(Received for publication, February 14, 1995; and in revised form, May 5, 1995)
From the
In vertebrate skeletal muscle, excitation-contraction coupling
may occur by a mechanical coupling mechanism involving protein-protein
interactions between the dihydropyridine receptor (DHPR) of the
transverse tubule membrane and the ryanodine receptor
(RYR)/Ca
In skeletal and cardiac muscle excitation-contraction (E-C) ( Different
E-C coupling mechanisms exist in vertebrate skeletal and cardiac
muscle. A major difference is that E-C coupling in cardiac muscle is
dependent on extracellular Ca The skeletal and cardiac
muscle DHPRs are related oligomeric protein complexes, which comprise
up to five subunits, all of which have cytoplasmic domains that could
potentially interact with the SR Ca Studies with skeletal and cardiac muscle chimeric cDNAs suggest that
the putative II-III cytoplasmic loop region of the DHPR The skeletal muscle
DHPR contains several consensus phosphorylation sites including a
serine residue, Ser
Figure 1:
In vitro phosphorylation of
purified CDCL, SDCL and SDCLS687A. A, purified peptides (2
µg of protein/lane) were separated by SDS-PAGE and stained with
Coomassie Brilliant Blue. The mobility of molecular size markers (in
kDa) is indicated on the left. B, an autoradiograph
of an SDS-PAGE gel shows in vitro PKA
The RYR was shown to bind
the neutral plant alkaloid ryanodine with nanomolar affinity in a
manner that correlates well with the functional state of the
Ca
Figure 2:
Effect of peptides on
[
The lack of
activation of [
Figure 3:
Effects of SDCL-P, SDCLS687A, and
CDCLS813A on [
The mutant CDCL peptide was as
effective as the phosphorylated and mutant SDCL peptides in inhibiting
the SDCL-activated RYR. Fig. 3A (closedcircles) shows that the addition of 20 µM CDCLS813A to incubation media containing 5 µM SDCL
resulted in a [ Previously,
we showed that BSA activated the RYR by a mechanism different from that
of the DHPR loop peptides(10) . In agreement with this
observation, the mutant peptides SDCLS687A and CDCLS813A did not
inhibit the increased [
Figure 4:
Effects of SDCL-P and SDCLS687A on
single channel activity of the purified skeletal Ca
Figure 5:
Specific binding of
The interaction of RYR with other DHPR
peptides was further examined in competition binding experiments. Fig. 6shows that the unphosphorylated, nonradioactively labeled
phosphorylated and mutant SDCLs, and CDCL decreased binding of the
Figure 6:
Effects of peptides on
Figure 7:
Effects of SDCL, SDCLf1, SDCLf2, and
SDCLf3 on [
Previous studies have shown that two of the five subunits
( The basal level of phosphorylation of the
skeletal muscle DHPR A Ser Previously, we showed that the II-III loop peptide
of the cardiac DHPR In conclusion, the data from this
study demonstrate that a peptide covering a stretch of 61 amino acids
in the II-III cytoplasmic loop region of the skeletal muscle DHPR
release channel of the sarcoplasmic
reticulum membrane. We have previously shown that the cytoplasmic
II-III loop peptides of the skeletal and cardiac muscle DHPR
1
subunits (SDCL and CDCL, respectively) activate the skeletal muscle
RYR. We now report that cyclic AMP-dependent protein kinase-mediated
phosphorylation of Ser
of SDCL yields a peptide that
fails to activate the RYR, as determined in
[
H]ryanodine binding and single channel
measurements. The phosphorylated SDCL bound to the skeletal muscle but
not cardiac muscle RYR, and the binding could be displaced by the
unphosphorylated SDCL. A mutant SDCL with a Ser
Ala substitution failed to activate the RYR, but was still able to
bind. Similarly, a Ser
Ala substitution in CDCL
yielded a peptide that failed to activate the skeletal RYR. Use of
three smaller overlapping peptides within the SDCL region identified an
amino acid region from 666 to 726 including Ser
, which
bound to and activated the skeletal muscle RYR. These results suggest
that cyclic AMP-dependent protein kinase-mediated phosphorylation of
the DHPR
subunit may play a role in the functional
interaction of the DHPR and RYR in skeletal muscle.
)coupling, a muscle action potential activates the
Ca
release channel in an intracellular Ca
storing membrane compartment, the sarcoplasmic reticulum (SR).
The SR Ca
release channels are also known as
ryanodine receptors (RYR) because of their ability to bind the plant
alkaloid ryanodine with high affinity and
specificity(1, 2) . In vertebrate skeletal muscle, the
SR Ca
release channels are thought to be linked to
another Ca
channel (L-type), also known as the
dihydropyridine receptor (DHPR), which is located in infoldings of the
surface membrane, the transverse (T-) tubule(3) .
, whereas skeletal
muscle E-C coupling is not. In cardiac muscle, the entry of
extracellular Ca
via a voltage-sensitive
DHPR/Ca
channel is required to trigger Ca
release from SR(4) . In contrast, in vertebrate skeletal
E-C coupling, the Ca
release channels are thought to
be regulated by a skeletal muscle DHPR isoform through protein-protein
interactions(5, 6) . In addition to a direct
activation by the DHPR, an involvement of Ca
in
activating the SR Ca
release channel during E-C
coupling has been described(6) .
release
channel(7) . Similarly, skeletal and cardiac muscle express two
structurally related Ca
release channels composed of
four identical
565-kDa polypeptides(1, 2) . In
addition to the DHPR, several endogenous effector molecules were shown
to regulate the RYR. These include small diffusible molecules such as
Ca
, Mg
, and ATP, and proteins such
as calmodulin, FK506-binding protein, and triadin(2) . An
involvement of triadin in mediating the functional interaction between
the skeletal muscle DHPR and RYR has been suggested(8) .
1 subunit
is a major determinant of the type of E-C coupling (skeletal or
cardiac) that occurs in muscle(9) . We expressed the II-III
loop regions of the skeletal and cardiac DHPR
1 subunits and
showed that both peptides activate the purified skeletal but not the
cardiac muscle Ca
release channel(10) .
Accordingly, the interaction was specific with respect to the skeletal
muscle RYR but not with respect to the peptides.
, in the II-III loop region of the
1 subunit which is rapidly phosphorylated by cyclic AMP-dependent
protein kinase (PKA)(11, 12) . Phosphorylation of the
skeletal muscle DHPR was shown to modulate L-type Ca
channel activity(13, 14) . Here, we report that
phosphorylation of Ser
of SDCL by PKA in vitro resulted in the formation of a peptide that failed to activate the
RYR. This result provides novel evidence for a role of DHPR
phosphorylation in regulating DHPR protein-RYR protein interactions in
skeletal muscle.
Materials
Catalytic subunit of cyclic
AMP-dependent protein kinase from bovine heart was purchased from Sigma
and [H]ryanodine (54.7 Ci/mmol) from DuPont NEN.
Unlabeled ryanodine was obtained from AgriSystems International (Wind
Gap, PA), Pefabloc (a protease inhibitor) from Boehringer Mannheim, and
phospholipids from Avanti (Birmingham, AL). All other chemicals were of
analytical grade.
Site-directed Mutagenesis
The site-directed
mutants SDCLS687A and CDCLS813A were generated by polymerase chain
reaction (PCR). Mutagenic primers, 5`-GGA GAC CCC TGG CCA TCT TCC
TGC-3` and 5`-CTT CTC CGG AGC GGC AGT CCT GGC CAG C-3`, were designed
to change Ser to Ala of SDCL (15) and Ser
to Ala of CDCL(16) , respectively. The megaprimers were
amplified with the mutagenic primers and 5` primers in the first round
PCR. The PCR products were agarose gel-purified and used as megaprimers
with the 3` primers to amplify mutant SDCL and CDCL in the second round
PCR. The PCR products were subcloned into the expression vector pET-11d
and the mutant SDCLS687A and CDCLS813A plasmids were confirmed by DNA
sequencing.
Preparation of DHPR-derived Peptides
SDCLf1
(Glu-Glu
), SDCLf2
(Pro
-Leu
) and SDCLf3
(Lys
-Leu
) cDNAs (15) were
amplified by PCR using SDCL as template and subcloned in the pET-11d
vector. The constructed plasmids were confirmed by DNA sequencing. All
peptides were expressed in Escherichia coli strain BL21(DE3)
and purified by DEAE-Sephacel column chromatography, followed by
hydroxyapatite column chromatography as described(10) .
In Vitro Phosphorylation
Catalytic subunit of
cyclic AMP-dependent protein kinase (PKA) from bovine heart (Sigma) was
reconstituted in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl,
20 mM dithiothreitol, and 50% glycerol and stored at -20
°C. Phosphorylation reaction was carried out by adding the peptides
(1 µg) to kinase reaction buffer A (20 mM Tris-HCl, pH
7.5, 100 mM NaCl, 12 mM MgCl, 1
mM dithiothreitol) plus 5 µCi of
[
-
P]ATP (
3000 Ci/mmol, DuPont NEN) and
5 units of PKA in a volume of 30 µl. Reaction mixtures were
incubated at room temperature for 30 min before the samples were
analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). The gel was
fixed in 12% trichloroacetic acid, dried, and exposed to an x-ray film.
The percentage of
P incorporation was determined in the
presence of 5-fold excess of ATP over SDCL by scintillation counting of
the dried gel containing the
P-labeled peptides. To
prepare
P-labeled SDCL as a probe for
P-SDCL
binding assay, SDCL (2 µg) was incubated with 50 µCi of
[
-
P]ATP and 15 units of PKA in 30 µl of
buffer A at room temperature for 30 min. The
P-labeled
SDCL was freed of [
-
P]ATP by gel filtration
on a G-25 Sephadex column. To prepare the cold phosphorylated SDCL, 0.5
mg of SDCL was incubated with 1.5 mM ATP and 500 units of PKA
in 1 ml of buffer A at room temperature for 1 h. Free ATP was removed
by dialysis against 20 mM Hepes, pH 7.4, 50 mM NaCl.
The phosphorylated SDCL was concentrated with a Centricon 10
concentrator (Amicon, Beverly, MA), frozen in liquid nitrogen, and
stored at -80 °C.
Isolation of SR Vesicles and Purification of
RYR
Heavy SR vesicles were prepared in the presence of protease
inhibitors from rabbit skeletal and canine heart muscle as
described(17, 18) . The RYR was purified in the
presence of CHAPS as a 30 S protein complex by sucrose gradient rate
centrifugation and reconstituted into proteoliposomes by removal of the
detergent by dialysis(19) .[
Unless
otherwise indicated, [H]Ryanodine Binding
H]ryanodine binding was
determined by incubating skeletal muscle SR vesicles at 12 °C for
20 h in 100 µl of 20 mM Na-Hepes, pH 7.4, 100 mM NaCl, 0.1 mM EGTA, 0.125 mM CaCl
, 5
mM AMP, 100 µM leupeptin, 0.5 mM Pefabloc, and 5 nM [
H]ryanodine.
Bound and free [
H]ryanodine were determined by a
filtration assay as described(19) . Nonspecific binding was
determined in the presence of a 1,000-fold excess of unlabeled
ryanodine.
Single Channel Recordings
Single channel
measurements were performed by fusing reconstituted proteoliposomes
containing the purified skeletal muscle RYR Ca release channel (19) with Muller-Rudin type planar lipid
bilayers containing phosphatidylethanolamine, phosphatidylserine, and
phosphatidylcholine in the ratio of 5:3:2, respectively (25 mg/ml
phospholipid in n-decane)(10) . Channels were
initially recorded in a symmetric KCl buffer solution containing 20
mM K-Pipes, pH 7.0, 0.25 M KCl, 0.15 mM CaCl
, and 0.1 mM EGTA. Before the addition of
the peptides, channel open probability (P
) was
lowered to < 0.1 by the addition of EGTA to the cis chamber. The data files were acquired and analyzed using a
commercially available software package (pClamp 6.0.1, Axon
Instruments, Burlingame, CA) in the continuous fetchex mode, using a
filter frequency setting at 2 kHz and sampling rate at 10 kHz. Channel
open probabilities were determined by 50% threshold analysis as
described previously(20) .
Proteoliposomes
containing the purified skeletal RYR and skeletal and cardiac muscle SR
vesicles were dot-blotted on nitrocellulose membranes. The membranes
were blocked by incubation for 1 h with 5% nonfat dry milk in binding
buffer B (10 mM Hepes, pH 7.4, 50 mM NaCl, 0.1 mM EGTA, and 0.125 mM CaClP-SDCL Overlay
). The membranes were
washed once with buffer B and placed on a piece of parafilm. The
binding reaction was carried out by overlaying the membranes with
buffer B supplemented with
P-labeled SDCL (5
10
cpm/ml), 1% bovine serum albumin (BSA), 100
µM leupeptin, and 0.5 mM Pefabloc. After
incubation for 12 h at room temperature, the membranes were washed
twice for 5 min with ice-cold buffer B, air-dried, and exposed to an
x-ray film. Bound
P-labeled SDCL was quantitated by
scintillation counting.
Phosphorylation of SDCL
In purified skeletal
muscle DHPR preparations, cAMP-dependent protein kinase rapidly
phosphorylates Ser in the cytoplasmic II-III loop region
of the DHPR
1 subunit (SDCL)(11, 12) . In the
present study, the effects of Ser
phosphorylation on SR
Ca
release channel function were examined, using the
phosphorylated and unphosphorylated SDCL as probes in
[
H]ryanodine binding and single channel
measurements. In some experiments, the cytoplasmic II-III loop peptide
of the cardiac DHPR
1 subunit (CDCL) served as a control, as CDCL
lacks a phosphorylation site corresponding to Ser
in
SDCL. SDCL and CDCL were expressed in E. coli, and purified by
DEAE-Sephacel and hydroxyapatite column chromatography(10) .
SDS-PAGE indicated that the two peptides were obtained with a purity
> 99% (Fig. 1A, lanes1 and 2). The purified peptides were subjected to in vitro phosphorylation by PKA as described under
``Experimental Procedures'' and analyzed by SDS-PAGE
and autoradiography. In the autoradiographs, a prominent
P-labeled band with a mobility corresponding to that of
SDCL was observed (Fig. 1B, lane2).
A corresponding
P-labeled band was not detected in the
lane containing CDCL (Fig. 1B, lane1). The molar ratio of phosphate to SDCL was determined
by scintillation counting to be 0.94 (data not shown), indicating only
one phosphorylation site in SDCL. To confirm the presence of a single
phosphorylation site in SDCL, a single point mutant with substitution
of Ser
to Ala was prepared. The derived peptide SDCLS687A
migrated on the gels at the same position as wild type SDCL (Fig. 1A, lane3), but could no
longer be phosphorylated by PKA (Fig. 1B, lane3). These results indicate that Ser
is
phosphorylated by PKA, and that it is the only PKA-dependent
phosphorylation site in SDCL.
P-phosphorylation of SDCL but not of CDCL and SDCLS687A. C, alignment of amino acid sequences of SDCL (Glu
to Leu
) (15) and CDCL (Asp
to
Gln
)(16) . Ser
, a phosphorylation
site, of SDCL, and Ser
of CDCL are marked with boxes.
Effects of Phosphorylated and Mutant SDCL Peptides on
[
To assess the effects
of phosphorylation on RYR function, a nonradioactively phosphorylated
SDCL (SDCL-P) was prepared. In control experiments, SDCL-P did not
incorporate any radioactivity when incubated with PKA and
[H]Ryanodine Binding
-
P]ATP. Phosphorylated peptides also
showed no significant incorporation of
P label following
their incubation with SR vesicles under conditions comparable to the
[
H]ryanodine binding measurements. These results
indicated that essentially fully phosphorylated SDCL peptides were used
in the functional studies described below.
release channel(1, 2) . An
increase in [
H]ryanodine binding affinity by
micromolar concentrations of SDCL and CDCL has been
described(10) . The effects of the unphosphorylated (SDCL),
phosphorylated (SDCL-P), and mutant (SDCLS687A) SDCL peptides on
[
H]ryanodine binding to SR vesicles are
illustrated in Fig. 2. As shown previously (10) , the
unphosphorylated SDCL increased [
H]ryanodine
binding in a dose-dependent manner. At 10 µM, SDCL
increased [
H]ryanodine binding by about 50% (Fig. 2, closedcircles). In contrast, the
phosphorylated SDCL was without a significant effect. A small
(
10%) increase in [
H]ryanodine binding was
observed at 10 µM SDCL-P (Fig. 2, triangles). The mutant peptide SDCLS687A was without an effect
on [
H]ryanodine binding to the SR vesicles (Fig. 2, opencircles).
H]ryanodine binding to skeletal SR vesicles. SR
vesicles (150 µg of protein/ml) were incubated with 5 nM [
H]ryanodine and the indicated amounts of
SDCL (closedcircles), SDCLS687A (opencircles), CDCL (closeddiamonds),
CDCLS813A (opendiamonds), and phosphorylated SDCL (triangles) as described under ``Experimental
Procedures.'' Values are the means ± S.D. of two or three
experiments carried out in duplicate. B
value
for [
H]ryanodine binding to skeletal SR vesicles
in the absence of peptides was 9.5 pmol/mg as determined by Scatchard
analysis. The control value (100%, without added peptides) corresponded
to about 10% of the B
value of high affinity
[
H]ryanodine binding.
H]ryanodine binding by the
phosphorylated and mutant peptides, shown in Fig. 2, could have
been due to a decrease in the binding affinity of the peptides and/or
the formation of inactive peptides. To distinguish between these
possibilities, we carried out binding studies (see below) and
determined the effects of the peptides on
[
H]ryanodine binding in the presence of SDCL and
CDCL. Fig. 3A shows that 5 µM SDCL
increased [
H]ryanodine binding to the skeletal SR
vesicles by about 50%, and the addition of the phosphorylated and
mutant peptides resulted in a decrease of
[
H]ryanodine binding close to control levels
(-SDCL). Half-maximal inhibition of the SDCL-activated binding
activity by
5 µM SDCL-P (Fig. 3A, triangles) and
4 µM SDCLS687A (opencircles) suggested that the three peptides bound to the
RYR with similar affinity.
H]ryanodine binding to skeletal SR
vesicles in the presence of SDCL or CDCL. Skeletal SR vesicles (150
µg/ml) were incubated in reaction mixture containing 5 µM SDCL (A) or 5 µM CDCL (B) and 5
nM [
H]ryanodine in the presence of the
indicated amounts of SDCLS687A (opencircles),
CDCLS813A (closedcircles), and SDCL-P (triangles). Values are the means ± S.D. of two or
three experiments carried out in duplicate. The control value (100%,
without added peptides) corresponded to about 10% of the B
value of high affinity
[
H]ryanodine binding.
Effects of Mutant CDCL Peptide on
[
We were surprised to
find that our mutant SDCL with SerH]Ryanodine Binding
Ala substitution
failed to activate the skeletal muscle RYR, because a Ser
Ala
mutation is in general considered to be a conserved mutation. This
result suggested that Ser
may be essential for RYR
activation. To confirm the role of Ser
in the formation
of an active SDCL peptide, we prepared a mutant CDCLS813A with a
Ser
Ala substitution. We choose to replace
Ser
of CDCL because of its close position to Ser
of SDCL in the amino acid sequence alignments (Fig. 1C). The mutant SDCLS813A was overproduced in E. coli and purified to homogeneity as described in ``Experimental Procedures'' (data not shown). Fig. 2shows that, in agreement with a previous
study(10) , wild-type CDCL is more potent than SDCL in
increasing [
H]ryanodine binding to the SR
vesicles. CDCL increased [
H]ryanodine binding by
about 75% at 10 µM concentration (Fig. 2, closeddiamonds). In contrast, a single point
mutation in CDCL (CDCLS813A) yielded a peptide that was without an
effect on [
H]ryanodine binding (Fig. 2, opendiamonds).
H]ryanodine binding level close to
the control level (-SDCL). Similarly, the mutant SDCL and CDCL
peptides were able to inhibit the CDCL-activated RYR (Fig. 3B). The addition of 5 µM CDCL
increased [
H]ryanodine binding by about 65%. This
increase was nearly fully prevented by the addition of an excess (20
µM) of the mutant SDCL and CDCL peptides.
H]ryanodine binding
activity of the RYR observed in the presence of BSA (data not shown).
Effects of Phosphorylated and Mutant SDCL Peptides on
Single Channel Activities
An antagonistic action of the
phosphorylated and mutant SDCL peptides was also observed in single
Ca release channel measurements. Single, purified
channels were recorded in symmetric 250 mM KCl medium. With
K
as the current carrier, single channel conductance
was
770 picosiemens (not shown). To optimize stimulation of
channel activity by SDCL, we limited the initial channel open
probabilities (P
) to <0.1 by adjusting the free
Ca
concentration in the cis chamber to about
5 µM by addition of EGTA (Fig. 4, toppanels). In Fig. 4A, the addition of 100
nM SDCL increased P
from 0.029 to 0.121 (middlepanel). The bottomtrace of Fig. 4A shows that the subsequent addition of 100
nM phosphorylated SDCL reduced channel activity about 2-fold (P
= 0.062) corresponding to a level about
twice that before the addition of SDCL. A comparable activation of
channel activity by 100 nM SDCL and about 2-fold inhibition of
the SDCL-activated channel by 200 nM SDCLS687A are shown in Fig. 4B. An inhibition of the SDCL-activated channels
by SDCL-P and SDCLS687A was observed in 8 (out of 8) and 9 (out of 9)
experiments, respectively (Table 1). The single channel data of Table 1are in good agreement with the
[
H]ryanodine binding measurements. Furthermore,
because of the use of purified RYRs, the single channel measurements
suggest that the phosphorylated SDCL and SDCLS687A antagonized the
action of SDCL by a direct interaction with the RYR.
release channel in the presence of SDCL. A, single-channel
currents, shown as upward deflections, were recorded in symmetrical
0.25 M KCl media containing 8.8 µM free
Ca
cis and 50 µM free
Ca
trans. Holding potential = 30 mV. Toptrace, control (P
=
0.029); middletrace, after addition of 100 nM SDCL cis (P
= 0.121); bottomtrace, after the further addition of 100
nM SDCL-P cis (P
=
0.062). B, single channel currents, shown as upward
deflections, were recorded in symmetrical 0.25 M KCl media
containing 8.8 µM free Ca
cis and 50 µM free Ca
trans.
Holding potential = 35 mV. Toptrace, control (P
= 0.025); middletrace, after addition of 100 nM SDCL cis (P
= 0.103); bottomtrace, after the further addition of 200 nM SDCLS687A cis (P
=
0.059).
Binding of
The phosphorylated SDCL inhibited activation of the RYR by
SDCL, suggesting that SDCL-P bound to the RYR. To examine their binding
to the skeletal muscle and cardiac muscle RYRs, we developed a protein
overlay assay using P-labeled SDCL to Skeletal
RYR
P-labeled SDCL as a probe. Skeletal
muscle and cardiac muscle SR vesicles containing a similar number of
high affinity [
H]ryanodine binding sites were
dot-blotted on a nitrocellulose membrane and overlaid with
P-labeled SDCL. In the autoradiographs, skeletal muscle SR
vesicles, but not cardiac SR vesicles, retained
P-labeled
SDCL (Fig. 5), suggesting that
P-labeled SDCL binds
specifically to the skeletal muscle RYR. Fig. 5also shows that
P-labeled SDCL binds to the purified skeletal RYR,
suggesting a direct interaction between the RYR and
P-labeled SDCL.
P-labeled
SDCL to the skeletal RYR. A nitrocellulose membrane was dot-blotted
with proteoliposomes containing the purified rabbit skeletal muscle RYR (RYR(s), 2 µg), rabbit skeletal muscle SR vesicles (SR(s), 30 µg), and canine cardiac muscle SR
vesicles (SR(c), 30 µg) and overlaid with 5
10
cpm/ml
P-labeled SDCL as described under
``Experimental
Procedures.''
P-labeled SDCL to the purified skeletal muscle RYR by 73%,
52%, 61%, and 53%, respectively (lanes 2-4 and 8). These data are consistent with binding of the peptides to
the same site(s) of the skeletal RYR. However, because of difficulties
in quantitating the binding data over a sufficient range of peptide
concentrations, binding to different sites cannot be ruled out at this
time.
P-labeled SDCL binding to skeletal RYR. Nitrocellulose
membranes were dot-blotted with the reconstituted skeletal muscle RYR
(0.8 µg) and overlaid with 5
10
cpm/ml
P-labeled SDCL in the absence and presence of 20-30
µM each of SDCL, SDCL-P, SDCLS687A, SDCLf1, SDCLf2,
SDCLf3, and CDCL as described under ``Experimental
Procedures.'' Toppanel, autoradiographs of RYR
overlaid with
P-labeled SDCL in the absence and presence
of peptides. Bottompanel, binding of
P-labeled SDCL to RYR. Values were corrected for
background binding (without RYR). Data are the means ± S.D. of
two experiments carried out in triplicate.
Identification of Sequences within SDCL Important for
Activation of the RYR
Three smaller overlapping peptides were
prepared to better define the region of SDCL that activates the RYR (Fig. 7, toppanel). The bottompanel of Fig. 7shows that the N-terminal portion
(SDCLf1, 61 amino acid residues) was as effective as SDCL (126 amino
acids) in increasing [H]ryanodine binding. The
middle portion (SDCLf2) was less effective and the C-terminal portion
(SDCLf3) was without a significant effect. Binding studies showed that
SDCLf1 decreased
P-labeled SDCL binding to the RYR, in
contrast to SDCLf2 and SDCLf3, which were largely ineffective in
inhibiting the binding of the
P-labeled peptide (Fig. 6, lanes5-7). These results
indicated that a small cytoplasmic region of the skeletal muscle DHPR
1 subunit ranging from Glu
to Glu
including Ser
specifically interacts with the
skeletal muscle RYR.
H]ryanodine binding to skeletal SR
vesicles. Toppanel shows amino acid regions of SDCL,
SDCLf1, SDCLf2 and SDCLf3. In bottompanel, SR
vesicles (150 µg of protein/ml) were incubated with 5 nM [
H]ryanodine and 10 µM each of
SDCL, SDCLf1, SDCLf2, or SDCLf3. Data are the means ± S.D. of
four experiments carried out in duplicate. Control value (100%, without
peptides) corresponded to about 10% of the B
value of high affinity [
H]ryanodine
binding.
1,
) of the skeletal muscle L-type Ca
channel (DHPR) have multiple phosphorylation sites for various
protein kinases including cAMP-dependent protein kinase(21) .
Phosphorylation altered L-type Ca
channel activity in
Ca
flux (13, 22) and single channel (14) measurements, and a large, voltage- and frequency-
dependent potentiation of L-type Ca
channel activity
was described in skeletal muscle, which was due to phosphorylation by
cAMP-dependent protein kinase(23) . The relevancy of these
observations regarding the mechanism of E-C coupling is difficult to
assess, because during normal skeletal muscle activity, the SR
Ca
release channel is thought to be opened via a
direct physical interaction with the DHPR, which acts as a
voltage-sensing molecule and not as a Ca
channel(5, 6) . However, morphological evidence (3) and ligand binding measurements (24) have
suggested that in skeletal muscle only a subgroup of RYRs may be
directly linked to DHPRs. This observation has raised the possibility
that Ca
released by DHPR-linked Ca
release channels could serve to amplify SR Ca
release by activating Ca
release channels not
linked to DHPRs. An increase of DHPR/Ca
channel
activity by protein phosphorylation could potentiate
Ca
-dependent SR Ca
release. In the
present study we used a DHPR-derived peptide shown previously to
activate the RYR, to provide novel evidence for an effect of DHPR
phosphorylation on the function of DHPR-linked Ca
release channels.
1 subunit has been estimated by in vitro back-phosphorylation to be 35-40%. This level could be
increased to 83-86% by increasing the intracellular cAMP
concentration(25) . Much of this activity in the predominant
(truncated) form of the DHPR
1 subunit could be localized to two
major phosphopeptides, which both contained
Ser
(12) . A rapid in vitro PKA-mediated
phosphorylation of Ser
also indicated that this
phosphorylation site may have an in vivo function. The present
study confirms that PKA phosphorylates Ser
and
furthermore shows that phosphorylation of Ser
results in
loss of activation of the RYR by SDCL. This loss of activation appears
to be due to the formation of an inactive peptide and not a decrease in
binding affinity.
Ala substitution is a highly
conserved mutation and, in general, is not considered to greatly affect
the structure of the unphosphorylated protein form. However,
replacement of Ser
of SDCL by Ala yielded a peptide
incapable of activating the RYR. This result suggests that the
-OH
of Ser
is essential to form an active SDCL peptide.
Mutagenesis studies with the cardiac loop peptide supported this
conclusion. Although there is no PKA-phosphorylatable Ser in CDCL,
amino acid sequence alignment of SDCL and CDCL reveals a Ser
of CDCL close to Ser
of SDCL. A single point
mutation with Ser
Ala substitution completely
abolished the activating effect of CDCL, suggesting that Ser
is also essential for the formation of an active cardiac loop
peptide. Our results indicate that
-OH of Ser
of
SDCL plays a crucial role in the interaction of the skeletal loop
peptide with the RYR. Phosphorylation of
-OH group of serine or
its replacement by a -H will alter this interaction, resulting in an
inactive peptide.
1 subunit (CDCL) activated the skeletal muscle
RYR(10) . This result was at variance with expression studies
showing that DHPR
1 subunit chimeras containing the skeletal
muscle II-III loop conferred skeletal muscle E-C coupling, whereas
chimeras containing the cardiac II-III loop showed cardiac E-C
coupling(9) . Activation of the skeletal muscle RYR by both
SDCL and CDCL raised the question of the specificity of this activation
in our in vitro studies. This study presents three lines of
evidence that favor a specific binding interaction. First, binding
studies showed that both SDCL and CDCL inhibited the binding of
P-labeled SDCL to the RYR. Second, phosphorylation of SDCL
resulted in formation of an inactive peptide without loss of binding to
the RYR. Third, a 61-amino acid sequence of SDCL was sufficient to
activate the skeletal muscle RYR.
1 subunit activates the skeletal muscle RYR in vitro. A
phosphorylation site in this sequence was found to play a crucial role
in regulating the RYR in vitro. These results suggest that
phosphorylation of Ser
by PKA may be relevant to the
function of the SR Ca
release channel in skeletal
muscle E-C coupling. The absence of a corresponding phosphorylation
site in the II-III loop peptide of the cardiac DHPR
1 subunit
suggests that this regulatory mechanism may be specific to skeletal
muscle.
1 subunit; CDCL, the cardiac muscle isoform
of SDCL; SDCLS687A, site-directed mutant SDCL with Ser
Ala substitution; CDCLS813A, site-directed mutant CDCL
with Ser
Ala substitution; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid; BSA, bovine serum albumin; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; Pipes,
1,4-piperazinediethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.