in the
junctional sarcoplasmic reticulum and is responsible for the
Ca
release from the Ca
store
(5, 6, 7, 8) . In the lumen of the
sarcoplasmic reticulum, the major protein is calsequestrin, an acidic
protein that binds to calcium with moderate affinity and high capacity
(9) . It is anchored to the luminal face of the junctional
sarcoplasmic reticulum
(10) and is thought to sequester and
concentrate calcium near its release site. Calsequestrin and the
ryanodine receptor/Ca
release channel are
functionally coupled. Activation of the ryanodine receptor induces
calcium dissociation from calsequestrin, allowing the free
Ca
to be released
(11) ; calsequestrin also
mediates the intraluminal Ca
control over the
activation of the ryanodine receptor/Ca
release
channel
(12, 13, 14) . Triadin is an abundant
membrane protein in the junctional sarcoplasmic reticulum, where it
co-localizes with the ryanodine receptor/Ca
release
channel
(15, 16, 17) . It contains a single
transmembrane domain that separates this protein into cytoplasmic and
luminal segments
(16) . Notably, only 47 amino acids of triadin
are cytoplasmic, with the bulk of this protein including the carboxyl
terminus being located in the lumen of the sarcoplasmic reticulum,
which contains a high concentration of positively charged amino acids
(16) . This membrane topology suggests that it plays an
important role in the lumen of the sarcoplasmic reticulum.
release channel and calsequestrin in the
lumen of the sarcoplasmic reticulum. Our results indicate that triadin
is the transmembrane protein that anchors calsequestrin to the
junctional face membrane. In addition, our data, along with previous
biophysical studies, suggest that triadin mediates the functional
coupling between the ryanodine receptor/Ca
release
channel and calsequestrin in the lumen of the sarcoplasmic reticulum.
Generation of Triadin-GST Fusion
Proteins
Triadin cDNA fragments
(16) corresponding to
amino acids 100-706 (luminal sequence) and amino acids 1-47
(cytoplasmic sequence) were amplified using polymerase chain reaction
and were subcloned into EcoRI sites of pGEX vectors. The
plasmid constructs were confirmed by sequencing. The GST fusion
proteins were expressed in Escherichia coli DH5 cells and
purified using glutathione-Sepharose 4B affinity column. The expressed
proteins can be recognized by all the available triadin antibodies
(15) .
Muscle Homogenization and Fusion Protein Affinity
Sepharose Binding Assay
Rabbit skeletal muscle was homogenized
in a 1:3 ratio in the homogenization buffer containing 20 m
M
Tris-HCl, pH 7.5, 1
M NaCl, 1.5% CHAPS, and protease
inhibitors: aprotinin (76.8 n
M), benzamidine (0.83
m
M), iodoacetamide (1 m
M), leupeptin (1.1
µ
M), pepstatin A (0.7 µ
M), and
phenylmethylsulfonyl fluoride (0.23 m
M). The homogenate was
incubated on ice for 2 h and then centrifuged at 30,000 rpm in a
Beckman ultracentrifuge using the Ti-45 rotor for 35 min. The
supernatant was collected and diluted 1:10 in the dilution buffer
containing 20 m
M Tris-HCl, pH 7.5, 1 m
M
CaCl, 1 m
M dithiothreitol, and protease inhibitors
as described above. The diluted homogenate was further incubated at 4
°C for 5 h and centrifuged again to remove precipitated materials
(mainly myosin). The solubilized skeletal muscle homogenate was
precleared using glutathione-Sepharose 4B for 3 h and then incubated
with the fusion protein-Sepharose for 4 h. After incubation, the
Sepharose was washed sequentially using Buffer I (20 m
M
Tris-HCl, pH 7.5, 150 m
M NaCl, 1 m
M CaCl
,
1 m
M, 0.5% CHAPS), and Buffer II (20 m
M Tris-HCl, pH
7.5, 150 m
M NaCl, 1 m
M CaCl
, 0.15%
CHAPS). Bound proteins were eluted using 20 m
M Tris-HCl, pH
7.4, 0.15% CHAPS, 0.75
M NaCl, 20 m
M EDTA and
analyzed by SDS-PAGE. Anti-calsequestrin monoclonal antibody VIIID1-2
(18) and sheep anti-rabbit skeletal muscle ryanodine receptor
antibody
(19) were used in the immunoblot assay. [
H]Ryanodine and
[
H]PN-200-100 Binding Assay-The
skeletal muscle homogenate was pre-equilibrated with 20 n
M [
H]ryanodine at 37 °C for 60 min in a buffer
containing 20 m
M Tris-HCl, pH 7.4, 0.15% CHAPS, 200
m
M KCl, 10 m
M ATP, and 20 µ
M free
Ca
. The Sepharose was incubated with the labeled
muscle homogenates as described above, and the
[
H]ryanodine bound to the Sepharose was counted.
As a positive control, Sepharose conjugated with anti-ryanodine
receptor monoclonal antibody XA7
(20) was used. For
[
H]PN-200-100 binding experiments, the skeletal
muscle homogenate was pre-equilibrated with 20 n
M [
H]PN-200-100 at 37 °C for 60 min. in a
buffer containing 20 m
M Tris-HCl, pH 7.4, 0.15% CHAPS, 150
m
M NaCl, and 200 µ
M diltiazem. The Sepharose was
incubated with the labeled muscle homogenate as described above. After
washing, the amount of [
H]PN-200-100 bound to the
Sepharose was counted. For nonspecific binding, 10 µ
M nitrendipine was added to the incubation buffer. As a positive control,
Protein G-Sepharose (Pharmacia) that was coupled with the anti-
1
subunit of the dihydropyridine receptor monoclonal antibody IIC12
(21) was used.
Inhibition of the Reassociation of Calsequestrin with
Junctional Face Membrane by Triadin Fusion Protein
Junctional
face membrane was prepared by 0.1% Triton X-100 and 0.75
M
NaCl extraction of the rabbit skeletal muscle triads
(11) . To
reconstitute the calsequestrin/junctional face membrane complex, the
junctional face membrane was incubated with calsequestrin that had been
purified using phenyl-Sepharose
(22) in a buffer containing 10
m
M Tris-HCl, pH 7.5, 0.1% Triton X-100, 1 m
M
CaCl, and 150 m
M NaCl. After incubation at 4
°C for 4 h, the sample was centrifuged using a Beckman TL-100
ultracentrifuge at 100,000 rpm for 15 min. The insoluble pellet, which
contained calsequestrin attached to junctional face membrane, was
collected and analyzed using SDS-PAGE. For triadin fusion protein
competition assay, various amounts of fusion protein were added to the
above incubation mixture. After incubation and centrifugation, the
pellets were separated on SDS-PAGE and transferred to nitrocellulose
for immunoblot assay using anti-calsequestrin antibody VIIID1-2. The
relative amount of calsequestrin on the immunoblot was estimated using
Molecular Dynamics 300S densitometer.
63 and
550 kDa (Fig. 1 A, arrowheads)
were eluted from the Sepharose that conjugated with the luminal domain
of triadin (L-triadin-Sepharose). The 63-kDa protein was identified as
calsequestrin, the calcium-binding protein in the lumen of the
sarcoplasmic reticulum; the high molecular mass protein was identified
as the ryanodine receptor/Ca
release channel (Fig.
1 A). Control experiments were also performed. As shown in
Fig. 1B, glutathione-Sepharose, glutathione-Sepharose
that contains GST alone (GST-Sepharose), or the cytoplasmic domain of
triadin (C-triadin-Sepharose), all failed to bind the ryanodine
receptor and calsequestrin (Fig. 1 B). Only
L-triadin-Sepharose was able to bind to these two proteins. The
ryanodine receptor and calsequestrin are minor components in the
solubilized whole muscle homogenates; however, they are the only major
proteins from the homogenates that bind to the L-triadin-Sepharose as
detected in the Coomassie Blue-stained gel (Fig. 1 A).
This further demonstrates the specificity of the interactions. Similar
results have been obtained with detergent-solubilized skeletal muscle
triads. Furthermore, we find that L-triadin-Sepharose binds to the
purified ryanodine receptor or purified calsequestrin (data not shown).
Also, the triadin fusion protein was able to bind to calsequestrin in a
protein overlay assay (data not shown). Thus, triadin interacts
independently with both the ryanodine receptor and calsequestrin.
Figure 1:
Association of the ryanodine receptor
and calsequestrin with the luminal portion of triadin. A,
after preclearance using glutathione-Sepharose, the CHAPS-solubilized
skeletal muscle homogenate was subjected to affinity chromatography on
L-triadin-Sepharose (30 µl). The starting material (75 µg),
skeletal muscle homogenate ( Homo.), flow-through
( Void), L-triadin-Sepharose without incubation with the
homogenate ( Beads(-H)), fusion
protein-Sepharose after incubating with muscle homogenates
( Beads(+H)), and the sample eluted from the Sepharose
( Elution) were analyzed on SDS-PAGE. The gels were either
stained with Coomassie Blue ( CB) or transferred to
nitrocellulose and stained with anti-calsequestrin ( Anti-CSQ)
or anti-ryanodine receptor antibody ( Anti-RyR).
B, specific interaction of the luminal portion of
triadin with the ryanodine receptor and calsequestrin. Chromatography
was performed on various affinity columns including the
glutathione-Sepharose ( Seph.), GST-Sepharose ( GST),
C-triadin-Sepharose ( C-triadin), and L-triadin-Sepharose
( L-triadin). The ryanodine receptor and calsequestrin
that bound to the Sepharose were analyzed by immunoblot
assay.
The association of the ryanodine receptor with triadin was
demonstrated further in [H]ryanodine receptor
binding experiments. The solubilized skeletal muscle homogenate was
prelabeled with [
H]ryanodine and incubated with
L-triadin-Sepharose. The amount of bound ryanodine receptor was
determined by counting the [
H]ryanodine on the
L-triadin-Sepharose. As shown in Fig. 2 A, the luminal portion
fusion protein bound the labeled ryanodine receptor in a dose-dependent
manner. The specificity of the binding was also examined. In a manner
similar to the ryanodine receptor monoclonal antibody XA7-Sepharose
(20) , L-triadin-Sepharose specifically bound the labeled
receptor, whereas the GST-Sepharose or C-triadin-Sepharose was not able
to bind to the labeled ryanodine receptor (Fig. 2 B).
Using the same bead assay, we examined the possible interaction of
triadin-Sepharose with the [
H]PN-200-100-labeled
dihydropyridine receptor. However, neither the cytoplasmic nor the
luminal portion of triadin was able to bind to the
[
H]PN-200-100-labeled receptor in the muscle
homogenate (Fig. 2 C). Our results demonstrate a specific
interaction between triadin and the ryanodine receptor that occurs in
the lumen of the sarcoplasmic reticulum. Clusters of negatively charged
residues in the ryanodine receptor located in the lumen of the
sarcoplasmic reticulum
(23, 24, 25) are likely
to be involved in the interaction with the positively charged triadin
(16) .
Figure 2:
Binding of the prelabeled rabbit skeletal
muscle ryanodine receptor by the luminal domain of triadin. A,
L-triadin-Sepharose binds [H]ryanodine-labeled
receptor in a dose-dependent manner. B, specificity of the
binding by the luminal domain of triadin is shown. Sepharose containing
different fusion proteins were used in the experiment. Anti-ryanodine
receptor monoclonal antibody XA7-Sepharose was used as positive control
in the assay. C, binding of the
[
H]PN200-100-labeled dihydropyridine receptor by
GST fusion proteins is shown. Anti-
1 subunit of the
dihydropyridine receptor monoclonal antibody IIC12 Sepharose was used
as positive control in the assay.
Calsequestrin binds Cawith moderate
affinity and high capacity
(9) , and the binding of
Ca
leads to dramatic conformational change of this
luminal protein
(26, 27) . We therefore examined the
effect of Ca
on the interaction between calsequestrin
and triadin. In the presence of CaCl
, L-triadin-Sepharose
was able to bind calsequestrin and remove calsequestrin from muscle
homogenate (Fig. 3, left panel). However, when EDTA
was present, the interaction was inhibited. Thus, calsequestrin binds
to the luminal domain of triadin in a Ca
-dependent
manner.
, calsequestrin reassociated with the
membrane fraction (Fig. 4 A, lane 2).
However, when increasing amounts of triadin-GST fusion protein were
added to the incubation buffer, less calsequestrin reassociated to the
junctional face membrane (Fig. 4 A, lanes 3-6).
The native triadin did not decrease during the competition process
(Fig. 4 A, bottom), and GST alone did not
inhibit the reassociation (Fig. 4 B). This result
suggests that the soluble triadin luminal portion fusion protein
interacts with calsequestrin and inhibits the reassociation of
calsequestrin to the membrane by competing with the native triadin in
the junctional face membrane. This study suggests that one functional
role of triadin is to serve as the transmembrane protein that anchors
calsequestrin to the luminal side of the junctional sarcoplasmic
reticulum near the ``SR feet'' (ryanodine receptor)
(10, 28) , where calsequestrin exerts its physiological
function.
Figure 4:
Effect of triadin luminal portion-GST
fusion protein on the reassociation of calsequestrin to junctional face
membrane. A, proteins of the junctional face membrane
( lane 1), and reassociated junctional face
membrane/calsequestrin complex in the absence of triadin fusion protein
( lane 2) and presence of increasing amounts (30, 60,
90, and 120 µg) of the fusion protein ( lanes 3-6)
were separated by SDS-PAGE and transferred to nitrocellulose.
Calsequestrin attached to the junctional face membrane was detected by
anti-calsequestrin antibody. The native triadin in the junctional face
membrane detected by anti-triadin antibody was shown at the bottom.
B, percentage of the reassociated calsequestrin in the
presence of various amounts of triadin luminal portion fusion protein
or GST as estimated by densitometry scanning of the
immunoblot.
It is known that the intraluminal
Ca-binding protein calsequestrin and the ryanodine
receptor/Ca
release channel are functionally coupled
(11, 12, 13, 14) . The ryanodine
receptor, when activated by Ca
release agents, such
as caffeine, induces dissociation of calcium from calsequestrin
(11) . Conversely, changes in luminal Ca
concentration lead to conformational changes in calsequestrin
(26, 27) , and this information can be transmitted to
the ryanodine receptor, thus affecting the activation of the ryanodine
receptor/Ca
release channel
(12, 13, 14) . However, despite the biophysical
evidence for functional coupling between the ryanodine receptor and
calsequestrin, there is no evidence for direct interaction between the
two proteins. It is thought that the interaction is mediated by a third
protein. Our data and previous studies suggest that triadin binds to
the ryanodine receptor
(17, 30) , and the luminal domain
of triadin also interacts with calsequestrin. Together, these results
suggest that triadin anchors calsequestrin to the junctional face
membrane, and thus may be involved in the functional coupling between
calsequestrin and the ryanodine receptor/Ca
release
channel in the lumen of the sarcoplasmic reticulum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.