Effects of dihydropyridine receptor II-III loop
peptides on Ca2+ release in skinned skeletal
muscle fibers
Graham D.
Lamb1,
Roque
El-Hayek2,
Noriaki
Ikemoto2, and
D. George
Stephenson1
1 School of Zoology, La Trobe University, Bundoora, Victoria
3083, Australia; and 2 Boston Biomedical Research Institute,
Boston, Massachusetts 02114
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ABSTRACT |
In
skeletal muscle fibers, the intracellular loop between domains II and
III of the
1-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca2+ release
channel in the sarcoplasmic reticulum. We examined the effects of
synthetic peptide segments of this loop on Ca2+ release in
mechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg2+ concentration ([Mg2+]; 1 mM), a
20-residue skeletal muscle DHPR peptide
[AS(20);
Thr671-Leu690; 30 µM], shown previously to
induce Ca2+ release in a triad preparation, caused only
small spontaneous force responses in ~40% of fibers, although it
potentiated responses to depolarization and caffeine in all fibers. The
COOH-terminal half of AS(20)
[AS(10)] induced much larger spontaneous
responses but also caused substantial inhibition of Ca2+
release to both depolarization and caffeine. Both peptides induced or
potentiated Ca2+ release even when the voltage sensors were
inactivated, indicating direct action on the Ca2+ release
channels. The corresponding 20-residue cardiac DHPR peptide [AC(20);
Thr793-Ala812] was ineffective, but its
COOH-terminal half [AC(10)] had effects similar to AS(20). In the presence of lower
[Mg2+] (0.2 mM), exposure to either
AS(20) or AC(10) (30 µM) induced substantial Ca2+ release. Peptide
CS (100 µM), a loop segment reported to inhibit Ca2+ release in triads, caused partial inhibition of
depolarization-induced Ca2+ release. In toad fibers, each
of the A peptides had effects similar to or greater than those in rat
fibers. These findings suggest that the A and C regions of the skeletal
DHPR II-III loop may have important roles in vivo.
excitation-contraction coupling; ryanodine receptor
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INTRODUCTION |
IN VERTEBRATE
SKELETAL MUSCLE, depolarization of the transverse-tubular (T-)
system triggers Ca2+ release from the sarcoplasmic
reticulum (SR) and subsequent contraction, without the need for inflow
of extracellular Ca2+. It is currently thought that the
dihydropyridine receptors (DHPRs) in the T-system membrane act as
voltage sensors, sensing T-system depolarization and then by some
protein-protein interaction activating adjacent ryanodine receptor
(RyR)/Ca2+ release channels in the apposing SR membrane
(21). The skeletal muscle DHPR is an oligomeric complex of
five subunits. The
1-subunit, which contains the
dihydropyridine binding site, evidently plays a crucial role in the
coupling with the RyR, as skeletal muscle-type coupling is absent in
dysgenic mouse muscle, which lacks the
1-subunit, but
can be restored by expression of that protein (34).
Additionally, it appears that the intracellular loop between repeats II
and III of the skeletal DHPR is essential for skeletal-type
excitation-contraction (E-C) coupling, because such coupling is lost if
the skeletal loop is replaced with the homologous segment of the
cardiac DHPR (33).
Lu et al. (16, 17) showed that the skeletal and cardiac
II-III loop peptides could each activate isolated skeletal RyRs and
increase ryanodine binding to skeletal but not cardiac RyRs. It was
subsequently found (3) that the stimulatory region of the
skeletal DHPR loop could be further localized to the sequence Thr671-Leu690, called peptide A. It has been
since shown that peptide A (at 1-30 µM) increases the open
probability of isolated RyRs and that it can also cause
voltage-dependent block of the channels (2). Recently, it
has been found that the COOH-terminal half of peptide A [called
AS(10)] is considerably more potent than
peptide A at inducing Ca2+ release and stimulating
ryanodine binding in skeletal muscle SR vesicles, with maximal
stimulation at ~20 µM and inhibition at higher concentrations
(4). It should be noted, however, that these experiments
showing the activating effect of the peptide A region were performed at
a free Mg2+ concentration ([Mg2+]) of
0.2-0.3 mM rather than at the physiological level of ~1 mM, and
such a lowered [Mg2+] may have augmented the stimulatory
effect of the peptide (12, 14, 19, 31). Furthermore,
experiments with chimeric DHPRs in dysgenic muscle indicate that a
quite different region of the skeletal II-III loop
(Phe725-Pro742) is crucial for skeletal-type
E-C coupling (22). Nevertheless, when a peptide
corresponding to this latter region was applied to isolated triads, it
did not induce any Ca2+ release, and it was found that
the region Glu724-Pro760 (called
peptide C) actually inhibited both peptide A-induced and
depolarization-induced Ca2+ release (3, 28).
Thus, from the studies to date, it is unclear which region(s) of the
DHPR II-III loop actually activate the RyRs in vivo.
Here we examine the effects of the skeletal and cardiac DHPRs II-III
loop peptides in mechanically skinned fibers, where voltage-sensor control of Ca2+ release is retained (11, 13),
and with important factors such as myoplasmic [Mg2+],
[ATP], and the SR Ca2+ load all maintained at
physiological levels. In this way we could investigate which peptides
are capable of inducing Ca2+ release in mammalian muscle
fibers under conditions close to those in vivo, which is an important
test of their proposed role in normal E-C coupling. Because only every
alternate RyR is associated with a DHPR tetrad in mammalian
skeletal muscle (5, 21), we hypothesize that exogenously
added loop peptides would have relatively free access to the key sites
on at least one-half of the RyRs in a skinned fiber and consequently
might activate Ca2+ release. (Here we assume that the
absence of an adjacent DHPR would leave room for the exogenous loop
peptide to bind to the normal coupling site on the RyR.) With this
skinned fiber technique, we are also able to test whether any of the
peptides interfere with normal E-C coupling, which is important for
assessing whether a peptide has an inhibitory action in vivo or
alternatively competes in some way with the normal coupling mechanism.
Finally, we can also examine the effects of the peptides in toad
skeletal muscle fibers, where there are two types of RyRs,
and
,
homologous to the principal mammalian skeletal RyR (RyR1) and the
smooth muscle brain RyR (RyR3), respectively, with the
-isoform
thought to be linked to the DHPRs (21, 23). The DHPR
II-III loop in amphibian skeletal muscle is very similar to that in
mammalian muscle, and the arrangement of charged residues in both the A and C regions is in fact identical in mammals and amphibia
(35). Thus it was of interest to see what effect the
mammalian skeletal II-III loop peptides have in amphibian fibers.
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METHODS |
Skinned fibers.
Mechanically skinned fibers were obtained and used as described
previously (10, 13). Briefly, Long Evans hooded rats were anesthetized with halothane (2% vol/vol) in a bell jar and killed by
asphyxiation, and then the extensor digitorum longus (EDL) or soleus
muscles were removed. Cane toads (Bufo marinus) were stunned
by a blow to the head and killed by pithing, and the iliofibularis muscles were removed. Single muscle fibers were mechanically skinned under paraffin oil, and a segment was attached to a force transducer (AME875; Horten, Norway) at 120% of resting length. The skinned fiber
was then placed within a 2-ml Perspex bath containing a potassium
hexamethylene-diamine-tetraacetate (K+-HDTA) solution (see
below) for 2 min to allow the sealed T-system to become normally
polarized, before being stimulated by rapid substitution of another
bath with an appropriate solution. Fibers were used with their
endogenous level of SR Ca2+ and were not additionally
loaded, except where indicated. All experiments were performed at room
temperature (23 ± 2°C).
Solutions.
All chemicals were obtained from Sigma, unless specified otherwise. The
K+-HDTA solutions used for rat and toad fibers contained
(in mM): 117 (toad) or 126 (rat) K+; 37 Na+; 50 HDTA2
(Fluka, Buchs, Switzerland); 8 total ATP; 8.6 total
magnesium; 10 creatine phosphate; 0.05 total EGTA; 60 (toad) or 90 (rat) HEPES; 1 N3
; pH 7.10 ± 0.01 and pCa (=
log10 [Ca2+]) 7.0. Skinned fibers were
depolarized by substitution of an Na+-HDTA solution, which
was identical to the corresponding K+-HDTA solution, except
that all K+ was replaced with Na+. The above
solutions all contained 1 mM free Mg2+ and had an
osmolality of 295 ± 5 (rat) or 255 ± 5 (toad)
mosmol/kgH2O. Solutions with a free [Mg2+] of
0.05 mM (toad) or 0.015 mM (rat) that were used to directly activate
the Ca2+ release channel were similar to the standard
K+-HDTA solution, except that they contained only 2.15 or
1.0 mM total Mg2+, respectively. The solution with 0.2 mM
free Mg2+ was made by appropriate mixture of the low and
normal [Mg2+] solutions. The loading solution used during
sequences of depolarization-induced responses was made by adding
Ca2+ to the standard K+-HDTA solution (50 µM
total EGTA) to give a pCa of 6.0; such a solution could be washed out
within 1 or 2 s, unlike loading solutions with heavier
Ca2+ buffering (e.g., 1 mM total EGTA; see below) that
require longer washout. Maximum Ca2+-activated force was
determined using a solution ("max") similar to standard
K+-HDTA solution, but with 50 mM Ca2+-EGTA (20 µM free Ca2+) replacing all HDTA and the total
Mg2+ reduced to 8.12 mM to keep the free
[Mg2+] at 1 mM (32). The Ca2+
sensitivity of the contractile apparatus was determined by exposing the
fiber to a sequence of solutions of progressively higher
[Ca2+] made by appropriate mixture of the 50 mM
Ca2+-EGTA solution and a similar solution with 50 mM free
EGTA (9, 32). Free [Ca2+] of solutions was
measured with a Ca2+-sensitive electrode (Orion Research,
Boston, MA).
Repeated Ca2+ load-release cycles
for low [Mg2+]- and caffeine-induced
release.
The Ca2+ content of the SR could be estimated from the size
of the force response when the SR was fully and rapidly depleted by
exposing the fiber to a low [Mg2+] K+-HDTA
solution (0.015 mM Mg2+) with 30 mM caffeine and 0.5 mM
free EGTA (pCa 8) present to chelate the released Ca2+
(25). (As the caffeine-low [Mg2+] solution
potently triggers Ca2+ release, the fiber was first
preequilibrated for 10 s in the standard K+-HDTA
solution with 0.5 mM EGTA to ensure EGTA was present within the fiber.)
Because fibers were skinned under paraffin oil, the SR initially
contained its normal endogenous level of Ca2+. After
depletion, the SR was reloaded with Ca2+ by exposure to the
standard K+-HDTA solution with 1 mM total EGTA at pCa 6.7, and then it could be subsequently depleted again. As found previously
(25), the force response obtained from such load/release
cycles was highly reproducible, and the time integral (i.e.,
"area") of the force response was approximately linearly related to
loading time (until saturating with prolonged loads), indicating that
it was indicative of the amount of Ca2+ loaded into the SR.
In the experiments here, the EDL fibers were reloaded with
Ca2+ to approximately the original endogenous level by
20 s loading under the above conditions.
To examine the ability of the peptides to trigger Ca2+
release at 0.2 mM Mg2+ (see Fig. 2), each EDL fiber was
fully depleted of Ca2+ and reloaded as above (with the
loading terminated by a 2-s exposure to a K+-HDTA solution
with 0.5 mM free EGTA) and then 1) equilibrated for 20 s in the standard K+-HDTA solution (1 mM Mg2+,
50 µM EGTA, pCa 7.0) with or without peptide, 2) exposed
for 15 s to the 0.2 mM Mg2+ solution (50 µM EGTA,
pCa 7.0) with or without peptide, and 3) once again fully
depleted of Ca2+ with the 30 mM caffeine-low
[Mg2+] solution ("full release" in Fig. 2). When
examining the effect of the peptides on caffeine-induced
Ca2+ release at set SR loading levels, the sequence was the
same as above, except that the fiber was exposed to a solution with 7 mM caffeine (at 1 mM Mg2+) instead of the 0.2 mM
Mg2+ solution. (In some experiments, the effect of caffeine
was examined without depleting and loading the SR to a set level, e.g.,
as in Fig. 3B.) Where noted, similar experiments were also
carried out with all solutions made from Na+-HDTA instead
of K+-HDTA, to ensure the T-system was chronically
depolarized and hence the voltage sensors were kept inactivated.
Peptides.
Peptides were synthesized on an Applied Biosystems model 431A
synthesizer employing N-(9-fluorenyl)methoxycarbonyl as the
-amino protecting group. Peptides were cleaved and deprotected with
95% trifluoroacetic acid. Purification was carried out by reverse-phase high-pressure liquid chromatography using a Rainin Instruments preparative C8 column; all peptides were purified to
homogeneity. It is unlikely that there was any substantial heterogeneity in the secondary structure of a given peptide type, because each peptide was only 10 or 20 residues long. The peptide regions of the DHPR
1-subunit II-III loop (Fig.
1) are classified as A or C in
conformity with previous usage (3), with subscripts s and
c denoting the rabbit skeletal and cardiac muscle DHPR, respectively,
and the number of amino acids indicating the full length A peptide
[i.e., AS(20) or
AC(20)] or the 10-amino acid segment at the
COOH-terminal end [i.e., AS(10) or
AC(10)]. Peptides were dissolved in
double-distilled water at 100-1,000 times the highest final
concentration. Tests with a Ca2+-sensitive electrode showed
no detectable Ca2+ contamination associated with the
peptides.

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Fig. 1.
Amino acid sequence of the various synthetic peptides encompassing
different regions of the II-III loop of the 1-subunit of
the skeletal and cardiac dihydropyridine receptors (DHPRs; denoted with
subscripts S and C, respectively), as well as decalysine (see
METHODS). The charges on the amino acids at neutral pH are
indicated.
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Force traces and analysis.
In force traces, unless otherwise indicated, the skinned muscle fiber
was bathed in the standard K+-HDTA solution (1 mM
Mg2+, 50 µM total EGTA, pCa 7.0). In the text, mean
values are given ± SE. Statistical probability (P) was
determined with Student's t-test or the nonparametric
Mann-Whitney test when the data were apparently not normally
distributed, with P < 0.05 considered significant.
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RESULTS |
Effect of A peptides in skinned fibers.
Previous experiments (3, 4) showing that peptides from the
A region of the skeletal muscle DHPR II-III loop induced rapid
Ca2+ release in triad preparations were performed in the
presence of ~0.2-0.3 mM free Mg2+. We first
sought to test whether the peptides were effective in inducing
Ca2+ release in mechanically skinned fibers under such
conditions before examining their effect at physiological
[Mg2+] (~1 mM). Because it was necessary to have the SR
loaded with Ca2+ to the same extent when examining each of
a number of successive treatments, each fiber was first emptied of its
endogenous SR Ca2+ and then subjected to repeated cycles in
which it was reloaded to a set level (close to the endogenous level),
exposed to a test treatment, and then fully depleted of
Ca2+ again (see METHODS). Lowering the
[Mg2+] from the standard level of 1 mM to 0.2 mM never
induced a force response in any rat EDL fiber before exposure to a
peptide (at least 2 repetitions in each fiber). In contrast, lowering
the [Mg2+] to 0.2 mM after 20 s preequilibration in
30 µM AS(20) [previously (3)
called peptide A] induced a substantial force response in every fiber
examined (mean 40.3% of maximum Ca2+-activated force;
Table 1). This stimulatory effect
of AS(20) was not readily reversed by washout
of the peptide (e.g., Fig. 2A
and Table 1), even over several load/release cycles, indicating that
some peptide continued to act on the stimulatory site on the release
channels. Similar results were found in two other fibers when the
voltage sensors were kept inactivated by having all solutions
Na+ based [response at 0.2 mM Mg2+ in
AS(20): 77% and 25% of maximum force in
those fibers]. Peptide AC(20), the matching
segment of the cardiac DHPR II-III loop (Fig. 1), did not cause any
response at 0.2 mM Mg2+ (Table 1), but its COOH-terminal
end [AC(10), 30 µM] triggered substantial
Ca2+ release, with its effect being more easily reversed by
washout than found with AS(20) (e.g., Fig.
2B).

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Fig. 2.
Peptides AS(20) and
AC(10) induce Ca2+ release in
mechanically skinned mammalian fibers at 0.2 mM Mg2+.
A: skinned extensor digitorum longus (EDL) fiber of the rat
was subjected to repeated load-release cycles in which the sarcoplasmic
reticulum (SR) was loaded with Ca2+ to a set level (close
to the endogenous level) and then the fiber exposed to a solution with
0.2 mM free Mg2+ with or without peptide
AS(20), before inducing release of the
remaining SR Ca2+ (full release) with a 30 mM caffeine-low
[Mg2+]-0.5 mM EGTA solution (see METHODS).
There was never any force response at 0.2 mM Mg2+ before
exposure to AS(20) (e.g., left),
but a large response was induced in presence of 30 µM
AS(20) (e.g., middle), and the
effect of the peptide could not be readily reversed by washout (e.g.,
right). B: exposure to 30 µM
AC(10) had an effect similar to
AS(20), but this was more readily reversed by
washout. C: there was no force response at 0.2 mM
Mg2+ in the presence of 30 µM
AS(10), but importantly the response on
subsequently depleting the SR with 30 mM caffeine (full release) was
markedly reduced, showing that the peptide was directly blocking the
release channels. After washout of AS(10), the
responsiveness to low [Mg2+] was heightened, indicating
persistence of the activating effect of the peptide. Different EDL
fibers in A, B, and C. Time scale: 2 s
throughout, except during wash period between 0.2 mM Mg2+
and full release, where it is 30 s. Maximum
Ca2+-activated force was ascertained in each fiber with a
heavily buffered Ca2+ solution with 20 µM free
Ca2+ (max).
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In contrast to the case with AS(20), there was
no significant force response at 0.2 mM Mg2+ in the
presence of 30 µM AS(10), the COOH-terminal
end of peptide AS(20) (Fig. 1; e.g., Fig.
2C and Table 1). However, when trying to trigger full
release of SR Ca2+ (with the 30 mM caffeine-low
[Mg2+] solution) straight afterward, the response was
invariably much smaller than would have been expected considering the
lack of Ca2+ release in the peptide (e.g., Fig. 2C,
n = 6), and this almost certainly indicates that the
peptide was blocking the release channels to some extent. Furthermore,
when the treatment cycle was subsequently repeated in the absence of
the peptide, the response to the full release solution was invariably
much larger, and in three of the six cases there was also an
appreciable response in 0.2 mM Mg2+ (e.g., Fig.
2C). The most straightforward explanation of these data is
that AS(10) stimulates Ca2+
release at 0.2 mM Mg2+ but that this effect is countered by
an additional blocking action of the peptide on the release channels
(see Effects of AC(20) and 10 amino acid peptides at
1 mM
Mg2+);
on washout, the blocking action is more easily reversed, revealing the
persistent activating effect of the peptide. A similar, though much
smaller, inhibitory effect may also occur with peptide
AS(20), because in some fibers that rate of
rise of the force response in 0.2 mM Mg2+ was even faster
after washout of the peptide than it was in the presence of the peptide
(e.g., Fig. 2A). In summary, peptides AS(20), AS(10), and
AC(10) [but not
AC(20)] all evidently stimulated Ca2+ release at 0.2 mM Mg2+, with
AS(10) also having a potent blocking effect.
Thus these data indicate that the A peptides have
Ca2+-releasing effects in skinned muscle fibers, in accord
with their reported effects in isolated triads (3, 4).
Effect of peptide AS(20) on
depolarization-induced responses in skinned fibers at 1 mM
Mg2+.
As described previously in both rat EDL fibers and toad iliofibularis
fibers (10-13), the E-C coupling mechanism is
retained in mechanically skinned fibers and is quite functional at the physiological intracellular [Mg2+] of 1 mM. The skinned
fibers retain the endogenous level of SR Ca2+ and require
no additional loading. The T-system seals over on skinning and
repolarizes if the fiber segment is bathed in a high [K+]
solution, and can then be rapidly depolarized by substituting a
solution in which all K+ is replaced with Na+
(see METHODS). Such stimulation elicits a rapidly rising
force response, reaching close to the Ca2+-activated
maximum, and lasting for only 1-3 s owing to inactivation of the
voltage sensors and reuptake of released Ca2+ into the SR
(10, 11, 13) (e.g., Fig. 3).
If after each depolarization the fiber is repolarized in the
K+ solution for
30 s to reprime the voltage sensors, many
similar depolarization-induced responses (~15 in toad fibers and
20-30 in rat EDL fibers) can be elicited before the fiber shows
substantial rundown. The time course, voltage dependence of activation
and inactivation, and modulation by pharmacological agents (e.g., D600)
(10-13) all strongly indicate that the responses in
these skinned fibers are controlled by the same basic E-C coupling
mechanism as in intact fibers.

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Fig. 3.
Effect of peptide AS(20) on
Ca2+ release in skinned fibers of the rat. Addition of 30 µM peptide AS(20) potentiated the force
responses to depolarization (A and B) and
caffeine application (B) in EDL fibers. C: in a
soleus fiber, AS(20) also induced substantial
spontaneous force responses lasting >20 s. Time scale in A
and B: 2 s during brief depolarizations (by
Na+ substitution) and caffeine application and 30 s
between stimulation and during the prolonged depolarization (dashed
line). Depol, depolarized.
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Under such conditions, addition of 30 µM peptide
AS(20) to the K+ (polarizing)
bathing solution induced small, spontaneous force responses (1-6%
of maximum Ca2+-activated force) lasting ~1-20 s,
within 30 s to 2 min of application, in ~40% of cases in
skinned EDL (fast-twitch) fibers of the rat (Table
2). The response to depolarization in the
presence of peptide AS(20) was on average
potentiated relative to preceding control responses in every fiber
examined (13/13) (Table 2), reaching approximately the maximum
Ca2+-activated level in each case. Broadly similar
responses were obtained on successive (typically 3-6)
depolarizations in the presence of the peptide (e.g., Fig.
3A). A similar potentiating effect was also seen with 5 µM
peptide AS(20) (not shown; 110% and 104% of
control response to depolarization, n = 2). Peptide AS(20) (30 µM) also potentiated the response
to caffeine application (5 or 7 mM; e.g., Fig. 3B), with the
mean force response increasing from 55 ± 6% to 92 ± 4% of
maximum Ca2+-activated force in the three fibers examined;
the peptide also caused similar potentiation of caffeine-induced
responses when the voltage sensors were kept inactivated using
Na+-based solutions (not shown). Ca2+ release
induced by lowering the free [Mg2+] to 0.05 mM or below
(12, 13) was also potentiated by
AS(20) (30 µM; not shown). The potentiation
of the force responses reflected increased Ca2+ release and
not an effect on the properties of the contractile apparatus, because
additional experiments with heavily buffered Ca2+ solutions
(see METHODS) showed that neither the maximum
Ca2+-activated force nor the Ca2+ sensitivity
of the contractile apparatus was significantly altered in any of the
three fibers examined.
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Table 2.
Effect of II-III loop peptides 1) in inducing spontaneous
Ca2+ release and 2) on the response to
depolarization in skinned EDL fibers of the rat at 1 mM
Mg2+
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It was also of interest to examine the effect of
AS(20) in soleus (slow-twitch) muscle fibers,
where the ratio of RyR Ca2+-release channels to DHPR
voltage sensors is reportedly higher than in EDL fibers
(1), so even more than 50% of the release channels should
not be directly coupled to DHPR molecules (see Introduction).
AS(20) (30 µM) caused spontaneous
Ca2+ release in three of the eight soleus fibers examined
(e.g., Fig. 3C), inducing force responses of between 2 and
16% of maximum force (mean 7 ± 5%). In many cases, the soleus
fibers did not respond to depolarization by Na+
substitution, possibly because the T-system was not well sealed or
polarized (note that the fibers always responded well to direct activation of Ca2+ release by caffeine or low
[Mg2+]). In the soleus fibers responding to
depolarization, the first depolarization-induced response in 30 µM
AS(20) was on average 156% of the preceding
control response (50 ± 16% and 32 ± 13% of maximum
Ca2+-activated force, with and without peptide,
respectively, n = 4). Thus
AS(20) caused comparatively greater
potentiation of depolarization-induced responses in soleus fibers than
in EDL fibers, although this predominantly reflects the relatively poor
control response in soleus fibers rather than the effectiveness of the
peptide per se. In every soleus fiber examined, peptide
AS(20) (30 µM) potentiated the response to
lowering [Mg2+] (to
0.015 mM) or applying 2 or 4 mM caffeine [mean response as a
percentage of maximum force increasing from 61 ± 1% to 85 ± 2% (n = 3) and from 21 ± 15% to 35 ± 19% (n = 3) for low [Mg2+] and caffeine,
respectively], showing that the peptide augmented direct stimulation
of the Ca2+-release channels.
Effects of AC(20) and 10-amino acid
peptides at 1 mM Mg2+.
The effects of other A-region peptides were also examined in EDL fibers
in the presence of physiological [Mg2+] (i.e., 1 mM).
AS(10) induced much larger spontaneous force
responses at 30 µM than did AS(20) (21% vs.
2% of maximum force; Table 2; e.g., Fig.
4), and the response to depolarization
decreased greatly over the first few repetitions (e.g., Figs. 4 and
5A and Table 2), indicative of
depletion of releasable Ca2+ and/or inhibition of release.
AC(20), the cardiac DHPR loop peptide, had
little if any effect, with only a single, brief spontaneous response
being observed (after 3-min exposure) in one of six EDL fibers examined
and with there being no potentiation of the depolarization-induced response (Table 2) or of the response to lowered [Mg2+]
(not shown). In contrast, AC(10), the
COOH-terminal end of AC(20), caused
significant potentiation of depolarization-induced responses (e.g.,
Fig. 4), and its actions quantitatively resembled those of
AS(20) (Table 2). Interestingly, the highly
positive peptide, decalysine (30 µM; Fig. 1), did not cause any
spontaneous responses or significantly alter the response to
depolarization (Table 2; Fig. 5A).

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Fig. 4.
Effects of peptides AS(10) and
AC(10) on depolarization-induced responses in
a rat EDL fiber. Depolarization-induced responses were slightly
potentiated in presence of 30 µM peptide
AC(10) (top). Peptide
AS(10) caused a progressive a decrease in the
depolarization-induced responses in the same fiber (bottom).
Washout of the peptide for 2 min and reloading (30 s) largely restored
the response to depolarization and low [Mg2+]. Time
scale: 2 s during depolarization (by Na+ substitution)
and low [Mg2+] and 30 s elsewhere.
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Fig. 5.
Effects of decalysine and peptide
AS(10) in a rat skinned fiber. A:
depolarization-induced responses were little affected by decalysine (30 µM). In contrast, peptide AS(10) induced a
spontaneous force response (shown on slow time scale) and a great
reduction in the response to both depolarization and low
[Mg2+]. B: trace from another EDL fiber
showing that the response to low [Mg2+] was inhibited in
the presence of AS(10) and could be largely
restored simply by washing out the peptide. Time scale: 2 s during
depolarization (by Na+ substitution) and low
[Mg2+] (0.015 mM) and 30 s elsewhere.
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We further examined the effects of AS(10) to
identify the basis of its inhibitory action on depolarization-induced
responses. Some of the progressive reduction in the response was
probably due to SR depletion resulting from the
Ca2+-releasing action of the peptide, because fibers
showing a spontaneous response during the initial minute of exposure to
30 µM AS(10) (i.e., before the first
depolarization) gave a significantly smaller response to the first
depolarization (41 ± 9% of preceding control response,
n = 4) than did fibers giving a spontaneous response afterward or not at all (78 ± 4% of control response,
n = 6). It was also apparent that
AS(10) had an inhibitory effect directly on
the Ca2+-release channels, because the response to lowering
[Mg2+] (3 fibers; e.g., Fig. 5B) or applying
10 mM caffeine (not shown, 2 fibers) was largely or completely
suppressed in the presence of 30 µM AS(10)
but could be restored simply by 30-60 s washout of the peptide
without any reloading of the SR. Further experiments indicated that
AS(10) also had a third type of effect. It was first shown that lower concentrations of
AS(10) caused a less-marked decline in the
depolarization-induced response: at 5 µM
AS(10) there was very little spontaneous
response (1% of control depolarization-induced response in 1 of 3 EDL
fibers), and the response to depolarization declined to 94 ± 1%
and 86 ± 4% of the control response on the first and third
depolarizations, respectively (n = 3, P < 0.05; cf. Table 2). The falling phase of the depolarization-induced responses was unchanged in the presence of
AS(10) (e.g., Fig. 6), which shows that the peptide
did not cause any appreciable reduction in SR Ca2+ uptake.
Experiments were then performed in which an EDL fiber was subjected to
a second depolarization 10 s after each control response, to
determine the extent to which the E-C coupling mechanism had reprimed
in that time (e.g., Fig. 6). In the absence of any peptide, a fiber
almost fully reprimed in 10 s, but in the presence 10 µM
AS(10), the repriming was substantially
slowed, with the response to the second depolarization 10 s later
being only ~5% of the response to the first (e.g., Fig. 6). This
effect could be reversed within 2 min by washout of the peptide.
Similar results were found in all three fibers examined in this way
with 5 or 10 µM AS(10). Thus part of the
marked decline in the response to successive depolarizations (60 s
apart) observed at 30 µM AS(10) (Table 2) is
probably due to incomplete repriming of the voltage sensors.

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Fig. 6.
Peptide AS(10) slows repriming
of depolarization-induced responses. This rat EDL fiber was subjected
to pairs of depolarizing stimuli 10 s apart, with 60 s
between successive pairs. Repolarizing the fiber in the K+
solution for 10 s resulted in almost full repriming of the
response when no peptide was present. In the presence of 10 µM
AS(10) there was little response to
depolarization following 10 s repriming in K+,
indicating that the peptide slowed repriming. Fiber depolarized in
Na+ solution where "Depol" bar is shown and repolarized
in K+ solution where bar is absent. Responses shown for
fourth pair of stimuli in presence of peptide (4 min total exposure)
and second after washout (2 min total). Time scale: 2 s
throughout.
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The effect of a very high concentration of
AS(10) was also examined in an attempt to get
the peptide to all sites within a fiber as quickly as possible. At 200 µM, peptide AS(10) elicited a single,
comparatively large spontaneous force response (~28 ± 9% of
maximum force) within 40 (±13) s in 6 of the 11 rat EDL fibers
examined, after which the response to depolarization was virtually
completely abolished (e.g., Fig.
7A). In the fibers not giving
any spontaneous force response in the peptide (5 of 11), the response
to depolarization was similarly abolished (mean response 2 ± 1%
and 0% of maximum Ca2+-activated force on first and third
depolarizations, n = 11). When the response to
depolarization had been completely abolished by the presence of 200 µM peptide AS(10), lowering the
[Mg2+] to 0.015 mM only induced a small force response
(6 ± 5% of maximum force) in the seven fibers examined, and
exposure to 10 mM caffeine similarly elicited little or no response (0 and 2%) in two other fibers (e.g., Fig. 7A). The total
Ca2+ content of these fibers was then assayed (6,
24), revealing that there was a considerable amount of
Ca2+ in stores within the fiber in every case. The assay
involved briefly equilibrating the fiber in a solution with a
particular concentration of a Ca2+ buffer (0.55 mM EGTA)
and then lysing all the membranous compartments by moving the fiber
into a Triton X-100/paraffin oil emulsion. As the force response in all
six fibers examined reached the maximum possible in the Triton/oil
environment (~50% of maximum in aqueous environment) (6,
24) (e.g., Fig. 7A), it was apparent that the SR
Ca2+ content must have exceeded the capacity of the
exogenous and nondiffusible endogenous Ca2+ buffers and
hence was
0.7 mM (expressed as mmol/l fiber volume; see Refs. 6 and
24). [Note that the endogenous total Ca2+ content of rat
EDL fibers is ~1 mM (6), and a content of 0.7 mM
Ca2+ is normally sufficient for depolarization to induce at
least 80% of the control depolarization-induced force response
(24).] Thus it is apparent that 1) relatively
little Ca2+ could have been lost from the SR in the period
following the single spontaneous force response in 200 µM peptide
AS(10), and 2) the Ca2+
release channels had become unresponsive to both T-system
depolarization and direct stimulation with caffeine and low
[Mg2+].

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Fig. 7.
Effects of high concentration of peptide
AS(10) in primed and inactivated rat EDL
fibers. A: addition of 200 µM peptide
AS(10) to a fiber primed in the standard
K+ solution induced a relatively large spontaneous force
response after 30 s, following which neither depolarization nor 10 mM caffeine could elicit any response. Fiber was then equilibrated in a
solution with 0.55 mM free EGTA (15 s) and lysed in an emulsion of
Triton X-100 and paraffin oil (tx/oil) to release all Ca2+
in the fiber. The resulting force response indicated that there was at
least 0.7 mM total Ca2+ in the fiber at the time of lysing,
which implies that the peptide had blocked Ca2+ release and
not simply depleted the SR of Ca2+. Time scale: 2 s
during depolarization and exposure to caffeine, and 30 s
elsewhere. B: when another EDL fiber was depolarized by
Na+ substitution and then kept in that solution for 1 min
to ensure the voltage sensors were inactivated, addition of 200 µM
peptide AS(10) induced a spontaneous force
response after ~30 s. When the fiber was returned to the
K+ (polarizing) solution, a further force response was
elicited, possibly indicating that the peptide had not exerted its full
stimulatory effect during the period in Na+ solution. Time
scale: 2 s during initial period of depolarization (continuous
bar) and 30 s elsewhere (including dashed bar).
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To determine whether the peptide was inducing spontaneous force
responses by directly activating the RyRs or simply by depolarizing the
T-system, we examined the effect of the peptide when the voltage sensors were inactivated. Each fiber was depolarized by Na+
substitution, generating a transient force response as usual, but then
the fiber was kept in the Na+ solution for 1 min to ensure
that the voltage sensors were inactivated (10, 11, 13),
before substituting a similar solution with 200 µM peptide
AS(10). In two of the five fibers examined
(e.g., Fig. 7B), a spontaneous force response (~5% of
maximum force) was elicited ~40 s after peptide application; this
response was considerably smaller than that seen when the peptide was
added to fibers normally polarized in the K+ solution (see
above), although the response was substantially more prolonged
(~20 s in Na+ vs. ~2-3 s in K+). In
one case when the fiber was returned to the K+ solution, an
additional spontaneous force response was elicited (e.g., Fig.
7B), probably indicating that the peptide was better able to
elicit Ca2+ release when the fiber was in the
K+ solution than in the Na+ solution (as has
been observed in SR vesicle studies; Ref. 20). In summary, it was
apparent that the peptide could elicit Ca2+ release even
when the voltage sensors were inactivated, indicating that the peptide
must have had a direct stimulatory effect on the RyRs.
Effect of peptide CS.
El-Hayek et al. (3) have reported that peptide
CS (Fig. 1), another segment of the II-III loop of the
1-subunit of the skeletal muscle DHPR (previously called
peptide C), did not elicit Ca2+ release or increase
ryanodine binding in skeletal muscle triads, and in fact antagonized
the ability of both AS(20) (3)
and depolarization (28) to activate Ca2+
release (at 0.2 mM Mg2+). In seeming contrast, experiments
with chimeras of cardiac and skeletal DHPRs expressed in dysgenic
muscle indicated that this section of the skeletal II-III loop was
essential for inducing normal skeletal-type activation of the
Ca2+-release channels (22). In the experiments
here, peptide CS (30 or 100 µM) did not induce any
spontaneous response in any rat EDL fiber examined or cause any
potentiation of Ca2+ release (Table 2) or evidence of SR
Ca2+ depletion. At 100 µM, peptide CS did,
however, cause significant inhibition of depolarization-induced
responses (e.g., Fig. 8 and Table 2).
This inhibitory effect could be reversed by washout in some fibers
(e.g., Fig. 8B), although in most fibers it was not well
reversed (e.g., Fig. 8A). The large response to low
[Mg2+] at the end of the trace in Fig. 8A
shows that the release channels were not noticeably blocked by the
exposure to peptide CS (also see below) and that
the SR was still loaded with Ca2+. Experiments in which
fibers were subjected to pairs of depolarizations 10 s apart (as
in Fig. 6) showed no significant difference in the repriming rate in
the absence or presence of 100 µM CS (n = 7). The presence of 100 µM CS also resulted in a
significantly smaller force response to 30 µM
AS(20) at 0.2 mM Mg2+ (24.0.% vs.
40.3%, P < 0.05; Table 1). [Note that treatments were compared across different fibers because it was not possible to
meaningfully compare the effects of the two treatments in the same
fiber, as the effect of peptide AS(20) could
not readily be reversed by washout.] The inhibitory effects of peptide
CS on the response to depolarization and peptide
AS(20) could not be due to some generalized
blocking action on the release channels, because the response to 7 mM
caffeine was unaffected by the presence of the peptide (32.8 ± 10.4% and 36.4 ± 7.7% of maximum force, with and without 100 µM CS in 3 EDL fibers).

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Fig. 8.
Peptide CS partially inhibits
depolarization-induced responses in rat EDL fibers. A: on
initial exposure to 100 µM peptide CS,
depolarization-induced responses decreased substantially and did not
recover on washout of the peptide. A further small but reversible
degree of inhibition was seen on reapplication of the peptide. Fiber
broke on maximum activation. B: example of fiber showing
reversible partial inhibition of responses on first exposure to peptide
CS. Time scale: 2 s during depolarization and low
[Mg2+] exposure and 30 s elsewhere.
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Effects of peptides in toad fibers.
When 30 µM peptide AS(20) was applied to
skinned iliofibularis fibers of the toad at 1 mM Mg2+, in 3 of 12 cases it elicited spontaneous force responses that were
2-16% of maximum force and lasted up to 20 s (e.g., Fig. 9A, note slow time scale),
showing that the peptide must have triggered appreciable
Ca2+ release (24). The force response
to depolarization decreased rapidly over the first few depolarizations
in AS(20), dropping to 79 ± 9% and
19 ± 4% of the preceding control level on the first and third
depolarizations, respectively (n = 12). In general, the
actions of AS(20) in toad fibers more closely
resembled those of AS(10) in rat EDL fibers,
rather than those of AS(20) (cf. Table 2).
AS(20) caused evident depletion of SR
Ca2+ in some fibers (e.g., Fig. 9B).
AS(10) (at 30 µM) caused more inhibition of
depolarization-induced responses than did
AS(20) (27 ± 15% and 0% of control on
first and third depolarizations, n = 5), and
AC(10) and decalysine had qualitatively
similar, although smaller effects. In contrast to the case with rat EDL
fibers, decalysine (30 µM) induced spontaneous Ca2+
release in all toad fibers examined (7 ± 2% of maximum force, n = 4).

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Fig. 9.
Effect of peptide AS(20) on
Ca2+ release in toad skinned fibers. A: addition
of 30 µM peptide AS(20) in this
iliofibularis fiber evoked substantial spontaneous Ca2+
release lasting over 20 s, caused a progressive decrease in the
response to depolarization, and reduced responses to low (0.05mM)
[Mg2+] and to 30 mM caffeine. B: in a
different fiber, the force response to depolarization was progressively
lost after addition of peptide AS(20) (30 µM) but was restored over 3-6 min by washing out the peptide and
repeatedly reloading the SR with Ca2+ (1-min
periods in pCa 6.0; see METHODS). The progressive decrease
in the depolarization-induced force responses evident after the second
load period indicates that the SR was leaking an appreciable amount of
Ca2+ at that time. Time scale in A and
B: 2 s during depolarization and exposure to low
[Mg2+] and caffeine and 30 s between stimulation
including periods of spontaneous response (spont. resp.).
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Finally, peptide CS at 30 µM did not elicit any
spontaneous Ca2+ release in any of the 7 toad fibers
examined or cause any apparent SR depletion or inhibition of
depolarization-induced responses (response to first and third
depolarizations in peptide: 97 ± 4% and 105 ± 4% of
preceding control in 7 fibers). These results are similar to the
findings with rat fibers at such a concentration. The effects of higher
concentrations of peptide CS were not examined.
 |
DISCUSSION |
Effects of A peptides in skinned fibers.
It is presently not fully understood how the DHPRs in the T-system
regulate the Ca2+-release channels in the SR. Studies in
myotubes with DHPR chimeras have indicated that the II-III loop of the
1-subunit of skeletal muscle DHPR plays an essential
role in the coupling process (33, 34). Other recent
studies have shown that II-III loop peptides of skeletal or cardiac
DHPR can directly activate the Ca2+-release channel in
isolated SR and bilayer preparations (2-4, 16, 17).
Here we examined the effects of particular peptides on Ca2+
release in a skinned fiber preparation that retains E-C coupling and in
which important factors, such as myoplasmic [Mg2+],
[ATP], and SR Ca2+ loading, were kept close to
physiological levels. In this way we could test which, if any, of the
peptides were capable of eliciting (or interfering with)
Ca2+ release under conditions approximating those in vivo,
with the assumption that the key activation site(s) would be accessible in at least one-half of the release channels (see Introduction). It was
found that 30 µM peptide AS(20),
which has been previously shown to activate Ca2+
release channels in isolated triads and bilayers
(2-4), induced only a small degree of
Ca2+ release in skinned fibers with functional coupling in
the presence of physiological [Mg2+] (1 mM) (Figs. 3 and
9 and Table 2), although it potentiated the response to both voltage
sensor activation and caffeine application (Fig. 3).
AS(20) apparently had access to the activation
site(s) on many RyRs, because it potently induced Ca2+
release in the presence of a lower [Mg2+] (0.2 mM; Table
1). The inhibitory effect of 1 mM Mg2+ observed in this
study is not in conflict with the finding that As(20) could activate isolated RyRs in
bilayers in the presence of 2 mM Mg2+ (2),
because the latter experiments were performed in the presence of a high
[Cl
] that reduced the affinity of the
Ca2+/Mg2+ inhibitory site for Mg2+
by fourfold or more compared with the conditions used here (15, 20).
Peptide AS(10), the COOH-terminal end of
peptide AS(20) (Fig. 1), had considerably
greater effects on Ca2+ release than did
AS(20) at the same concentration (30 µM),
inducing substantial spontaneous force responses (~20% of maximum
force) in the majority of cases (Fig. 5A and Table 2). The
spontaneous force responses were only slightly larger with 200 µM
AS(10), but very much smaller at 5 µM. The
ability of 200 µM AS(10) to elicit a single,
comparatively large spontaneous force response, and yet not cause
radical depletion of SR Ca2+ after application for several
minutes (Fig. 7A), indicates that at later times the peptide
no longer potently activated the RyR. This must have been due at least
in part to a separate inhibitory action of the peptide on the RyR (see
later), because the peptide clearly interfered with Ca2+
release to caffeine and lowered [Mg2+] (e.g., Figs.
2C, 5B, and 7A). Peptide
AS(10) evidently also interfered with
repriming of the E-C coupling mechanism, possibly because it affected
the T-system potential (see later). Such an action, however, could not
account for the ability of AS(10) to induce
spontaneous Ca2+ release when the voltage sensors were
inactivated (Fig. 7B), showing that the peptide must have
had some relatively direct stimulatory effect on the RyR itself. The
fact that the spontaneous force responses were larger when a fiber was
kept polarized in the K+ solution than when inactivated in
the Na+ solution (e.g., Fig. 7) is probably due in part to
the apparently greater level of RyR activation that can be achieved in
the presence of K+ compared with Na+ (Fig.
2B in Ref. 20), although the peptide's possible
depolarizing action may have also contributed to the difference.
In contrast to AS(20) and
AS(10), peptide
AC(20), which is the segment of the cardiac
DHPR II-III loop corresponding to AS(20) (Fig.
1), had little effect on Ca2+ release, but its
COOH-terminal end, AC(10), had somewhat
greater effect, potentiating Ca2+ release in a
manner comparable with AS(20) (Table 2).
AC(10), like
AS(20), induced considerable
Ca2+ release at 0.2 mM Mg2+ (Fig. 2,
Table 1), and AS(10) was also evidently
stimulatory, although this effect was confounded by the peptide's
additional inhibitory action (Fig. 2C). Truncating
AS(20) to AS(10), or
AC(20) to AC(10),
removes negatively charged amino acids closest to the region of
greatest density of positively charged amino acids (Fig. 1). It is also
noteworthy that the ability of the peptides to induce Ca2+
release in rat fibers is in the same rank order as the net positive charge of consecutive stretches of charged amino acids
[AS(10) (5+) > AS(20) (3+)
AC(10) (3+) > AC(20) (1
)]. This could mean that the
activity of the peptides is determined in large part by the presence of
a region of positively charged amino acids, as suggested by Zhu et al.
(36). This could not be the only important factor,
however, because decalysine, which has an even higher positive charge
than peptide AS(10), had little if any effect
in rat fibers (Fig. 5A and Table 2), just as the peptide RKRRK had little effect in isolated triads (4). Thus it
seems that other factors in addition to charge distribution influence the ability of the peptides to induce Ca2+ release
(4).
Inhibitory effects of AS(10).
In addition to its stimulatory effect,
AS(10) had a direct inhibitory effect on
Ca2+ release (Figs. 2C, 5B, and
7A). This dual action is similar to that observed when
applying AS(20) to Ca2+ release
channels in bilayers (2). The inhibitory effect in that
study was greatest for potentials that induced movement of the
positively charged peptide into the channel, and the results were
consistent with the peptide blocking the channel pore (2), as has been found with other positive peptides (18). We
also note that Dulhunty et al. (2) did not detect any such
inhibitory effect of AS(20) when inducing
Ca2+ release from SR vesicles, which is consistent with the
lack of any marked inhibition with 30 µM
AS(20) in this study. Thus it seems that the
release channels are not as susceptible to block by the peptides under
physiological conditions where Ca2+ is flowing out of the
SR down its electrochemical gradient. Nevertheless, it was evident
that, when the more strongly charged peptide
AS(10) was present at high enough
concentrations (
30 µM), Ca2+ release did become
inhibited. This may have been due to blockage of the channel pore (and
possibly only occurs on channel opening). Alternatively, the peptide
may have inhibited the release channel by acting at a modulatory site
on its cytoplasmic face. Many divalent and polyvalent cations have been
shown to act at a low-affinity Ca2+/Mg2+
inhibitory site, with the half-inhibitory concentration being ~1 µM
for trivalent metal cations (and ruthenium red) and ~100 µM for
divalent metal cations (e.g., Ca2+ or Mg2+) in
skeletal RyRs, and ~10-fold higher in cardiac RyRs (14, 15, 19,
20, 31). The peptides may also have such an action as ryanodine
binding and Ca2+ release in skeletal muscle triad decrease
with >30 µM AS(20) or
AS(10) and >10 µM polylysine, with there
being little or no inhibition in cardiac RyRs, even with 100 µM
polylysine (4). Certainly, a pore-blocking effect alone
would not seem to account for the much greater inhibitory effect of the
peptides on skeletal compared with cardiac RyRs (4). A
peptide corresponding to the COOH-terminal region of the DHPR also
inhibits RyR activation (30), and, given that this region
also has concentrations of positively charged residues, its actions may
be similar to those of AS(10).
Irrespective of exactly how the peptides inhibit the RyRs, it is
noteworthy that this effect of AS(10) could be
reversed within ~30 s by washout of the peptide (e.g., Fig.
5B), revealing a persistent activating effect of the peptide
(Fig. 2C). This too is in good agreement with the finding
that isolated RyRs were actually more active after washout of
AS(20) than in its presence, which was interpreted as meaning that the activating effect of the peptide was
less easily reversed by washout than the inhibitory effect of the
peptide (2).
It also appeared that AS(10) had an additional
inhibitory action, in which it slowed repriming of the E-C coupling
mechanism (e.g., Fig. 6). The very small response after 10 s
repriming compared with 60 s repriming is not readily
explained by direct inhibition of the release channel. Such slow
repriming would be expected if the T-system was depolarized to some
extent by the presence of AS(10), and this
might arise from the peptide-blocking K+
channels in the T- system membrane, given that
AS(10) in many ways resembles K+
channel inactivation peptides, which act from the cytoplasmic side and
have a strongly positively charged region and an adjacent hydrophobic
region (18). Alternatively, it is possible that AS(10) slowed repriming of the voltage
sensor/DHPRs by acting directly on those molecules or by interfering
with their function via their coupling with the release channels.
Effects of peptide CS.
Peptide CS, which corresponds to a part of the skeletal
DHPR II-III loop thought to be critical for initiating normal
skeletal-type coupling (22), never induced detectable
Ca2+ release or evidence of SR depletion in any rat or toad
fiber (e.g., Fig. 8 and Table 2). In fact, peptide CS (at
100 µM) caused partial inhibition of both depolarization-induced
responses (e.g., Fig. 8 and Table 2) and peptide
AS(20)-induced Ca2+
release (Table 1). These effects were not due to some generalized inhibitory action on the Ca2+ release channels, because the
response to caffeine (7 mM) was not affected by the presence of peptide
CS. Furthermore, the inhibition of depolarization-induced
responses was not due to peptide CS in some way
depolarizing the T-system, because the repriming rate was not
noticeably altered. These results with peptide CS are in
good agreement with the findings in triads (3, 28); the extent of the inhibition is smaller in the present study, but this is
probably largely the result of quantifying responses in terms of peak
force [which is indicative of total Ca2+ release
(24)], rather than the rate of Ca2+ release
(3, 28). Diffusional limitations would also have been
greater in the skinned fibers than in the isolated triads. The fact
that peptide CS interfered with activation by both the normal E-C coupling mechanism and the application of
AS(20) is consistent with there being some
commonality in the two methods of activation (3, 4) but
does not mean that the two are identical. It is possible that
exogenously added peptide CS occludes a common binding site
on the release channel that is critical to both E-C coupling and
peptide A activation. Given that even the first depolarization-induced
response in the presence of peptide CS was reduced (Fig. 8
and Table 2), it seems that peptide CS exerted its
inhibitory action either without the voltage sensors having to be
activated or extremely quickly on such activation. The exact basis of
the inhibitory effect of CS, however, is not apparent.
Relative effect of peptides in toad fibers.
The A peptides, which correspond to segments of the mammalian DHPR
II-III loop, also induced Ca2+ release in toad muscle
fibers, with the extent of spontaneous release being, if anything,
somewhat larger than in EDL fibers (e.g., Fig. 9A). This
could reflect greater potency of the peptides on either the
- or the
-isoform of the RyR present in amphibian muscle (23) or
that the peptides activate the
-isoform, which in turn triggers
substantial Ca2+-induced Ca2+ release through
the
-isoform. It is also likely that the toad fibers show greater
net release and depletion of SR Ca2+ than rat fibers
because the Ca2+ pump in the SR is less able to recover
released Ca2+, given that we find that the rat fibers can
refill the SR about twice as fast as can the toad fibers at the same
[Ca2+] (e.g., at pCa 6.7 or 6.0) (unpublished
observations). It is pertinent that the mammalian A peptides have
stimulatory effects in toad fibers, because the amphibian skeletal DHPR
II-III loop, which presumably physically activates the
-RyR in vivo,
has A and C regions with high homology and identical charge
distribution compared with the mammalian skeletal counterparts
(35). In fact, the A region in frog (TSAQKAKAEERKRKKLARAN)
closely resembles a chimera of AS(20) with the
COOH-terminal end of AC(20) (cf. Fig. 1). The
fact that the A region of the mammalian II-III loop triggers some
Ca2+ release in toad fibers much as it does in mammalian
fibers under the same conditions suggests that there is a binding site
for the A peptide on the RyRs in amphibia (presumably on the
-isoform) similar to that on RyR1.
Relevance to E-C coupling.
The ability of the A region to trigger Ca2+ release under
some circumstances and the fact that it, like normal E-C coupling, is
partially inhibited by peptide CS could suggest that the A region plays a role in normal E-C coupling. Movement of the A region of
the II-III loop would be consistent with a number of other findings. It
has been found that heparin, a strongly negatively charged molecule
that binds to the A region of the II-III loop (8),
interferes with E-C coupling in toad skinned fibers if and only if the
DHPR/voltage sensors are activated in its presence (9).
This could mean that that region of the loop only moves into the
triadic gap when the DHPRs are activated, and this would also be
consistent with other studies indicating that the associated serine
residue (Ser687) is more readily phosphorylated if the
DHPRs are repetitively activated (26, 29). In addition, it
has also been shown that E-C coupling involves a nonlinear surface
potential change associated with the DHPRs, and one possible cause of
this could be the exposure of positive charges on the intracellular
face of the DHPR that were occluded in the resting conformation
(7). Thus there are a number of different lines of
evidence that are consistent with DHPR/voltage sensor activation
involving the movement of the A region of the II-III loop.
Nevertheless, we point out that this does not mean that the A region
itself initiates Ca2+ release.
It is possible that the ability of the A peptide to activate the
release channels is unrelated to the normal E-C coupling mechanism,
perhaps resulting from peptide binding to a region of the RyR that is
normally inaccessible to the intracellular loop of the DHPR.
Importantly, it was found here that the A peptides induced only a very
small degree of Ca2+ release in the skinned fibers at
physiological [Mg2+] (1 mM), with appreciable release
only occurring at a substantially lower [Mg2+] (Table 1).
The rate of Ca2+ release in the fibers here at 1 mM
Mg2+ was probably underestimated owing to the occurrence of
simultaneous Ca2+ reuptake into the SR, but nevertheless it
must still have been far lower than during normal E-C coupling. As the
skinned fibers evidently have functional E-C coupling, it cannot be
argued that peptides are comparatively ineffective simply because some
cofactor normally present in vivo has been lost. This lack of
effectiveness of AS(20) at physiological
[Mg2+] may imply that normal E-C coupling involves some
different or additional action to the binding of
AS(20) to the release channel. Alternatively,
it is possible that the applied peptides are acting at the same site on
the RyR as the II-III loop does in vivo, but that the exact interaction
is not the same because the peptide is not held in its normal
orientation or conformation by structural connections with the rest of
the DHPR. It may be a relatively rare occurrence for loop peptides to
be simultaneously bound to each of the four subunits of a RyR tetramer,
and, if this is a prerequisite for channel opening, it could explain
why the peptides only rarely induced substantial Ca2+
efflux. Here it is noteworthy that single cyclic nucleotide-gated (CNG)
channels, which also have a tetrameric structure, are activated in a
highly nonlinear manner with increasing levels of bound ligand, having
an open probability of 0, 0.01, 0.33, and 1.0 with 1, 2, 3, and 4 bound
nucleotides, respectively (27). Furthermore, when fewer
than four ligands were bound, the CNG channels showed substate activity
(27) that seems highly comparable to that observed with
AS(20) stimulation of isolated RyRs
(2). Thus the poor ability of A peptides to induce
Ca2+ release in the skinned fibers at physiological
[Mg2+] could simply be due to such highly nonlinear
activation behavior. Another possibility is that lateral interactions
between adjacent RyRs result in the array of RyRs acting in concert,
which could make exogenously applied peptides a less effective stimulus
than synchronously activating all the DHPRs facing the RyR array,
as presumably happens in vivo. Finally, we cannot exclude the
possibility that the applied A peptides fail to reach the activating
site on even the uncoupled RyRs, although it is evident that
AS(20) must have reached stimulatory sites on
some RyRs because it was effective at inducing Ca2+ in 0.2 mM Mg2+ (Fig. 2A and Table 1).
The finding that peptide CS did not induce any detectable
Ca2+ release in the fibers here does not readily fit with
that region of the II-III loop having a direct stimulatory effect on
the release channels, as would have been expected from the simplest
interpretation of the results of experiments with DHPR chimeras
(22). Because peptide CS actually
interfered with the coupling mechanism, it is apparent that this
peptide must have had access to at least some regions of the DHPR-RyR
complex within the triad junction, presumably including the putative
coupling site(s) on at least half of the RyRs
(5). It is possible that the ability of
CS to interfere with the coupling mechanism was due to the
exogenous peptide competing with the in situ C-region peptide for the
activation site, but with the exogenous peptide being poorly
stimulatory. Alternatively, binding of the CS region may
not be sufficient by itself to fully stimulate the Ca2+
release channel, even though it may be a prerequisite for proper coupling.
One possibility is that normal E-C coupling involves some coordinated
movement or interaction of both the C and A regions of the II-III loop,
as suggested previously (28). This could be consistent
with the finding that a chimera of the cardiac DHPR with the skeletal
CS region was able to give skeletal-type E-C coupling
(22), because it was found that
AC(10) could also induce Ca2+
release under some circumstances (Tables 1 and 2 and see Ref. 4).
Another perhaps related possibility is that the peptide C region has a
repulsive action on the RyR that destabilizes the closed state of the
channel, with this interaction only being possible because the DHPR and
RyR are held in fixed apposition in situ (5).
In conclusion, this study has shown that the A and C regions of the
DHPR II-III loop have activating and inhibitory actions, respectively,
on Ca2+ release in functioning muscle fibers, showing that
these regions may be important in normal E-C coupling.
 |
ACKNOWLEDGEMENTS |
We are grateful to Maria Cellini and Aida Yousef for technical assistance.
 |
FOOTNOTES |
This work was supported by the National Health and Medical Research
Council of Australia (Grant 9936582) and by grants from the National
Institutes of Health.
Address for reprint requests and other correspondence: G. D. Lamb, School of Zoology, La Trobe Univ., Bundoora, Victoria, 3083 Australia (E-mail: zoogl{at}zoo.latrobe.edu.au).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 December 1999; accepted in final form 11 April 2000.
 |
REFERENCES |
1.
Delbono, O,
and
Meissner G.
Sarcoplasmic reticulum Ca2+ release in rat slow- and fast-twitch muscles.
J Membr Biol
151:
123-130,
1996[ISI][Medline].
2.
Dulhunty, AF,
Laver DR,
Gallant EM,
Casarotto MG,
Pace SM,
and
Curtis S.
Activation and inhibition of skeletal RyR channels by a part of the skeletal DHPR II-III loop: effects of DHPR Ser687 and FKBP12.
Biophys J
77:
189-203,
1998[Abstract/Free Full Text].
3.
El-Hayek, R,
Antoniu B,
Wang J,
Hamilton SL,
and
Ikemoto N.
Identification of calcium release-triggering and blocking regions of the II-III loop of the skeletal muscle dihydropyridine receptor.
J Biol Chem
270:
22116-22118,
1995[Abstract/Free Full Text].
4.
El-Hayek, R,
and
Ikemoto N.
Identification of the minimum essential region in the II-III loop of the dihydropyridine receptor
1 subunit required for activation of skeletal muscle-type excitation-contraction coupling.
Biochemistry
37:
7015-7020,
1998[ISI][Medline].
5.
Franzini-Armstrong, C,
and
Jorgensen AO.
Structure and development of E-C coupling units in skeletal muscle.
Annu Rev Physiol
56:
509-534,
1994[ISI][Medline].
6.
Fryer, MW,
and
Stephenson DG.
Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle.
J Physiol (Lond)
493:
357-370,
1996[Abstract].
7.
Jong, D-S,
Stroffekova K,
and
Heiny JA.
A surface potential change in the membranes of frog skeletal muscle is associated with excitation-contraction coupling.
J Physiol (Lond)
499:
787-808,
1997[Abstract].
8.
Knaus, HG,
Scheffauer F,
Romanin C,
Schindler H-G,
and
Glossmann H.
Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels.
J Biol Chem
265:
11156-11166,
1990[Abstract/Free Full Text].
9.
Lamb, GD,
Posterino GS,
and
Stephenson DG.
Effects of heparin on excitation-contraction coupling in skeletal muscle of toad and rat.
J Physiol (Lond)
474:
319-320,
1994[Abstract].
10.
Lamb, GD,
and
Stephenson DG.
Calcium release in skinned muscle fibres of the toad by transverse tubule depolarization or by direct stimulation.
J Physiol (Lond)
423:
495-517,
1990[Abstract].
11.
Lamb, GD,
and
Stephenson DG.
Control of calcium release and the effect of ryanodine in skinned muscle fibres of the toad.
J Physiol (Lond)
423:
519-542,
1990[Abstract].
12.
Lamb, GD,
and
Stephenson DG.
Effect of Mg2+ on the control of Ca2+ release in skeletal muscle fibres of the toad.
J Physiol (Lond)
434:
507-528,
1991[Abstract].
13.
Lamb, GD,
and
Stephenson DG.
Effects of intracellular pH and [Mg2+] on the excitation-contraction coupling in skeletal muscle fibres of the rat.
J Physiol (Lond)
478:
331-339,
1994[Abstract].
14.
Laver, DR,
Baynes TM,
and
Dulhunty AF.
Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms.
J Membr Biol
156:
213-229,
1997[ISI][Medline].
15.
Laver, DR,
Owen VJ,
Junankar PR,
Taske NL,
Dulhunty AF,
and
Lamb GD.
Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia.
Biophys J
73:
1917-1923,
1997.
16.
Lu, X,
Xu L,
and
Meissner G.
Activation of the skeletal muscle calcium release channel by a cytoplasmic loop of the dihydropyridine receptor.
J Biol Chem
269:
6511-6516,
1994[Abstract/Free Full Text].
17.
Lu, X,
Xu L,
and
Meissner G.
Phosphorylation of dihydropyridine receptor II-III loop peptide regulates skeletal muscle calcium release channel function.
J Biol Chem
270:
18459-18464,
1995[Abstract/Free Full Text].
18.
Mead, FC,
Sullivan D,
and
Williams AJ.
Evidence for negative charge in the conduction pathway of the cardiac ryanodine receptor channel provided by the interaction of K+ channel N-type inactivation peptides.
J Membr Biol
163:
225-234,
1998[ISI][Medline].
19.
Meissner, G,
Darling E,
and
Eveleth J.
Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides.
Biochemistry
25:
236-244,
1986[ISI][Medline].
20.
Meissner, G,
Rios E,
Tripathy A,
and
Pasek DA.
Regulation of skeletal muscle Ca2+ release channel (ryanodine receptor) by Ca2+ and monovalent cations and anions.
J Biol Chem
272:
1628-1638,
1997[Abstract/Free Full Text].
21.
Melzer, W,
Herrmann-Frank A,
and
Lüttgau HC.
The role of Ca2+ ions on excitation-contraction coupling of skeletal muscle fibres.
Biochim Biophys Acta
1241:
59-116,
1995[ISI][Medline].
22.
Nakai, J,
Tanabe T,
Konno T,
Adams B,
and
Beam KG.
Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling.
J Biol Chem
273:
24983-24986,
1998[Abstract/Free Full Text].
23.
Ogawa, Y.
Role of ryanodine receptors.
Crit Rev Biochem Mol Biol
29:
229-274,
1994[Abstract].
24.
Owen, VJ,
Lamb GD,
Stephenson DG,
and
Fryer MW.
Relationship between depolarization-induced force responses and Ca2+ content in skeletal muscle fibres of rat and toad.
J Physiol (Lond)
498:
571-586,
1997[Abstract].
25.
Posterino, GS,
and
Lamb GD.
Effects of reducing agents and oxidants on excitation-contraction coupling in skeletal muscle fibres of rat and toad.
J Physiol (Lond)
496:
809-825,
1996[Abstract].
26.
Röhrkasten, A,
Meyer HE,
Nastainczyk W,
Sieber M,
and
Hofmann F.
cAMP-dependent protein kinase rapidly phosphorylates serine-687 of the skeletal muscle receptor for calcium channel blockers.
J Biol Chem
263:
15325-15329,
1988[Abstract/Free Full Text].
27.
Ruiz, M,
and
Karpen JW.
Single cyclic nucleotide-gated channels locked in different ligand-bound states.
Nature
389:
389-392,
1997[ISI][Medline].
28.
Saiki, Y,
El-Hayek R,
and
Ikemoto N.
Involvement of the Glu724-Pro760 region of the dihydropyridine receptor II-III loop in skeletal muscle-type excitation-contraction coupling.
J Biol Chem
274:
7825-7832,
1999[Abstract/Free Full Text].
29.
Sculptoreanu, A,
Scheuer T,
and
Catterall WA.
Voltage-dependent potentiation of L-type Ca2+ channels due to phosphorylation by cAMP-dependent protein kinase.
Science
364:
240-243,
1993.
30.
Slavik, K,
Wang J-P,
Aghdasi B,
Zhang J-Z,
Mandel F,
Malouf N,
and
Hamilton SL.
A carboxy-terminal peptide of the
1-subunit of the dihydropyridine receptor inhibits Ca2+- release channels.
Am J Physiol Cell Physiol
272:
C1475-C1481,
1997[Abstract/Free Full Text].
31.
Soler, F,
Fernandez-Belda F,
and
Gomez-Fernandez J.
The Ca2+ release channel in junctional sarcoplasmic reticulum: gating and blocked by cations.
Int J Biochem
24:
903-909,
1992[ISI][Medline].
32.
Stephenson, DG,
and
Williams DA.
Calcium-activated force in fast- and slow-twitch skinned muscle fibres of the rat at different temperatures.
J Physiol (Lond)
317:
291-302,
1981.
33.
Tanabe, T,
Beam KG,
Adams BA,
Niidome T,
and
Numa S.
Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling.
Nature
346:
567-569,
1990[ISI][Medline].
34.
Tanabe, T,
Beam KG,
Powell JA,
and
Numa S.
Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA.
Nature
336:
134-139,
1988[ISI][Medline].
35.
Zhou, J,
Cribbs L,
Yi J,
Shirokov R,
Perez-Reyes E,
and
Rios E.
Molecular cloning and functional expression of a skeletal muscle dihydropyridine receptor from Rana catesbeiana.
J Biol Chem
273:
25503-25509,
1998[Abstract/Free Full Text].
36.
Zhu, X,
Gurrola G,
Jiang MT,
Walker JW,
and
Valdivia HH.
Conversion of an inactive cardiac dihydropyridine receptor II-III loop segment into forms that activate skeletal ryanodine receptors.
FEBS Lett
450:
221-226,
1999[ISI][Medline].
Am J Physiol Cell Physiol 279(4):C891-C905
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