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INTRODUCTION |
In cardiac and skeletal muscle, the dihydropyridine receptor
(DHPR)1 of the external
membrane and the Ca2+ release channel/ryanodine receptor
(RyR) of sarcoplasmic reticulum (SR) are key components of
excitation-contraction (E-C) coupling, the series of events that link
an electrical stimulus (depolarization) to a mechanical contraction
(1). Skeletal and cardiac muscle express different subtypes of DHPR and
RyR, which account for different E-C coupling mechanisms. In the heart,
a small influx of Ca2+ through DHPRs triggers the opening
of RyRs (2). In skeletal muscle, however, external Ca2+ is
not required for Ca2+ release (3). Contractions are instead
triggered by membrane depolarizations, and because Ca2+
release may be arrested immediately upon repolarization, a mechanical coupling between the DHPR and the RyR is thought to mediate E-C coupling (4, 5).
Compelling evidence indicates that the skeletal DHPR subtype is
indispensable to elicit a Ca2+-independent (skeletal-type)
contraction (6, 7) and that the 138-amino acid cytoplasmic loop between
repeats II and III of the
1 subunit participates in this
process (6, 8). In experiments with isolated peptides, the II-III loop
activates purified RyRs (9), and a small fragment of the II-III loop (Thr671-Leu690) induces Ca2+
release from SR vesicles (10). In dysgenic myotubes, skeletal-type E-C
coupling is partially restored by a chimeric DHPR that is entirely
cardiac except for a short segment of skeletal II-III loop
(Phe725-Pro742) (8). Although apparently
contradictory in the identity of the activating region, these results
suggest that specific domains of the II-III loop directly interact with
the RyR to change its conformational state and produce Ca2+
release. Therefore, in skeletal muscle, the II-III loop stands as the
strongest candidate among regions of the DHPR to bind to RyRs. However,
the precise amino acid residues of the II-III loop that trigger
Ca2+ release remain unknown. Furthermore, other DHPR
segments (11) or subunits (12) have not been discarded as points of contact.
We have previously shown that Imperatoxin A (IpTxa), a
33-amino acid peptide from the scorpion Pandinus imperator,
is a high-affinity activator of RyRs (13, 14). The biological
significance of IpTxa is unknown, because the apparent
target for this membrane-impermeable peptide is located
intracellularly. Because some peptide toxins activate intracellular
signaling pathways by mimicking surface receptors (15, 16), we tested
the hypothesis that IpTxa activates RyRs by mimicking a
domain of the DHPR that is critical to trigger Ca2+
release. We found that IpTxa and a synthetic peptide with
an amino acid sequence corresponding to a segment of the II-III loop (Glu666-Leu690) (10) activate RyRs in a similar
manner and appear to compete for a common binding site on the channel
protein. Both peptides bind to RyRs via a structural domain consisting
of a cluster of basic amino acids
(Arg681-Lys685 of the II-III loop and
Lys19-Arg24 of IpTxa) followed by a
hydroxylated amino acid (Ser687 of the II-III loop and
Thr27 of IpTxa). Thus, IpTxa
presents an interesting case of toxin mimicry of effector proteins that
may be used to identify regions of the RyR that trigger
Ca2+ release. If the peptide segment emulated by
IpTxa is an actual participant in the DHPR/RyR interaction,
IpTxa may also be exploited to identify regions of the RyR
involved in E-C coupling.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]Ryanodine (60-80 Ci/mmol) was
from NEN Life Science Products, agelenin and Tx2-9 were from The
Peptide Institute, Inc. (Osaka, Japan), bovine brain
phosphatidylethanolamine and phosphatidylserine were from Avanti Polar
Lipids (Birmingham, AL), and Fmoc-amino acids were from Applied
Biosystems. Polyclonal skeletal RyR antibody was from Upstate
Biotechnology. Peroxidase-conjugated secondary antibody and
-conotoxin were from Calbiochem. The chemiluminescence detection kit
was from Boehringer Mannheim. Pre-cast linear gradient polyacrylamide
gels were from Bio-Rad. All other reagents were of high-purity reagent grade.
[3H]Ryanodine and
125I-IpTxa Binding
Assay--
[3H]Ryanodine (7 nM) was
incubated for 90 min at 36 °C with 40-50 µg of rabbit skeletal SR
vesicles in medium containing 0.2 M KCl, 10 µM CaCl2, and 10 mM Na-Hepes (pH
7.2) in the absence and presence of peptides. Free ligand and bound
ligand were separated by rapid filtration on Whatman GF/B glass fiber
filters, as described previously (13, 14). Native IpTxa
(10-µg batches) was purified according to established procedures (13,
14) and iodinated to a specific activity of 60-80 Ci/mmol with the
Bolton-Hunter© method following the specifications of the manufacturer
(New England Nuclear). The binding of
125I-IpTxa to skeletal SR and Chinese hamster
ovary (CHO) cell homogenates was performed under conditions identical
to those described for [3H]ryanodine, except that the
protein concentration was 0.1-0.2 mg/ml in the case of CHO cells.
Bmax and KD of the
125I-IpTxa-receptor complex were obtained by
fitting data points with the following equation: B = Bmax × 125I-IpTxa/(KD + 125I-IpTxa), where B is the specific
binding of 125I-IpTxa.
Transfection of CHO Cells with RyR--
CHO cells were
transfected by lipofection with plasmid pRRS11, the rabbit skeletal
muscle RyR (RyR1), as described previously (17). Expression of the RyR
was confirmed by immunoblot analysis using monoclonal antibodies
against the skeletal RyR and by [3H]ryanodine binding.
Control and transfected cells were homogenized in 500 mM
sucrose, 1 mM EGTA, and 10 mM Hepes-Tris (pH
7.4) and spun at 44,000 × g for 30 min (17). The
pellet was recovered and used for [3H]ryanodine binding experiments.
Synthesis of Peptides--
Linear analogs of IpTxa
and the II-III loop were synthesized by the solid-phase methodology
with Fmoc amino acids in an automated peptide synthesizer and subjected
to the same cyclization and HPLC purification method as described
previously (14). Analytical HPLC, amino acid analysis, and mass
spectrometry confirmed the structure and the purity of the synthetic
peptides. Photoactivatable IpTxa was prepared by inserting
p-benzoyl-phenylalanine, a photoactivatable cross-linker
(18), in place of Leu7 during the synthesis of
IpTxa. Photoactivatable IpTxa was subjected to
the same cyclization and HPLC purification method as described for
synthetic IpTxa (14).
Planar Bilayer Recording of RyRs--
Recording of single RyR in
lipid bilayers was performed as described previously (13, 19). Single
channel data were collected at steady voltages (+30 mV) for 2-5 min in
symmetrical 300 mM cesium methanesulfonate, 10 µM CaCl2, and 10 mM Na-Hepes (pH
7.2). IpTxa and the II-III loop peptide were added to the
cis chamber, which corresponded to the cytosolic side of the
channel (13, 19). The addition of the peptides to the trans
(luminal) side of the channel was without effect. In some experiments,
we added 10 mM CaCl2 to the trans
solution. At 0 mV, Ca2+ was the only charge carrier in
these experiments, and both peptides were effective in inducing a
subconductance state of about one-fourth of the full conductance level.
However, the low signal:noise ratio obtained under these conditions
made the analysis of the kinetic effect difficult. For the experiments
presented here, we omitted Ca2+ in the trans
solution. Signals were filtered with an 8-pole low pass Bessel filter
at 2 kHz and digitized at 5 kHz. Data acquisition and analysis were
done with Axon Instruments software and hardware (pClamp v6.0.2,
Digidata 200 AD/DA interface), as described previously (13, 19). The
current values for the full and subconductance states were obtained
from Gaussian fits to the all point amplitude histograms, as described
previously (19).
Purification of SR Vesicles and Determination of Ca2+
Release--
SR vesicles were purified from rabbit white fast skeletal
muscle, as described previously (13, 19). Maximal
[3H]ryanodine binding site density was typically 3-5
pmol/mg protein. Ca2+ release from SR vesicles was measured
by the method of Palade (20), with slight modifications. Briefly, SR
vesicles (60 µg of protein in 20 µl of reaction medium) were placed
in a cuvette containing 980 µl of 95 mM KCl, 20 mM K-MOPS (pH 7.0), 7.5 mM sodium
pyrophosphate, 250 µM Antipyrylazo III, 1.5 mM MgATP, 25 µg of creatine phosphokinase, and 5 mM phosphocreatine. The mixture was allowed to equilibrate
for 3 min at 37 °C under constant stirring. Free Ca2+
was monitored by measuring A710-790 nm using a
diode array spectrophotometer (Hewlett-Packard Model 8452A). Vesicles were actively filled by three to five consecutive additions of 10 nmol
of CaCl2 before the addition of IpTxa or the
II-III loop peptide. The total amount of Ca2+ loaded was
quantified by the addition of 5 µM of the
Ca2+ ionophore A23187 at the end of each experiment.
Cross-Linking of Photoactivatable IpTxa,
SDS-Polyacrylamide Gel Electrophoresis, and Western Blot Analysis of
RyR--
SR microsomes (0.4 mg/ml) were incubated with 30 nM photoactivatable IpTxa (see above) in buffer
containing 10 µM free Ca2+, 200 mM KCl, and 10 mM Na-Hepes (pH 7.2) in the
absence and the presence of 50 µM IpTxa.
After 60 min at 36 °C, 1-ml aliquots were spread over 1-cm-diameter
plastic wells and irradiated at short range with ultraviolet light (360 nm) for 30 min. Samples were washed twice with incubation buffer by
centrifugation in a table-top minifuge at 12,000 rpm. Pellets were then
resuspended in Laemmli buffer (0.25 M Tris, pH 6.8, 0.4 M dithiothreitol, 8% SDS, 40% glycerol, and 0.04%
bromphenol blue) and subjected to SDS-polyacrylamide gel
electrophoresis on two identical linear gradient acrylamide gels
(4-12%). Proteins contained in one gel were stained with Coomassie
Blue, whereas proteins in the other gel were transferred to
nitrocellulose membranes for Western blot analysis. Blots were probed
first with a rabbit polyclonal RyR1 antibody (dilution, 1:3,000) and
then with an anti-rabbit peroxidase-conjugated secondary antibody.
Dried gels were then exposed to x-ray film for 2 days.
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RESULTS |
Selective Activation of [3H]Ryanodine Binding by
IpTxa among Ca2+ Channel Toxins--
The amino
acid sequence of IpTxa (14) exhibits no significant
homology with the well-characterized Na+ and K+
channel scorpion toxins (data not shown). However, IpTxa
does share 45% and 42% sequence identity with agelenin (21) and Tx2-9 (22), respectively, two spider toxins that block presynaptic (P-type)
Ca2+ channels (Fig.
1A). The Cys residues, which
stabilize the three-dimensional structure by forming disulfide bridges
(16), are similarly arranged in these three peptides (gray
boxes). Indeed, they may be used as a frame to align the amino
acid sequence of
-conotoxin MVIIC, a snail peptide that blocks
P-type Ca2+ channels (23), and to reveal regions of
homology (open boxes). Fig. 1B shows that,
despite the demonstrated structural kinship among these peptides, only
IpTxa is capable of enhancing [3H]ryanodine
binding. ED50, the concentration of IpTxa
required to produce a half-maximal effect (6.4 ± 3.1 nM, mean ± S.D.; n = 18), is only
slightly higher than that exhibited by [3H]ryanodine
among ligands of RyRs (24). This selective and high-affinity effect
suggests that IpTxa possesses a unique structural motif that activates RyRs, which is not present even in structurally related
peptides.

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Fig. 1.
A, regions of homology between
IpTxa and other Ca2+ channel-blocking
peptides. The amino acid sequences of -conotoxin MVIIC (23),
agelenin (21), IpTxa (14), and Tx2-9 (22) were aligned
using the Cys residues, which are critical to maintain the
three-dimensional structure of the peptides. Gaps (-) have been
introduced to maximize homology. B, selective effect of
IpTxa on [3H]ryanodine binding. The binding
of [3H]ryanodine (7 nM) to skeletal SR
vesicles (30-40 µg) was conducted in 0.2 M KCl, 10 µM CaCl2, and 10 mM Na-Hepes (pH
7.2) for 90 min at 36 °C. Control binding (100%) was defined as the
specific binding of [3H]ryanodine in the absence of
peptide toxins and corresponded to 0.71 ± 0.06 pmol/mg protein.
Data are the mean ± S.D. of n = 16 (IpTxa) and an average of n = 3 (other
peptides).
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Physical Interaction of IpTxa with RyRs--
To test
whether IpTxa may be used independently as a high-affinity,
specific ligand for RyRs, we radiolabeled IpTxa and
conducted binding experiments in the absence of ryanodine. Fig.
2A shows that the radiolabeled
derivative of IpTxa retained high affinity (KD = 11 ± 3 nM) and bound to
skeletal SR with a maximal receptor site density
(Bmax) of 16.1 ± 1.9 pmol/mg protein
(n = 3). In the same tissue, the
Bmax for [3H]ryanodine was
3.7 ± 0.6 pmol/mg protein. Thus, assuming all 125I-IpTxa binding occurs to the RyR, the
125I-IpTxa:[3H]ryanodine binding
site stoichiometry is 4.3:1. Because one [3H]ryanodine
molecule binds with high affinity to the tetrameric RyR (24), this
ratio suggests that about four IpTxa molecules bind to
every RyR tetramer. In CHO cells transfected with the skeletal RyR
(Fig. 2B, + RyR1), the
125I-IpTxa:[3H]ryanodine binding
site stoichiometry is 4.6:1 (n = 2). In naïve CHO cells, there is neither 125I-IpTxa (Fig.
2B, Untransfected) nor
[3H]ryanodine binding (data not shown).

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Fig. 2.
IpTxa binds directly and with
high affinity to skeletal RyR. Specific binding of
125I-IpTxa to (A) skeletal SR or to
(B) CHO cells transfected with the skeletal RyR (+ RyR1) or to CHO cells without gene transfection
(Untransfected). The binding of
125I-IpTxa to skeletal SR was carried out
exactly as described for [3H]ryanodine. In the case of
CHO cells, protein concentration was 10-20 µg. Results are from one
of three (skeletal SR) or two (CHO cells) similar experiments performed
in duplicate. Experimental error was 10%.
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To confirm that IpTxa physically interacts with the RyR
monomer in skeletal SR, we prepared photoactivatable IpTxa,
a synthetic derivative of IpTxa in which Leu7
was replaced by the light-sensitive cross-linker
p-benzoyl-phenylalanine (18). The photoreactive derivative
retained high affinity for the RyR (KD = 12 ± 4 nM; n = 3; data not shown). Fig. 3A shows a SDS-polyacrylamide
gel electrophoresis profile of SR proteins that were radiated with
ultraviolet light after incubation with 30 nM
photoactivatable 125I-IpTxa in the absence
(lane 1) and the presence (lane 2) of 50 µM unlabeled IpTxa. An immunoblot analysis
using a skeletal RyR polyclonal antibody recognized only the high
molecular weight band of SR proteins (Fig. 3B). The
autoradiogram of the SDS-gel (Fig. 3C) shows clear labeling
of the band corresponding to the RyR (lane 1). Other bands
are also labeled, most likely from a nonspecific interaction with the
toxin, because labeling persists in the presence of excess
IpTxa (lane 2). Together with data from Fig. 2,
these results indicate that IpTxa makes direct
protein-protein interactions with the RyR with a stoichiometry of four
IpTxa molecules per single RyR channel.

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Fig. 3.
Covalent labeling of the
125I-IpTxa receptor. A,
photoactivatable 125I-IpTxa (30 nM)
was incubated with 50 µg of SR vesicles for 60 min at 36 °C in the
absence (lane 1) and the presence (lane 2) of 50 µM IpTxa. Vesicles were illuminated at short
range with ultraviolet light (360 nm) for 30 min, washed twice by
centrifugation, and resuspended in sample buffer for SDS-polyacrylamide
gel electrophoresis in a 4-12% polyacrylamide gel. B, the
identity of the RyR was investigated by Western blot analysis using a
rabbit polyclonal antibody against the skeletal RyR (Upstate
Biotechnology). Unlabeled SR proteins were run in parallel to those
shown in A and transferred to nitrocellulose membranes as
described under "Experimental Procedures." C,
autoradiography of the gel in A. T.D., tracking
dye. Results are representative of n = 3 experiments
(cross-linking) or n = 4 experiments (Western
blots).
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Functional Effect of IpTxa and a Short Segment of the
II-III Loop--
The functional effect of the IpTxa-RyR
interaction was tested in planar lipid bilayer and Ca2+
release experiments. Fig. 4A
shows that 50 nM IpTxa added to the cytoplasmic
(cis) side of the skeletal RyR induced the appearance of a
subconductance state corresponding to ~25% of the full conductance as previously shown (25). Although of small amplitude, the
subconductance state displayed a mean open time that was >100-fold
longer than that of unmodified channels. Ion flow would therefore be
expected to be greater for an IpTxa-modified channel,
despite its lower conductance. Fig. 4B shows that
IpTxa elicited Ca2+ release from actively
loaded SR vesicles in a dose-dependent manner. The effect
of IpTxa was blocked by ruthenium red, consistent with
Ca2+ release occurring through RyRs. Strikingly similar
results were observed with a 25-amino acid synthetic peptide with
primary sequence (Glu666-Leu690; Fig.
5A) overlapping that of
peptide A (Thr671-Leu690), a segment of the
II-III loop that activates RyRs (10). The 25-amino acid segment of the
II-III loop (henceforth termed "the II-III loop peptide"), like
IpTxa, induced the appearance of a small-amplitude and
long-lifetime subconductance state (Fig. 4C) and elicited
Ca2+ release from SR (Fig. 4D). Thus, albeit
with different affinity, the two apparently unrelated peptides exhibit
similar functional effects on RyRs.

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Fig. 4.
Functional effect of IpTxa and
the II-III loop on skeletal RyRs. Rabbit skeletal RyR channels
were reconstituted in planar lipid bilayers (11) and activated by 10 µM (cis) cytosolic Ca2+ in the
absence (Control) and ~1 min after the addition of 50 nM IpTxa (A) or 3 µM
II-III loop (B) to the cis side. The peptides
induced the appearance of subconductance states of long lifetime
(arrows). Mean open times ( ), which were calculated from
open time histograms, were 0.7 ± 0.1 ms (83%) and 4.2 ± 1.4 ms (17%) in control; 0.8 ± 0.1 ms (63%), 3.8 ± 1.4 ms
(21%), and 319 ± 58 ms (17%, substate) after IpTxa
(n = 3); and 0.9 ± 0.2 ms (73%), 3.6 ± 1.5 ms (19%), and 243 ± 34 ms (8%, substate) after II-III loop
peptide (n = 3). The addition of peptides to the
(trans) luminal side of the channel was without effect.
Holding potential was +30 mV for all traces. c, closed;
o, open. Ca2+ release from actively loaded SR
vesicles by IpTxa (B) or the II-III loop peptide
(D) was measured with the spectrophotometric
Ca2+ indicator Antipyrylazo III, as described previously
(20). Arrows indicate the addition of 10 nmol of
Ca2+ to the 1-ml reaction medium. Thapsigargin (1 µM) was added simultaneously with the peptides to block
Ca2+ uptake by the SR. When equilibrium was reached, the
Ca2+ ionophore A23187 (5 µM) was added to
assess SR Ca2+ content (asterisks).
Calibration bars, 60 s (horizontal) and 5 µM Ca2+ (vertical).
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Fig. 5.
Amino acid sequence and structural domains of
IpTxa and the II-III loop peptide. A, complete
amino acid sequence of IpTxa and the segment of the II-III
loop used in this study. No regions of homology were observed, except
for a cluster of basic amino acids (boxes) followed by Ser
or Thr (ovals). B, simplified scheme showing the
proposed structural analogy between IpTxa and the II-III
loop. Vertical lines represent regions of the peptides
without significant homology. Rectangles correspond to the
cluster of basic amino acids (Lys19-Arg24 and
Arg681-Lys685, respectively), and
ovals correspond to the hydroxylated amino acid
(Thr26 and Ser687, respectively).
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Structural Analogy between IpTxa and the II-III Loop
Peptide--
A one-to-one residue alignment between IpTxa
and the II-III loop peptide does not reveal significant homology in
their amino acid sequence (Fig. 5A). However, both
IpTxa and the II-III loop peptide display a structural
motif consisting of a cluster of basic amino acids (boxes,
Lys19-Arg24 and
Arg681-Lys685, respectively) followed by Thr or
Ser, two hydroxylated amino acids (ovals, Thr26
and Ser687, respectively). In IpTxa, the
cluster of basic amino acids is interrupted by Cys21 and
encompasses the sequence KCK, which is also found in agelenin and Tx2-9
(Fig. 1A). Therefore, it is likely that the KCK sequence alone does not suffice to activate RyRs and that Cys21
stabilizes the peptide structure without intervening in protein-protein interactions with the RyR. Hydroxylated amino acids in a position close
to Thr26 of IpTxa are also found in agelenin
(Ser28) and in Tx2-9 (Thr23 and
Thr24), but none is preceded by a cluster of basic amino
acids. Likewise, the motif RRG, which appears in IpTxa and
-conotoxin MVIIC, is only followed by a hydroxylated amino acid in
the former peptide. Indeed, similar to Ser687 of the II-III
loop (27), the distinctive arrangement of Thr26 of
IpTxa with the preceding residues produces a
phosphorylation consensus for several protein kinases (28). As
presented in Fig. 5B, the structural motif consisting of a
cluster of positively charged amino acids followed by a hydroxylated
residue is not found in other peptide toxins or in other regions of the
DHPR, including the
-subunit. Thus, IpTxa and the II-III
loop peptide share a specific arrangement of amino acid residues that
may be responsible for their similar functional effect on RyR channels.
Competition Experiments between IpTxa and the II-III
Loop Peptide--
To test whether the analogous functional effects
produced by IpTxa and the II-III loop peptide (Fig. 4)
result from activation of the same modulatory site on the RyR, we
carried out competition experiments between the two peptides. Fig.
6A shows that the II-III loop
peptide incrementally decreases the capacity of IpTxa to activate RyRs. The ED50 for II-III loop-inhibition of the
IpTxa effect was 1.3 ± 0.7 µM
(n = 3), in agreement with the value calculated from
direct activation of [3H]ryanodine binding by the II-III
loop peptide (Fig. 7B). In
contrast, a scrambled II-III loop (a synthetic peptide with amino acid
composition identical to the II-III loop peptide but in random
sequence) was incapable of stimulating [3H]ryanodine
binding (data not shown) or of abolishing the IpTxa effect
(Fig. 6B). Thus, the effects of the II-III loop peptide require a defined amino acid sequence and are unrelated to peptide mass
or electrical charge. In other competition studies, the II-III loop
peptide displaced the binding of 125I-IpTxa to
SR vesicles with an ED50 of 36 ± 4 µM
(Fig. 5C). This reduced affinity may be due to displacement
of 125I-IpTxa from sites of nonequivalent
affinity, or it may result from positive allosteric interaction between
the II-III loop peptide and 125I-IpTxa.
Nevertheless, the II-III loop-125I-IpTxa
competition appears to be specific, because the scrambled II-III loop
had no significant effect at concentrations up to 300 µM
(Fig. 6C).

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Fig. 6.
Activation of RyRs by IpTxa and
competition with the II-III loop peptide. A, inhibition of
IpTxa effect by the II-III loop peptide.
[3H]Ryanodine (7 nM) was incubated with
skeletal SR in the presence of IpTxa alone
(Control) or IpTxa plus the indicated
concentrations of the II-III loop. For each curve, 100% represents the
specific binding of [3H]ryanodine in the absence of
IpTxa. Data points were fitted with the following equation:
B = Bmax/1 + (KD/[IpTxa]). B, binding of
[3H]ryanodine was measured in the presence of
IpTxa alone (Control) or IpTxa plus
the indicated concentrations of the peptide RLKSQKMRATGKAERARSEK
(scrambled II-III loop). C, displacement of
125I-IpTxa binding by the II-III loop. The
binding of 125I-IpTxa (30 nM) to
skeletal SR was carried out exactly as described for
[3H]ryanodine binding. Nonspecific binding was determined
in the presence of 50 µM IpTxa and subtracted
from each data point. All data points are the mean of three independent
determinations performed in duplicate. Experimental error was 5%
(A and B) and 10% (C).
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Fig. 7.
Parallel mutations in IpTxa and
the II-III loop peptide lead to analogous effects. A and
B, effect of substituting Thr or Ser. Specific binding of
[3H]ryanodine to skeletal SR was measured in the absence
(100%) or in the presence of (A) native or synthetic
IpTxa ( and , respectively) or derivatives T26A or
T26E or (B) the II-III loop peptide (B,
Control) or derivatives S687A and S687E. Results are the
mean ± S.D. of n = 4-6 independent
experiments.
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Effect of Mutations in IpTxa and the II-III Loop
Peptide--
If, by analogy with other peptide toxin-ion channel
associations (29, 30), the high-affinity IpTxa-RyR
interaction is mediated by electrostatic forces, then mutations in the
binding domain of IpTxa should alter its electrostatic
potential and the measured affinity constant of the
IpTxa-RyR complex. Likewise, if IpTxa and the
II-III loop peptide bind to the skeletal RyR via a common structural
motif, then corresponding mutations should evoke parallel changes in
affinity for both peptides. Fig. 7A shows that synthetic
IpTxa (a synthetic peptide with an amino acid sequence
identical to that of native IpTxa; Ref. 14) activates [3H]ryanodine binding to skeletal SR with potency and
affinity identical to native IpTxa (5 ± 3 nM; n = 6). Other synthetic derivatives of
IpTxa in which Thr26 was replaced with Ala
(T26A) or with the negatively charged residue Glu (T26E) increased
[3H]ryanodine binding with an affinity 12- and 160-fold
lower (ED50 = 60 ± 5 and 800 ± 78 nM nM, respectively). Mutations to the II-III loop peptide elicited qualitatively similar results (Fig.
7B). The replacement of Ser687 with Ala (S687A)
or with Glu (S687E) decreased the affinity of the II-III loop peptide
from 0.81 ± 0.2 µM (control) to 4.2 ± 1.1 and
22 ± 4 µM, respectively. Therefore, substituting
Thr or Ser of IpTxa and the II-III loop peptide with
nonpolar or charged amino acids has a substantial impact on the ability
of both peptides to activate RyRs. However, neither mutation results in
a complete loss of peptide activity.
Mutations within the cluster of basic amino acids elicit more dramatic
effects. In IpTxa, replacing Arg23 with Glu
(R23E) abolished IpTxa stimulation of
[3H]ryanodine binding (Fig.
8A). The corresponding
substitution in the II-III loop peptide, Arg684 to Glu
(R684E), also abolished its effect on [3H]ryanodine
binding (Fig. 8B). In contrast, replacing either
Lys8 of IpTxa or the comparable amino acid
Lys675 of the II-III loop peptide with Glu (K8E and K675E,
respectively) yielded ED50 values of 19 ± 4 nM and 1.5 ± 0.3 µM, respectively. Thus, the dramatic effect of mutations within the cluster of basic amino acids cannot be solely attributed to a change of electrical charge because mutations distant to the cluster but producing the same
electrical change have relatively minor effects.

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Fig. 8.
Effect of substitutions within
the cluster of basic amino acids. Specific
[3H]ryanodine binding to skeletal SR was measured in the
absence (100%) or the presence of IpTxa derivatives K8E
and R23E (A) or the II-III loop derivatives K675E and R684E
(B). Results are the mean ± S.D. of n = 4 independent experiments.
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DISCUSSION |
The molecular mechanism by which depolarization of the skeletal
T-tubule membrane induces Ca2+ release from the SR remains
an outstanding problem in E-C coupling, with a physical DHPR/RyR
interaction being the most plausible hypothesis. Whereas the
identification of the RyR region(s) involved in this interaction is in
progress (31, 32), there is already substantial evidence to invoke the
participation of the II-III loop of the
1 subunit of the
DHPR (6, 8-10). As discussed below, there is still controversy
regarding the precise structural domain(s) of the II-III loop involved
in this interaction. In this study, we identified a unique structural
motif involved in activation of RyRs in IpTxa and in a
short segment of the II-III loop. Although the participation of this
structural motif in E-C coupling awaits further testing, our results
provide a structural framework for a mechanical model in which specific
amino acids of the II-III loop are capable of interacting with RyRs and
triggering Ca2+ release.
Analysis of the amino acid sequence of IpTxa and the effect
of mutations strongly suggest that the structural motif encompassing Lys19-Arg24, the cluster of basic amino acids,
followed by Thr26, the hydroxylated amino acid, is involved
in IpTxa binding to the RyR. Our results, however, do not
allow us to ascertain the weight of individual amino acids within this
structural domain for participation in binding. For instance, agelenin,
Tx2-9, and
-conotoxin, three structurally related peptides that are
incapable of increasing [3H]ryanodine binding (Fig. 1),
bear resemblance to IpTxa in several regions, except in the
proposed structural domain. However, agelenin and Tx2-9 do maintain the
KCK motif, and thus the actual involvement of these residues in the
binding of IpTxa remains undetermined. Either the KCK motif
is independent of the binding site or, in analogy to other peptide
toxins (29, 30), it may be involved in IpTxa docking
without participating in activation. The pair of Arg residues following
the KCK motif is clearly unique to IpTxa. As shown in Fig.
8, replacing Arg23 with Glu (R23E) totally abolished the
capacity of IpTxa to activate RyRs. Because a similar
substitution in a region distant to this cluster (K8E; Fig. 8) was
without major functional consequences, the lack of effect of R23E was
probably the result of local changes in the electrostatic potential of
the toxin's binding site, rather than global conformational changes.
Lastly, the involvement of Thr26 was demonstrated by
hydrophobic (T26A) and charged (T26E) substitutions (Fig. 5). Although
the mutated peptides retained their ability to activate RyRs, there was
a significant loss of affinity for both peptides.
Replacing equivalent amino acids in the II-III loop peptide produced
results that were qualitatively similar to those obtained with
IpTxa mutants (Figs. 7 and 8). These data, plus the
competition experiments in which the II-III loop peptide inhibited the
effect of IpTxa (Fig. 6A) and displaced the
binding of 125I-IpTxa (Fig. 6C),
strongly suggest that both peptides activate RyRs by a physical
interaction with the channel protein via the aforementioned structural
domain. If this structural domain of the II-III loop is important for
the activation of RyR in vivo, then IpTxa is a
peptide mimetic of an effector protein, and its structure-activity
information may have direct implications for the mechanism of E-C
coupling in skeletal muscle. In RyRs reconstituted in lipid bilayers,
IpTxa and the II-III loop peptide induced the appearance of
a subconductance state, again supporting the notion that both peptides
lead to the same conformational changes in the channel protein.
The small-amplitude, long-lifetime subconductance state produced by
IpTxa and the II-III loop peptide was seen in
Cs+-conducting (Figs. 4 and 5) and in
Ca2+-conducting RyRs (data not shown; see Ref. 25) and is
clearly different from that produced by FK506 and ryanodine. FK506 and other immunosupressants that strip RyRs of the accessory protein FKBP12
produce rapidly fluctuating subconductance states that represent
approximately one-half and one-fourth of the full conductance state
(33, 19). Ryanodine irreversibly locks the channel in a subconductance
state of variable amplitude, depending on the charge carrier (34).
However, the effects of IpTxa and the II-III loop peptide
are also different from those produced by the whole 138-amino acid
II-III loop, which activates RyRs by increasing the frequency of full
openings without inducing the appearance of a subconductance state (9,
27). These results argue against the II-III loop fragment studied here
being an exact functional equivalent of the whole II-III loop. They
suggest instead that the II-III loop-RyR interaction is more complex,
perhaps encompassing several points of contact that, when acting
concertedly, modify the RyR kinetics in a way that is more similar to
the activation produced in vivo. In skeletal muscle cells,
the DHPR-RyR interaction involves orthograde (DHPR
RyR) as well as
retrograde (RyR
DHPR) signals (32). Thus, providing that the II-III
loop peptide studied here is an actual participant in the E-C coupling
mechanism, this fragment and IpTxa may be regarded as
partial effectors of the DHPR-RyR interaction whose critical task is to
relay the signal that initiates Ca2+ release.
Using synthetic peptides corresponding to small segments of the II-III
loop, El-Hayek et al. (10) found that only the
amino-terminal portion (peptide A,
Thr671-Leu690) was capable of activating RyRs.
More recently, the same group (35) reduced the length of the activator
peptide to 10 residues (Arg681-Leu690) that
encompassed the structural domain studied here. Interestingly, a
pentapeptide corresponding to the cluster of basic amino acids was
insufficient to activate RyRs (35), and our own results indicate that a
scrambled II-III loop peptide is also without effect (data not shown).
Therefore, the distinctive distribution of the positively charged
residues and their relation to the hydroxylated amino acid are critical
determinants of the structural domain that binds and activates RyRs.
Lu et al. (27) found that phosphorylation of
Ser687 of the skeletal II-III loop (or substitution by Ala)
abolished the capacity of the whole loop to activate RyRs. Leong and
MacLennan (36) used the II-III loop in an affinity column and found
that replacement of Lys677 or Lys682 with Glu
decreased the capacity of the II-III loop to interact with peptide
fragments of the skeletal RyR. These results are in line with the
hypothesis that the amino-terminal region of the II-III loop binds to
and activates the skeletal RyR. However, expressing chimeras of cardiac
and skeletal DHPR in dysgenic myotubes, Nakai et al. (8)
found that Phe725-Pro742 was the most important
region of the skeletal DHPR to elicit a skeletal-type contraction. This
region is located almost at the middle of the II-III loop, more than 30 residues downstream from peptide A. Although the results with DHPR
chimeras are apparently contradictory to those obtained with isolated
peptides, it is still possible that the structural domain of peptide A
postulated here as being important for activation of RyRs participates
in the transmission signal from DHPR to RyR. A potential scenario would
be that Phe725-Pro742, which determines
skeletal-type E-C coupling (8), binds to the skeletal RyR at rest to
inhibit Ca2+ release and that depolarization of the
T-membrane rearranges the II-III loop such that peptide A interacts
with and activates the RyR. In agreement with this hypothesis, El-Hayek
et al. (10) found that the activating effect of peptide A
was antagonized by Glu724-Pro760, a fragment of
the skeletal II-III loop encompassing the amino acid sequence
identified by Nakai et al. (8). However, even in this
simplified scenario, outstanding issues remain unsolved. For example,
skeletal-type contractions do not require entry of external
Ca2+, whereas activation of RyRs by peptide A (10),
IpTxa (13), or the whole II-III loop (9) requires
Ca2+, at least at suboptimal levels. Again, this may
reflect the fact that these peptides are only partial effectors of the
transmission signal, with other segments of the DHPR conferring
Ca2+ independence to the DHPR-RyR interaction.
Toxin mimicry of surface receptors is not unprecedented. Mastoparan, a
peptide from wasp venom, is a potent secretagogue that mimics an
intracellular loop of G protein-coupled receptors (15). As previously
shown (16), the capacity of foreign peptides to activate intracellular
signals may yield insights into the molecular mechanisms of signal
transduction in the target cells. In the case of IpTxa, the
high affinity of 125I-IpTxa demonstrates a
direct protein-protein interaction with the RyR and reveals a ~4:1
stoichiometry of IpTxa/RyR binding sites (Fig. 2). Assuming
that IpTxa is a surrogate high-affinity ligand for the
II-III domain that triggers Ca2+ release, these results
suggest that in the intact muscle, up to four II-III loops directly
gate one RyR. This would be consistent with the current structural
model of E-C coupling in skeletal muscle (26), where every other foot
protein (RyR) faces a tetrad of T-tubule particles representing four
DHPR molecules. Another inference based on the above assumption is that
the II-III loop but not the carboxyl terminus of the
1
subunit (11) or the
-subunit (12) is structurally endowed to trigger
Ca2+ release. This is because the structural domain
identified as being responsible for activating RyRs (Fig. 4) is
contained within the II-III loop sequence only. However, as mentioned
above, our results do not rule out the possibility of multiple sites of
interaction between the DHPR and the RyR because inhibitory sites of
contact have been detected in other segments of the
1
subunit (11) and even within the II-III loop itself (10).
Regardless of its potential use as a peptide probe to study the
molecular determinants of E-C coupling, it is important to keep in mind
that IpTxa triggers conformational changes in the channel
protein that ultimately lead to Ca2+ release (Fig. 4). The
specificity, high affinity, and reversibility of the
IpTxa-RyR interaction may thus be exploited to identify amino acids of the RyR directly responsible for channel opening.