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INTRODUCTION |
Excitation-contraction
(EC)1 coupling in muscle
cells is the signaling process by which electrical stimuli arriving
at the transverse tubule membrane transmit information to the
sarcoplasmic reticulum (SR) to release intracellular Ca2+
necessary for muscle contraction. Skeletal type and cardiac type EC
coupling differ in their dependence on extracellular Ca2+
entry. Functional interaction between dihydropyridine receptors (DHPRs)
within the transverse tubule and Ca2+ release
channels/ryanodine receptors (RyRs) within SR defines the type of EC
coupling and its underlying mechanism. During cardiac EC coupling, a
small influx of Ca2+ through cardiac DHPRs is required to
open RyR2 (1-5), whereas skeletal type EC coupling does not require
entry of external Ca2+. Instead, membrane depolarization
triggers the opening of RyR1 through a mechanism involving
conformational coupling of skeletal DHPRs (
1s-subunit)
and RyR1 (6-8).
Expression of cDNAs encoding cardiac/skeletal muscle chimeric
1-DHPRs in dysgenic myotubes, which lack endogenous
1s-DHPR, identified the a site within the cytoplasmic
loop between repeats II and III (the cytosolic II-III loop; amino acids
666-791) essential for the physical coupling with RyRs and skeletal EC
coupling (9, 10). Experiments with the full-length skeletal II-III loop
peptide showed specific activation of RyR1 but not RyR2 channels
incorporated into planar lipid bilayers and in radioligand binding
studies with [3H]ryanodine (11, 12). The domain within
the skeletal II-III loop peptide essential for RyR1 activity was
further refined to the region between Arg681 and
Leu690 termed peptide A (pA) (13, 14). Studies of how pA
modifies single channel gating behavior revealed both activating and
inhibitory properties on RyR1 depending on the concentration, the free
cis Ca2+ concentration, and the holding
potential (12, 15). These actions of pA were also found to extend to
RyR2, suggesting a modulatory influence downstream of the skeletal
1s-DHPR/RyR1 interaction (12). However, expression of
1s-DHPR chimeras in dysgenic myotubes has shown that the
pA region of the II-III loop was not essential for engaging skeletal
type EC coupling (16), whereas a 46-amino acid fragment
(Leu720-Gln765) was essential for
bidirectional signaling (9, 17-19). Although the action of pA and
related peptides may not be directly involved in mediating
bidirectional signaling in skeletal EC coupling, they are very useful
in defining basic properties of RyR gating.
The mechanism by which pA alters RyR1 channel function has not yet been
fully defined. Imperatoxin A (IpTxa), a 33-amino acid peptide isolated from the scorpion Pandinus imperator,
was shown to be a high affinity agonist of RyR1 channels (20, 21).
Nanomolar IpTxa increased high affinity
[3H]ryanodine-binding and induced rapid
Ca2+ release from SR vesicles. More recent studies have
shown that IpTxa prolonged the duration of Ca2+
sparks in frog skeletal muscle (22, 23) and significantly increased the
amplitude and the rate of Ca2+ release in developing
skeletal muscle (24). In measurements of single channel currents,
IpTxa stabilized long living subconductance states in
skeletal and cardiac muscle ryanodine receptor channels having 43 and
28% of the native full transition at the holding potential of
40 and
+40 mV, respectively (25). IpTxa was proposed to mimic the
actions of pA based on their similar RyR1-activating properties,
possibly through a common effector site (26). Mutations within
IpTxa and peptide A have indicated that several basic amino acids followed by a hydroxyl-containing amino acid is the critical domain needed for the binding of both peptides to RyR1 (26).
Recently, maurocalcine (MCa), a novel peptide isolated from the venom
of the scorpion Scorpio maurus palmatus, has been found to
possess 82% sequence identity with IpTxa (27, 28). MCa shares the highly basic structural domain identified in peptide A and
IpTxa important for interactions with RyR1. Preliminary work has shown that synthetic MCa (sMCa) activates RyR1 channels by
inducing long living subconductance states (27). Both
IpTxa- and sMCa-modified channels can reversibly transit
between subconductance states and fast gating states. The actions of
these peptides were also found to be additive with those of ryanodine,
resulting in additional substates from the ryanodine-locked
half-conducting state. However, the predominant subconductance induced
by sMCa differs from that of IpTxa. At a holding potential
of +40 mV, the predominant substates induced by sMCa and
IpTxa are 48 and 28% of the full conductance,
respectively, suggesting that the structural difference in these
peptides may induce slightly different channel conformations that
stabilize different unitary conductances (25, 27).
In the present investigation, we define in detail the mechanism by
which sMCa alters RyR1 function and its relationship to the actions of
pA. We find that sMCa and pA stabilize distinct subconductance states
through an interaction with the RyR1 channel complex that cannot be
explained by simple competition at a common effector site. In
combination, the peptides reveal that the sMCa-modified RyR1 channel
gates in stable proportional conductances. Gating in proportional
conductances was also observed in the presence of sMCa and ryanodine
and of sMCa and channels deficient in FKBP12. These results reveal that
RyR1 channels gate in stable proportional conductances in the presence
of allosteric modulators that mediate actions through distinct effector
sites and are consistent with an iris model regulating a single
permeation pore.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]Ryanodine was obtained from
PerkinElmer Life Sciences with specific activity of 57 Ci/mmol and
purity of >90%. Unlabeled ryanodine was purchased from Calbiochem.
Purified natural phospholipids were obtained from Avanti Polar Lipids
(Alabaster, AL) or Northern Lipids (Vancouver, Canada). Peptide A was a
generous gift from Dr. Noriaki Ikemoto (Boston Biomedical Research
Institute, Boston, MA). sMCa was made by the solid phase peptide
synthesis method (27), purified with HPLC and ion exchange
chromatography, and verified with HPLC, amino acid analysis, and mass
spectrometry. The structural authenticity of pA utilized in the present
investigation was confirmed by two methods, mass spectrometry and amino
acid sequence analysis. The peptide was found to have a major peak at
the anticipated molecular weight (Mr = 2330) and
have the amino acid sequence reported for the DHPR II-III loop
(Thr671-Leu690). All other chemicals were
commercially obtained at the highest purity available.
Preparation of Skeletal Muscle SR Membranes--
Membrane
vesicles enriched in RyR1·FKBP12 complex and SERCA pumps were
prepared from rabbit fast twitch skeletal muscle based on the method of
Saito et al. (29). Briefly, freshly ground muscle was
homogenized in ice-cold buffer containing 5 mM
imidazole-HCl, pH 7.4, 300 mM sucrose, 10 µg/ml
leupeptin, and 100 µM phenylmethylsulfonyl fluoride.
Differential centrifugation was performed to obtain a heavy SR
fraction, and junctional SR was collected from the 38-45% (w/w)
interface of a discontinuous sucrose gradient. The junctional SR was
then resuspended to 10-15 mg/ml (30), frozen in liquid N2,
and stored at
80 °C until needed.
Macroscopic Ca2+ Transport
Measurement--
Ca2+ transport across SR vesicles was
measured with the membrane-impermeant Ca2+-sensitive dye,
antipyrylazo III, using a diode array spectrophotometer (model 8452;
Hewlett Packard, Palo Alto, CA). Skeletal SR vesicles (50 µg/ml) were
added to 1.15 ml of ATP-regenerating buffer consisting of 95 mM KCl, 20 mM potassium MOPS, 7.5 mM sodium pyrophosphate (31), 250 µM
antipyrylazo III, 12 µg of creatine phosphokinase, 5 µM
phosphocreatine, and 1 mM MgATP, pH 7.0 (final volume of 1.2 ml). Transport assays were performed at 37 °C in
temperature-controlled cuvettes with constant stirring. SR vesicles
were loaded with sequential additions of CaCl2 that
constituted ~80% of their loading capacity. Net Ca2+
fluxes across SR vesicles were measured by monitoring extravesicular changes in free Ca2+ by subtracting the absorbance of
antipyrylazo III at 710 nm from absorbance at 790 nm at 2-4-s
intervals. The loading capacity of the SR vesicles were determined by
the sequential addition of 24 or 12 nmol of Ca2+ until the
vesicles cannot uptake any Ca2+ or start calcium-induced
calcium release. The maximum amount of Ca2+ accumulated by
SR vesicles defines the loading capacity. At the end of each
experiment, the total intravesicular Ca2+ was determined by
the addition of 3 µM of the Ca2+ ionophore
A23187 and the absorbance signals were calibrated by the addition of 12 or 24 nmol of CaCl2 from a National Bureau of Standards
stock solution. Test compounds were either added before loading
(pretreatment) or after the last addition of Ca2+ was
accumulated by the vesicles.
[3H]Ryanodine Binding Assay--
The specific
binding of [3H]ryanodine to 12 µg of SR protein
was performed with two distinct assay protocols, each with a slight modification of the original method (32, 33). Protocol A used a buffer
composed of 3 nM [3H]ryanodine, 200 mM KCl, 10 mM HEPES, ~7 µM free
Ca2+, pH 7.2, and incubation was performed at 36 °C for
1.5 h. Protocol B used a buffer composed of 1 nM
[3H]ryanodine, 140 mM KCl, 15 mM
NaCl, 50 µM free Ca2+, 20 mM
PIPES, 10% sucrose, pH 7.4, and incubation was at 37 °C for 3 h. Additions of test compounds were made to the radiolabeled assay
buffer, singly or in combination, prior to the addition of SR.
To test whether sMCa and pA interacted in a classic competitive manner
to modulate RyR1 conformation, the complete dose response for sMCa
toward enhancing specific [3H]ryanodine binding was
assessed in the absence and presence of pA (15 or 30 µM)
under the buffer conditions described above.
Separation of bound and free [3H]ryanodine was performed
by filtration through a Whatman GF/B glass fiber filter using a Brandel (Gaithersburg, MD) cell harvester. Filters were washed two times with 3 ml of ice-cold buffer containing 20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, 50 µM
Ca2+, pH 7.1. Filters were then soaked overnight in 5 ml of
scintillation mixture (Ready Safe; Beckman), and bound radioactivity
was determined by scintillation spectrometry. Nonspecific binding was
determined in the presence of a 1000-fold excess of unlabeled
ryanodine. Each experiment was performed in duplicate or triplicate and
repeated at least two times.
BIAcore Analysis of Peptide Interactions with RyR1--
Real
time surface plasmon resonance (SPR) experiments were performed on a
BIAcore biosensor system 1000 at 25 °C. Biotinylated sMCa was
immobilized on the sensor chip surface coated with streptavidin (sensor
chip SA). Purified RyR1 was prepared from heavy SR vesicles solubilized
in the presence of CHAPS (34) and isolated by sucrose density gradient
of the solubilized SR proteins as previously described (35). Before SPR
experiments, purified RyR1 was dialyzed overnight at 4 °C in a
buffer containing 150 mM NaCl, 10 mM HEPES, pH
7.4, 0.005% polysorbate in order to reduce the sucrose and NaCl concentration.
In these conditions, RyR1 has been characterized by an apparent
sedimentation coefficient of 30 S that corresponds to a homotetramer of
~565-kDa subunits (36, 37). sMCa-RyR1 interaction was measured by
injection of purified RyR1 at 2 µg/ml (~1 nM in its
tetrameric form) in a buffer containing 10 mM HEPES, 150 mM NaCl, 2 mM EGTA, 2 mM
CaCl2 (pCa 5), 0.005% polysorbate 20, pH 7.4. Nonspecific binding was measured by injecting RyR1 over a control
surface saturated with biotin instead of biotinylated sMCa. To study
the possible competition of sMCa and pA at a mutually exclusive site, RyR1 was preincubated for 30 min with 20 µM pA prior to
injection onto the biosensor chip containing immobilized sMCa.
Single Channel Recording--
Cs+ current through
single RyR1 channels incorporated into planar bilayer lipid membranes
was measured in an asymmetric CsCl (10:1 or 1:10
cis/trans) solution. The bilayer lipid membrane was formed from a mixture of phosphatidylethanolamine and
phosphatidylcholine (5:2, w/w) at 50 mg/ml in decane, across a
150-300-µm aperture in a 1.0-ml polystyrene cup. SR vesicles were
added to the cis side of the chamber at a final
concentration of 0.1-10 µg/ml. The cis solution contained
500 mM CsCl, 7 µM CaCl2, 20 mM HEPES, pH 7.4, and the trans solution
contained 50 mM CsCl, 7 µM free Ca2+, 20 mM HEPES, pH 7.4. After a single
fusion event, the vesicles are quickly removed by perfusion with 7 volumes of identical buffer without vesicles. Under conditions of a
10:1 (cis/trans) Cs+ gradient, a
holding potential of +30 to +40 mV drives the current from the
cis to the trans chamber. To drive the current
from trans to cis, the CsCl in the cis
solution was lowered to 50 mM by perfusion, and the CsCl in
the trans side was raised to 500 mM by bolus
addition from a 4 M CsCl stock. Measurements of
trans to cis current were made at a holding
potential of
40 mV. Single channel current was measured under voltage
clamp using a Dagan 3900A integrating patch clamp (Dagan Instruments,
Minneapolis, MN). Holding potentials were with respect to the
trans (ground) chamber, and positive current was defined as
current flowing from cis to trans. Current signals were captured at 10 kHz and filtered at 1 kHz using a four-pole
Bessel filter. Data were digitized with a Digidata 1200 interface (Axon
Instruments, Burlingame, CA) and stored on a computer for subsequent
analysis. The experiments were performed at room temperature and were
replicated at least three times. Unless otherwise stated, test
chemicals were sequentially added to the cis solution after
an initial period of recording control channel behavior.
Single channel activity was analyzed with pCLAMP 6.0 (Axon
Instruments). Open events for full conductance and subconductances were
defined as intervals at which the currents exceeded 50% of maximum
open level and the defined subconductance level, respectively. Open
probability (Po) for unmodified channels
(control channels) was calculated from 60-90 s of continuous record
using the PStat program. For peptide A-modified channels, the overall
open probability is calculated from the whole recording time, whereas
the within group open probability is calculated only from the groups of
time points when channels are at rapid gating states. Current
levels were analyzed by mean-variance analysis, and peaks in the all points amplitude histogram were fitted with Gaussian functions. Dwell
open and closed times for relatively fast gating events (duration of <50 ms) were calculated from least-square fits of biexponential function using the PStat software. Arithmetic average time is used for analyzing the long subconductance states induced by
sMCa and the long closed time induced by pA, because there are
not enough events to fit with a reasonable biexponential function.
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RESULTS |
Maurocalcine Induces Ca2+ Release from SR Vesicles by
Activating RyR1--
Fig. 1 compares the
amino acid sequences of MCa, IpTxa, and pA, showing a
common basic domain terminating with an amino acid possessing a
hydroxyl-containing side chain that has been proposed to contribute
essential structure for activating RyR1 (26). It is therefore
conceivable that MCa shares many properties with IpTxa and
pA in the manner in which it modulates RyR1. The present work
therefore elucidates the mechanism(s) by which sMCa and pA alter the
function of SR Ca2+ transport and RyR1 channel
function.
To assess how MCa alters Ca2+ fluxes across skeletal muscle
SR vesicles, macroscopic Ca2+ transport measurements were
performed under pyrophosphate-supported active loading conditions.
After SR vesicles were sequentially loaded with Ca2+ to
~80% of their capacity and extravesicular Ca2+ level
returned to the base line (~150 nM free
Ca2+), sMCa (20 nM) quickly induced
Ca2+ release (Fig.
2A). The addition of ryanodine
receptor inhibitors, ruthenium red (5 µM), or ryanodine
(500 µM) at the plateau of Ca2+ release,
blocked RyR1 channels, and led to Ca2+ reuptake into
vesicles, suggesting that sMCa induced Ca2+ release by
selective activation of RyR1. The actions of sMCa on SR
Ca2+ release were concentration-dependent, with
an EC50 of 17.5 nM (Fig. 2, B and
C). The loading capacity of SR vesicles has been shown to be
determined by a balance between Ca2+ uptake through
ATP-dependent SERCA pumps and Ca2+ release
through ruthenium red-sensitive and -insensitive efflux pathways (38).
Fig. 2D shows that sMCa (20 nM) significantly decreased the loading capacity of SR vesicles, and this effect was
abolished by ruthenium red at a concentration previously shown to
completely block RyR1 in this assay (39). This provides additional evidence that sMCa selectively activates RyR1 with no measurable effects on either leak channels or SERCA pumps.

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Fig. 2.
Maurocalcine activates ryanodine receptor
channels in a concentration-dependent manner.
A, measurements of Ca2+ release across skeletal
muscle SR membrane vesicles were performed as described under
"Experimental Procedures." Vesicles were loaded with the sequential
addition of Ca2+ (60 nmol of Ca2+, three times;
24 nmol of Ca2+, one time) up to ~80% of their loading
capacity. After extravesicular Ca2+ returned to base line,
the addition of 20 nM sMCa induced rapid Ca2+
release. Ryanodine (500 µM, trace
2) and ruthenium red (5 µM, trace 3) added at
the plateau phase of Ca2+ release blocked ryanodine
receptor channels and led to Ca2+ reaccumulation into SR
vesicles, suggesting that sMCa induced Ca2+ release through
RyR1 channels. B, Ca2+ release was induced by 0 (trace 1), 5 nM (trace
2), 10 nM (trace 3), 15 nM (trace 4), 20 nM
(trace 5), and 30 nM
(trace 6) of sMCa. C, summary plot of
initial Ca2+ release rate induced by sMCa. The
EC50 is 17.5 nM. D, the loading
capacity of skeletal muscle SR without (open
bars) and with 20 nM sMCa (filled
bars) was determined in the absence (left) or
presence (right) of 2 µM ruthenium red
(RR). The loading capacity in the absence of sMCa was
considered as 100%. The data were represented as mean ± S.E.
from at least three measurements performed with two different
preparations. Significant level was set as p < 0.05 (Student's t test).
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pA Inhibits Ca2+-, Caffeine-, and sMCa-induced
Ca2+ Release from SR Vesicles--
pA was initially
reported to enhance the binding of [3H]ryanodine to RyR1
in the concentration range of 1-50 µM (13). Here, an
unexpected result was that pA (2-40 µM) failed to induce
Ca2+ release from actively loaded SR membrane vesicles, but
instead
5 µM caused the base line of the dye signal to
gradually decline (Fig. 3A,
traces 3 and 4). This observation
provided the impetus to examine the possible RyR1-inhibitory activity
of pA. Indeed, pA (2-10 µM) introduced into the
transport medium 2 min before bolus addition of 60 µM
Ca2+ dose-dependently inhibited
Ca2+-induced Ca2+ release (Fig. 3A).
pA also inhibited caffeine-induced Ca2+ release in a
concentration-dependent manner (IC50 = 1.5 µM; Fig. 3B). It is worth noting that 5 µM pA completely inhibited caffeine-induced Ca2+ release, in agreement with the recent report in which
micromolar pA was shown to decrease Po of single
channels reconstituted in bilayer lipid membranes (12). However, unlike
the reported activating effect of submicromolar pA on single channel
Po, we did not observe that pA, from
submicromolar to micromolar concentrations (0.1-40 µM),
was able to induce Ca2+ efflux from actively loaded SR.
Additional experiments with a combination of pA and thapsigargin were
performed to determine whether pA enhances Ca2+ release
under passive conditions (in the absence of SERCA-mediated reuptake of
Ca2+). After active Ca2+ loading of the SR
vesicles was complete, pA (0.1-10 µM) failed to enhance
thapsigargin-induced Ca2+ release (not shown). Consistent
with an inhibitory action on RyR1, 5-20 µM pA increased
the loading capacity of SR in a concentration-dependent manner (Fig. 3C), and in support of distinct mechanisms of
action, pA (
10 µM) was found to inhibit sMCa-induced
Ca2+ release in a dose-dependent manner (Fig.
3D).

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Fig. 3.
Peptide A inhibits Ca2+ release
induced by ryanodine receptor activators. A, pA
inhibits Ca2+-induced Ca2+ release
(CICR). Different concentrations of pA (0, trace
1; 2 µM, trace 2; 5 µM, trace 3; 10 µM,
trace 4) were added 2 min before adding 60 µM Ca2+. B, pretreatment with pA
inhibits caffeine (4 mM)-induced Ca2+ release.
The pA concentrations tested were 0 (trace 1), 1 µM (trace 2), 2 µM
(trace 3), and 5 µM
(trace 4). C, pA increased
Ca2+ loading capacity in skeletal muscle SR vesicles in a
concentration-dependent manner. The loading capacity
without pA is considered as 100%. The p value for comparing
the loading capacity in the presence and absence of 5, 10, and 20 µM of pA was 0.002, <0.001, and 0.004, respectively
(Student's t test). D, pA inhibits the
sMCa-induced Ca2+ release. pA (0, trace
1; 2 µM, trace 2; 5 µM, trace 3; 10 µM,
trace 4) was added 2 min before adding sMCa (20 nM).
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Since it is generally agreed that the amount of high affinity
[3H]ryanodine binding measured in radioligand receptor
analysis reflects the degree of channel activity, we examined how sMCa (0.01-300 nM) (Fig.
4A) or peptide A (0.03-50
µM) (Fig. 4B) influenced occupancy. In
accordance with the results obtained from macroscopic Ca2+
transport experiments, sMCa dose-dependently increased
[3H]ryanodine binding with an EC50 of 1.2 nM. By comparison, the dose response exhibited by pA was
bell-shaped, with an EC50 of 266 nM and an
IC50 of 23.5 µM. Considering their sequence
homology, the mechanism by which sMCa and pA interact with RyR1 to
produce seemingly antagonistic effects on SR Ca2+ release
was further investigated using two methodological approaches, [3H]ryanodine binding analysis and real time SPR
analysis. If sMCa and pA compete for one or more common, mutually
exclusive effector sites (i.e. classic competition), then
increasing the concentration of sMCa would be expected to overcome the
inhibition of [3H]ryanodine binding produced by a fixed
concentration of pA. Fig. 5A
shows that the presence of 15 or 20 µM pA reduced the
ability of sMCa to enhance [3H]ryanodine binding by 50 and 100%, respectively, even at a concentration of sMCa nearly
1,000-fold EC50 (1 µM). Thus, indirect
analysis using [3H]ryanodine-binding as an indicator of
channel conformation indicated that, in combination, the peptides did
not exhibit classic competitive interaction at RyR1. The SPR technique
permitted more direct (without the use of [3H]ryanodine)
examination of whether sMCa and pA interact by competing for a common
site on RyR1. SPR sensorgrams revealed that RyR1 failed to interact
with biotin, whereas RyR1 interacted strongly with biotin-sMCa (Fig
5B, traces a and b,
respectively). In consonance with results using
[3H]ryanodine-binding analysis, 1 nM RyR1
oligomer that had been pretreated for 30 min with a great excess of pA
(20 µM) maintained its ability to strongly interact with
immobilized biotin-sMCa (Fig. 5B, trace
c), and the amplitude of the interaction was not significantly lower than control (Fig. 5C, n = 3 separate SPR sensorgrams obtained with and without pretreatment of
RyR1 with pA). Taken together, these results indicate that the
antagonistic actions of sMCa and pA on RyR1 function were not mediated
by simple competitive displacement at a common binding site.

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Fig. 4.
Maurocalcine and peptide A have different
effects on [3H]ryanodine binding. The radioactive
ryanodine binding assays were performed with protocol B as described
under "Experimental Procedures." The experiments were repeated in
two different preparations each in triplicate. Data are presented as
mean ± S.E.
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Fig. 5.
Maurocalcine and peptide A do not interact on
RyR1 in a mutually exclusive, competitive manner. A,
the enhancement of high affinity (1 nM) binding of
[3H]ryanodine to junctional SR membranes by sMCa was
measured in the presence and absence of 15 or 30 µM pA.
sMCa at concentrations nearly 1,000-fold EC50 failed to
overcome inhibition produced by pA. B, SPR sensorgrams of
the interaction of purified RyR1 with biotin (trace
a) or biotin-sMCa (trace b).
Trace c represents the interaction of purified
RyR1, preincubated for 30 min in the presence of 20 µM
pA, with immobilized sMCa. The solid bar
indicates the addition of RyR1 to the sensor. C, amplitude
histograms of interaction signal of purified RyR1 with sMCa in the
absence or presence of 20 µM pA (n = 3 determinations in each experimental condition).
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sMCa and pA Modulate RyR1 Behavior by Stabilizing Distinct
Conducting States--
Macroscopic Ca2+ transport,
radioligand binding, and SPR analyses suggested that sMCa and pA exert
their effects on RyR1 through distinct mechanisms. We more directly
explored whether the two peptides modify channel gating behavior by
interacting at distinct effector sites or with multiple common effector
sites. Based on sequence homology, the latter hypothesis was tested by
determining whether the actions of the two peptides were additive by
analyzing their influence singly and in combination on single RyR1
channels reconstituted into planar lipid bilayers.
Fig. 6A shows 16 s of
continuous record of a native RyR1 channel rapidly gating between
closed and full open states. The addition of sMCa (50 nM) to the cis (cytoplasmic) solution resulted
in the enhancement of channel open probability
(Po from 0.03 to 0.79) by inducing a predominant
long lived subconductance state at 48% of the channel's native
full-open level. Micromolar ryanodine irreversibly locks RyR channels
in a highly stable conformation approximating 50% of the full
conductance that infrequently closes but never opens to the full state
(40, 41). By contrast, sMCa modified the characteristic rapid gating
transitions into a long lived 48% subconductance that frequently
transitioned to periods of fast gating between full open and closed
conformations (Fig. 6B). Reversible association and
dissociation of sMCa from RyR1 probably accounted for transitions
between modified and native gating modes. The amplitude of the
subconductance gating is independent of the concentration of sMCa (5 nM to 1 µM, n = 15). The
major subconductance of 48% was achieved with sMCa at both positive (Fig. 6, A and B) and negative (Fig. 6,
C and D) holding potentials, and the
I/V relationship of the sMCa-modified channel was linear and
did not exhibit rectification of Cs+ current (not shown).
The major subconductance in sMCa-modified channels is not changed by
allosteric modulators of channel gating that stabilize full conductance
openings such as bastadin 10 (42) and ATP. sMCa stabilizes
subconductances with amplitude of 45 and 46% of the full conducting
level in the presence of bastadin 10 (n = 3) and ATP
(n = 3), respectively. The average mean open time of
the sMCa-modified 48% conductance state was >700 ms
(n = 8 channels). On rare occasions, gating transitions
to 29 and 16% subconductances were recorded with the sMCa-modified
channels as previously reported (27). Because of their scarcity,
subconductances below 48% were not analyzed in detail. To further
address reversibility of the sMCa-modified channel, the cis
chamber was perfused with 10 volumes of buffer lacking sMCa (Fig.
7). Removal of sMCa from the
cis chamber quickly restored the fast gating behavior
characteristic of control channels, and reintroduction of the scorpion
peptide immediately induced the long living subconductance states
characteristic of sMCa-modified channels. These results indicate that
the action of sMCa on RyR1 is fully reversible.

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Fig. 6.
Maurocalcine induces long lived substates at
+40 and 40 mV holding potential. 16 s of continuous
measurement of a single channel current without (A) and with
5 nM of sMCa (B) were recorded in the asymmetric
solution using Cs+ as current carrier. The cis
buffer contains 500 mM CsCl, 20 mM HEPES, 50 µM Ca2+, and the trans buffer
contains 50 mM CsCl, 20 mM HEPES, ~7
µM free Ca2+. The holding potential is +35
mV. The closed and full conductance opening levels are labeled as
C and O, respectively, and the subconductance
levels are indicated by the dotted lines between
closed and open states. In separate experiments, 16 s of
continuous measurements of single channel current at a holding
potential of 40 mV were recorded without (C) and with 5 nM of sMCa (D) in cis buffer
containing ~7 µM free Ca2+.
Traces a-d represent 2 s of recordings at
indicated positions from A-D, respectively.
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Fig. 7.
The effect of maurocalcine is fully
reversible. Shown are four recordings from one representative
experiment (n = 4) with one single ryanodine receptor
channel. Single channel currents were recorded at +40 mV as in Fig. 6
with 200 µM Ca2+ in the cis
solutions.
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pA modified the gating properties of RyR1 channels in a manner distinct
from sMCa. pA (0.5-20 µM) induced long lived closed states interspersed with bursts of channel gating activity (Fig. 8). pA decreased the overall
Po of the RyR1 channels by stabilizing very long
closed states (Po from 0.26 to 0.060, n = 10 channels). The average closed time of
events >10 ms was 45 ms for unmodified channels compared with 463 ms
for pA-modified channels. The percentage of time accounted by long
closures (>10 ms) was greatly increased by pA, from 37 to 91%.
However, the mean closed times for pA-modified channels during
the burst of rapid gating activity were not significantly altered
(control
c1 = 0.8 ms, 61%;
c2 = 4.9 ms, 39% versus peptide A-modified
c1 = 0.8 ms, 64%;
c2 = 3.8 ms, 35%; n = 10 channels). Distinct modes of channel gating were observed within a
burst of activity, and the subconductance states were easily defined. pA induced prominent transitions to subconductances having 65% (s1)
and 86% (s2) of the full conductance (Fig. 8B), and these events were summarized in the amplitude histogram shown in Fig. 10A (second panel). Within the active
gating periods, the pA-modified channels exhibited ~3-fold higher
Po compared with the unmodified channel (Fig. 8,
compare A and B). pA-modified channels exhibited longer mean open times (
o1 = 0.73 ms (66%) and
o2 = 3.9 ms (34%)) compared with unmodified channels
(
o1 = 0.46 ms (83%) and
o2 = 2.7 ms
(17%)).

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Fig. 8.
Peptide A induces multiple subconductances
and stabilizes closed states. 16 s of
continuous recording of a single RyR1 channel without (A)
and with 5 µM peptide A (B) shows typical
grouped gating behavior and subconductances (65 and 86% of full
conductance) induced by peptide A. Traces a and
b show 2 s of record expanded from within the indicated
position of A and B, respectively.
Po, overall open probability;
Pburst, open probability within the gating
groups.
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sMCa and pA Modify RyR1 Channels in an Additive Manner--
A
possible molecular interaction between sMCa and pA on RyR1 was further
investigated by sequentially adding both of the peptides to the
cytoplasmic side of reconstituted channels. The addition of 5 µM pA modified the typical rapidly gating RyR1 channel
(Fig. 9, first
trace) to a burst gating mode (Fig. 9, second
trace). In addition to occasional transitions to the full
open state, two prominent subconductances within the gating episodes
were 65% (s1 state) and 86% (s2 state) of full conductance level
(Fig. 8, second trace). The subsequent addition
of 50 nM sMCa induced virtually no observable long lived
openings characteristic of sMCa-modified channels. (Fig. 9,
third trace). Increasing the sMCa to 1 µM (in the presence of pA) induced long lived
subconductance states, indicating that both sMCa and pA can co-occupy
the RyR1 channel complex (Fig. 9, fourth trace).
Most interestingly, the presence of pA and sMCa in combination
stabilized subconductance states that were not seen with either sMCa or
pA alone, indicating the simultaneous binding and additive effects of
pA and sMCa. Specifically, in n = 8 channels,
pA-modified channels demonstrated gating transitions to 65% (s1), 86%
(s2), or 100% of the full chord conductance during a burst of gating
events. Binding of sMCa further modified these transitions to 28, 37, or 43% of the full conductance, respectively, all of which were
proportional to the pA-modified states with a factor of 0.43 (Figs. 9
and 10). The modulatory effect of sMCa
reveals a fundamental property of how allosteric modulators influence
RyR1 channel conductances. Instead of stabilizing a particular
subconductance state, sMCa imposes proportional control of channel
conductance relative to the existing gating transitions. The concept of
proportional gating was further analyzed by defining the ratio between
the conductances of the sMCa-modified states and the counterpart states
without sMCa as the "proportional gating factor" (
). For any
given gating state with conductance S, the binding of sMCa
induces a subconducting state of product
× S
(noted as
S; e.g.
S1,
S2, and
full in Fig. 10). In order to
calculate a proportional gating factor, amplitude histograms were
constructed from local transitions that included gating events in the
presence of pA and in the presence or absence of sMCa. The examples of
those transitions chosen were shown by the solid
lines (a-c) at the bottom of the
fourth panel in Fig. 9. Fig. 10A shows
representative histograms derived from within bursts of gating
activity in the same channel. The addition of sMCa to a pA-modified
channel induced subconductance states that shifted proportionally to
those produced by pA (traces 3-5). The
proportional gating factors were calculated for every transition in
sMCa/pA-modified channels (from n = 8 channels) and
were grouped according to their respective current levels (Fig.
10B). Although there was a reasonable amount of variance in
the calculation because the histograms were derived from a limited
number of events taking place during bursts of activity, the calculated
proportional gating factors for all groups were closely centered at
0.43 for all of the current transitions.

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Fig. 9.
Maurocalcine and peptide A influence RyR1
channel gating in an additive manner. Shown are four recordings of
a representative (n = 8) experiment with the sequential
addition of 5 µM peptide A, 50 nM sMCa, and 1 µM sMCa. Single channel currents were recorded at +35 mV
as in Fig. 5 with 50 µM Ca2+ in the
cis solutions. The additive nature of sMCa and pA on
subconductance behavior indicates that they are binding to distinct
effector sites. The subconductance states induced by further binding of
sMCa are labeled as full and S1, where is the proportional gating factor calculated as the ratio between the
conductance of sMCa-modified and adjacent sMCa-unmodified states. See
the legend to Fig. 10 for more details.
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Fig. 10.
The subconductance level induced by
maurocalcine is dependent on the preexisting gating state.
A, the amplitude histograms were constructed from selected
fragments of single channel recordings to show the subconductances
induced by peptide A only and together with sMCa (1 µM).
The major conductances in peptide A-modified channels are 65% (S1
state), 86% (S2 state), and 100% (full) of the native full
conductance. The subconductance states induced by further binding of
sMCa are labeled as S1, S2, and
full, accordingly. The proportional gating factor ( )
is calculated as the ratio between the conductance of sMCa-modified and
adjacent sMCa-unmodified states. B, summary plot of all the
transitions and their proportional gating factors ( ) grouped
according to their conductance states (n = 8).
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Occasionally, native channels in the absence of pharmacological
modifiers exhibit periods of gating transitioning from the closed state
to a subconductance level (Fig. 11,
first trace), probably due to the loss of FKBP12
from the channel complex during preparation. This provided an
opportunity to test the hypothesis that sMCa also imposes a
proportional gating factor on channels modified by means other than pA.
Once sMCa was added to channels displaying subconductance behavior,
additional subconductances proportional to the original current levels
with a proportional gating factor of 0.45 were easily identified within
the trace (Fig. 10, second and third
panels). This mode of modulation was also demonstrable with
ryanodine-modified channels (27). Once sMCa was introduced to
channels locked in the characteristic ryanodine-modified half-state, it produced additional subconductances of the
ryanodine-modified channel with proportional factors of 0.57 and 0.39 (27). These results demonstrated that sMCa imposed a proportional
gating factor on the native channel conductance, subconductances
stabilized by allosteric ligands, and subconductances induced by
altered protein-protein interactions. Therefore, proportional gating
appears to reflect a universal property of how sMCa modifies channel
gating behavior.

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Fig. 11.
Effects of maurocalcine in native channels
with stable subconductance gatings. A, the native
ryanodine receptor channel has stable subconductance states at 80%
(Sub1) and 60% (Sub2) of its full conductance.
The addition of 5 nM sMCa (middle and
bottom panels) induced subconductances
(e.g. Sub1 and Sub2)
proportional to their current conductance states with the proportional
gating factor at 0.45 (n = 2). B, the
amplitude histograms of the corresponding current traces in
A.
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DISCUSSION |
sMCa and pA Modulate RyR1 Channels through Different
Mechanisms--
Using a macroscopic Ca2+ transport assay,
[3H]ryanodine binding analysis, SPR techniques, and
measurements of single channel currents, we have investigated the
molecular mechanisms by which sMCa and pA modulate RyR1. sMCa is a high
affinity activator of the channels. Like its closely related scorpion
toxin, IpTxa, sMCa rapidly induces Ca2+ release
from SR vesicles through a ruthenium red and ryanodine-sensitive pathway, increases high affinity [3H]ryanodine binding,
and increases channel activity by stabilizing long lived subconductance
states. However, their interactions with RyR1 are not identical, since
the predominant subconductance induced by sMCa is different from that
of IpTxa. At the holding potential of +40 mV, the
predominant substates induced by sMCa and IpTxa are 48 and
28% of the full conductance, respectively. The rectifying effect of
IpTxa is not obvious in sMCa-modified channels, since the
substates induced by sMCa at negative potential also hold 48% of the
native full conducting current. One possible explanation is that in the
present experiments the influence of sMCa on channel behavior was
tested with asymmetric Cs+ as current carrier, whereas
comparable studies with IpTxa were performed with symmetric
K+ (25). However, the structural difference in these
peptides may also account for this minor difference in their function.
pA was initially proposed to mimic the essential site for the physical
coupling between DHPR and RyR (13, 14). It has been shown to enhance
the binding of [3H]ryanodine, elicit Ca2+
release from SR vesicles, and induce long living subconductance of RyR
channels in the concentration range of 1-50 µM. The
actual physiological role of this portion of the II-III loop in EC
coupling, however, is challenged by in situ studies with
skeletal/cardiac muscle DHPR chimeras expressed in dysgenic myotubes.
These studies have identified a different region (amino acid residues
720-765) to be the critical site for DHPR and RyR interaction in
skeletal muscle (9). Also, skeletal type EC coupling was unaffected when an
1s-DHPR with a scrambled sequence corresponding
to pA (residues 681-690) was expressed by dysgenic myotubes (16). Since both active and inhibitory effects of peptide A on RyR channels have been reported, the mechanism of the interaction of peptide A with
RyR1 must be complex (12, 15). In contrast to previous reports by
El-Hayek et al. (13), we exclusively observed the inhibitory
effect of pA. pA did not induce Ca2+ release from SR
vesicles under active or passive assay conditions; rather, it inhibited
Ca2+ release induced either by Ca2+, caffeine,
or sMCa. pA also increased Ca2+ loading capacity in a
concentration-dependent manner. Taken together, these
observations indicate that a prominent action of pA is to inhibit
RyR1-mediated SR Ca2+ release.
Moreover, pA modified single RyR1 channel gating behavior in a manner
distinct from that of sMCa. pA stabilized long closed states of RyR1
and produced characteristic burst gatings separated by very long closed
intervals, consistent with the observations that micromolar pA inhibits
channel activity (12, 15). pA stabilized openings to multiple
subconductances, and the levels were distinct from those produced by
sMCa. In our study, pA was inhibitory to channels with moderate
activities (i.e. Po = 0.05-0.4), whereas pA-induced burst activity may become stimulatory in channels with very low activity (i.e. Po = 0.001-0.006) as reported by other groups. Although the prominent
inhibitory actions of pA raise doubt about its physiological role
within
1s-DHPR in engaging EC coupling as originally suggested, its
unique property of stabilizing multiple subconductance states makes it
a valuable tool for studying the mechanism of action of sMCa. The
behavior of pA-modified single channels provided a plausible
explanation for the bell-shaped dose-response curve observed with
[3H]ryanodine binding analysis. pA increased mean open
time within burst events, a state favorable for
[3H]ryanodine binding. Once bound to high affinity sites,
[3H]ryanodine has been shown to dissociate very slowly
(t1/2 > 30 min) (32). On the other hand, increasing
concentrations of pA decrease the overall activity of ryanodine
receptor channels and can account for the decrease of
[3H]ryanodine binding. The first effect dominates at low
micromolar concentration of pA, and the inhibitory effect becomes
increasingly dominant as concentration increases. The ability of pA to
enhance gating activity of purified RyR1 was recently shown to be
dependent on the presence of FKBP12 (43). These results suggest that
the net effect of pA on RyR1 gating not only depends on the
concentration of pA but also on the conformational state of RyR1
defined by the interaction of RyR1 with other SR proteins.
sMCa Imposes a Proportional Gating Factor on Channel
Conductances--
sMCa reversibly associates with RyR1 to stabilize
long living subconductance states. In this paper, we identify that
toxin-modified RyR1 exhibits proportional gating behavior that is
additive to preexisting channel gating behavior, regardless of the
predominating conductance or how it was achieved (e.g.
dissociation of FKBP12 or pharmacological modification with ryanodine
or pA). Ligand-induced subconductance gating of RyR has been proposed
to be mediated either by conformational alterations or partial
occlusions of the conduction pathway (44). For example,
nanomolar ryanodine has been proposed to produce a long lived 50%
conductance conformation and was interpreted as predominantly the
result of partial occlusion, whereas higher concentrations fully
occlude a single conductance pore. In the present study, we found that
sMCa predominantly stabilizes a gating transition to 48% of the full
conductance amplitude, and unlike ryanodine, the current level does not
decrease with increasing sMCa concentration. The actions of sMCa can be
best explained by a model that stabilizes RyR1 conformation by an
allosteric mechanism that indirectly limits the conduction pore rather
than by direct occlusion of the conduction pore by the scorpion
peptide. In support of this interpretation, the relative amplitude of
subconductance gating induced by sMCa is virtually unchanged under both
positive and negative membrane potential. Considering the high sequence homology and functional similarity between IpTxa and sMCa,
it is highly likely that both peptides bind to the same effector site
on RyR1. IpTxa was shown by cryoelectron microscopy to bind to a cytoplasmic region far from the center of the conducting vestibule
of RyR1, further supporting an allosteric mechanism for the toxins
(46).
The three-dimensional structure of RyR1 determined by cryoelectron
microscopy has revealed a mushroom-shaped structure consisting of a
large square cytoplasmic domain with four peripheral clamp-shaped domains (domains 5-10 as defined by Radermacher et al.
(47)) connected by "handle" domains (domain 3) (45, 47, 48). The transmembrane (TM) region composed of four subdomains (each from one
subunit of RyR) forms the "stem" of the mushroom-shaped structure of the channel. Studies comparing open RyR1 modified by
AMP-PCP/Ca2+ (46) or ryanodine/Ca2+ (48) with
RyR1 in the closed state have revealed long range conformational
changes in the TM domain and opening of a central pore induced by these
activating reagents. The TM domain in the open state was found to be
twisted counterclockwise by ~4°, possibly by shifting four TM
subdomains in an iris-like manner (48). The proportional gating of RyR
induced by sMCa is consistent with the central pore model and an
iris-like mechanism. IpTxa was shown to bind (one toxin
molecule per channel subunit) to a cytoplasmic cavity between domain 3 and domain 7/8 (47), which is presumably also the binding site for
sMCa. Domain 3 and domain 7/8 are connected to the central conducting
vestibule through short "bridges." Based on this structural
information, occupation of the toxin sites may transmit long range
conformational changes via a flexible bridging region that introduces a
constriction onto the conduction pathway. In this manner, binding of
sMCa to any one of the four RyR1 subunits can modify the conformational
change of all four subunits.
The combined effect of sMCa and pA on channel conductance is the simple
product of the individual effect of each peptide alone, suggesting that
each peptide can independently occupy distinct class of effector sites
on RyR1 and exert its effect additively. Based on the high sequence
homology between MCa and IpTxa, it is likely that these
scorpion peptides bind to overlapping sites within the clamp region.
The exact locations of regions of RyR1 that recognize pA remain to be
elucidated. Biochemical data from [3H]ryanodine-binding
analysis and measurements of SPR presented here strongly suggest that
sMCa and pA can occupy binding sites on RyR1 in tandem and that their
interaction does not follow simple competition at a single class of
mutually exclusive sites. Although the purified RyR1 utilized in SPR
experiments is likely to be in its tetrameric form, the experiments
aimed at assessing competition used a low concentration of RyR1 (1 nM) and a great excess of pA (20 µM) that is
150 times the previously reported IC50 for interaction of
RyR1 and pA in solution (43). Under these assay conditions, it is
therefore reasonable to assume that RyR1 is saturated with peptide
regardless of its oligomeric form. Despite the saturating levels of pA,
SPR indicated no diminution in the ability of RyR1 to interact with
immobilized sMCa. However, we cannot totally discount possible
allosterism between the sMCa and pA binding sites among the four
symmetrical clamp regions of the oligomer, since higher concentrations
of sMCa are needed to induce RyR1 long lived subconductance states in
the presence of pA (Fig. 9). Moreover, the inhibitory actions of pA
clearly dominate over the activating actions of sMCa. Most likely, this is the result of the long-lived channel closures induced by pA. The
most plausible explanation is that sMCa and pA limit the
counterclockwise twist of RyR1 (i.e. to less than the 4°
for the full open state predicted by results from cryoreconstruction)
(45) through their interactions with distinct effector sites, thereby
limiting the final conductance of the channel (Fig.
12). Consistent with this interpretation, the proportional gating factor imposed by sMCa was
constant in the presence and absence of diverse channel modulators that
both do not change the unitary conductance of the channel such as
bastadin 10 (42) and ATP and do promote subconductances (e.g. FKBP12-deficient channels). Even ryanodine-modified
chan nels, which are thought to undergo profound conformational changes within the conducting pathway, are subject to proportional gating upon
binding of sMCa. In conclusion, the proportional gating imposed by
sMCa-bound RyR1 appears to be additive with the subconductances favored
in the presence of pA and reflects a common mechanism by which peptides
interacting with the clamp domains can allosterically limit channel
conductance, and it is consistent with an iris model of a single
conduction pore for RyR1.

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Fig. 12.
Conceptual model of the actions of
maurocalcine and peptide A in modifying RyR1 gating. Four TM
subdomains (one per subunit of RyR1) represented by four
leaves in the model are orientated to close the conducting
pathway of RyR1 in the absence of activating reagents. Channel
activators like Ca2+, ATP, and bastadin 10 stabilize an
open conformation with four TM subdomains twisted ~4° from the
closed state in an iris-like manner as proposed by Serysheva
et al. (45). sMCa imposes a proportional gating
factor on RyR1 channels by binding to the hinge region between domain 3 and domain 7/8 and allosterically limits (indicated by the
dashed arrow) the degree of twist of four TM
subdomains, thereby stabilizing the subconductance gating. pA probably
interacts at distinct sites, although not necessarily within the clamp
domains, such that the actions of sMCa and pA are additive.
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