(Received for publication, July 13, 1995; and in revised form, July 27, 1995)
From the
In an attempt to identify and characterize functional domains of
the rabbit skeletal muscle dihydropyridine receptor subunit II-III loop, we synthetized several peptides
corresponding to different regions of the loop: peptides A, B, C, C1,
C2, D (cf. Fig. 1). Peptide A
(Thr
-Leu
) activated
ryanodine binding to, and induced
Ca
release from, rabbit skeletal muscle triads, but
none of the other peptides had such effects. Peptide A-induced
Ca
release and activation of ryanodine binding were
partially suppressed by an equimolar concentration of peptide C
(Glu
-Pro
) but were not affected by
the other peptides. These results suggest that the short stretch in the
II-III loop, Thr
-Leu
, is responsible
for triggering SR Ca
release, while the other region,
Glu
-Pro
, functions as a blocker of
the release trigger. A hypothesis is proposed to account for how these
subdomains interact with the sarcoplasmic reticulum
Ca
release channel protein during
excitation-contraction coupling.
Figure 1:
The location and
amino acid sequence of the various synthetic peptides (A, B, C, C1, C2,
and D) encompassing different regions of the II-III loop of the
subunit of the rabbit skeletal muscle dihydropyridine
receptor.
The electrical signal elicited at the T-tubule ()membrane is transmitted to the sarcoplasmic reticulum (SR)
to induce Ca
release, which in turn leads to muscle
contraction(1, 2, 3, 4, 5, 6, 7, 8) .
According to the current widely accepted view, upon T-tubule
depolarization a portion of the dihydropyridine receptor (DHPR), the
voltage-sensing protein in the T-tubule, undergoes a conformational
change to make contact with the ryanodine receptor (RyR) to open its
Ca
release
channel(9, 10, 11, 12, 13) .
The idea that the cytoplasmic loop linking Repeats II and III of the
subunit of the DHPR, the so-called II-III loop, may
play an essential role in this process has emerged from an earlier
finding that this portion of the DHPR is the critical determinant of
the skeletal muscle-type Ca
current(14) .
This view has been further supported by recent findings that the
expressed II-III loop (both skeletal and cardiac isoforms) enhanced the
ryanodine binding to the skeletal muscle RyR(15) . The site
important for activation of ryanodine binding was localized in the
region encompassing residues Glu
-Glu
(16) , which contains the phosphorylatable serine
687(17) . Furthermore, a recent study with dysgenic myotubes
expressing the chimeric (skeletal/cardiac) DHPR has shown that the
critical determinant of the skeletal muscle-type Ca
transient is localized in the stretch of residues
Glu
-Pro
(18) . In this study,
using synthetic peptides corresponding to different regions of the
II-III loop of rabbit skeletal muscle DHPR
subunit,
we identified the region responsible for triggering Ca
release and another region for blocking the release. The
implication of these findings on the E-C coupling mechanism is
discussed.
In an attempt to identify the subdomains of the II-III loop
of the subunit of the DHPR that play important roles
in excitation-contraction coupling, we synthetized several peptides
corresponding to different regions of the loop as shown in Fig. 1and investigated the effect of each of those synthetic
peptides on [
H]ryanodine binding to, and
Ca
release from, rabbit skeletal muscle triads. Fig. 2A depicts the extent of ryanodine binding
activation/inhibition (expressed as percent of control) induced by
various concentrations of these peptides. Of all the peptides
investigated up to a concentration of 50 µM, only peptide
A produced significant activation of ryanodine binding. Increasing
concentrations of peptide A progressively increased ryanodine binding
to a maximal level (about 230% of control). However, peptides B, C, C1,
C2, and D produced virtually no effect on the ryanodine binding.
Mirroring the ryanodine binding experiments (Fig. 2A),
only peptide A induced an appreciable SR Ca
release (Fig. 2B). Thus, at a maximally activating
concentration (20 µM, see the inset to Fig. 2B), peptide A induced a significant amount of
Ca
release from SR. However, equimolar concentrations
of all the other peptides induced virtually no Ca
release.
Figure 2:
Effects of various synthetic peptides of
the II-III loop on [H]ryanodine binding (A) and SR calcium release from skeletal muscle triads (B). A, 50 µg of SR triads were incubated with 8
nM [
H]ryanodine in the absence and
presence of the indicated peptide concentrations and 10 µM free Ca
. Data represent the mean ± S.D.
of three or more experiments carried out in duplicate.
[
H]Ryanodine binding in the absence of peptides
(control) was 1.07 ± 0.12 pmol/mg. B, SR Ca
release from triad vesicles induced by 20 µM
peptide. Inset shows the effect of increasing concentrations
of peptide A-induced activation of Ca
release. Data
represent the average of three experiments with two different
preparations.
Peptide A produced no appreciable effects on ryanodine binding to microsomes isolated from porcine cardiac muscle (percent of control: at 20 µM peptide A, 102 ± 7 (n = 3); at 50 µM, 104 ± 11 (n = 4)). This is in agreement with the recent report that the expressed II-III loop activates the skeletal muscle RyR but not the cardiac RyR isoform(15) .
Under the same conditions as
above, in which peptide A produced significant activation, the whole
II-III loop expressed in Escherichia coli(22) had
virtually no effects on ryanodine binding nor induced Ca release, unless 5 mM AMP was added as done in the
original study by Meissner and co-workers(15) . This suggests
that there might be an inhibitory domain counteracting the peptide A
region within the II-III loop. Indeed, as shown by the experiments in Fig. 3, A and B, the presence of 50 µM peptide C, but not the other peptides (B, C1, C2, or D), produced
significant suppression of the activation of ryanodine binding induced
by 50 µM peptide A (Fig. 3A). Again
mirroring the ryanodine binding experiments, an equimolar concentration
(20 µM in this case) of peptide C produced significant
inhibition of SR Ca
release induced by peptide A.
However, peptides B, C1, C2, and D had no effect. It is particularly
interesting that neither peptide C1 nor C2, which represent the two
subdomains of peptide C, had any Ca
release blocking
effect by themselves. This indicates that both C1 and C2 subdomains
must be linked to exert the blocking function.
Figure 3:
Partial inhibition of peptide A-induced
activation of [H]ryanodine binding (A)
and SR Ca
release by peptides B and C, C1, C2, or D (B). A, [
H]ryanodine binding
was performed as described under ``Experimental Procedures''
in the presence of equimolar mixtures (50 µM) of the
selected pair of peptides as indicated. Data represent the mean
± S.D. of three experiments. Comparison of the mean values was
done using an unpaired Student's t test method. Asterisk indicates p < 0.05 versus A. B, Ca
release was monitored after mixing
primed triads with a solution containing an equimolar mixture (20
µM) of the selected pair of peptides. Data represent the
average of three or four experiments done using two different
preparations. The amount of calcium released was calculated for each
curve (see ``Experimental Procedures'') and tabulated. Data
are means ± S.D. The numbers in parentheses represent the number of experiments. Asterisk indicates p < 0.05 versus A.
Several important new
properties of the II-III loop of the DHPR are revealed in this study.
Most importantly, we could localize the critical site for activating
the RyR/Ca release channel to peptide A
(Thr
-Leu
), which represents
approximately one-third of the recently reported 61-residue ryanodine
binding activating peptide of the II-III loop(16) . Another
important aspect of this study is the finding of peptide C, which
antagonized the effect of peptide A on Ca
release or
ryanodine binding. These results suggest that there are at least two
functionally important subdomains in the II-III loop: an activator that
is responsible for the stimulation of the RyR/Ca
release channel in E-C coupling and a blocker that antagonizes
the activator. These results suggest an intriguing hypothesis as
follows. In the resting state, the putative signal receptor site in the
RyR is occupied by the blocker domain of the loop. Upon depolarization,
the blocker domain (corresponding to peptide C) is removed from the
site; then the activator domain (corresponding to peptide A) is allowed
to interact with the site to trigger SR Ca
release.
In the present study, the activation of SR Ca
release
by peptide A was produced presumably by competitive binding with the
blocker domain to the signal receptor (in the case of coupled RyR) or
by direct binding (in the case of uncoupled RyR). Peptide C1
(Phe
-Gly
) used in the present study
covers the 17-residue (Glu
-Pro
)
region reported to be a critical determinant for the skeletal
muscle-type regulation(18) , which requires a physical contact
of the II-III loop to the RyR(11) . On this basis, we
tentatively propose that the C1 subdomain may behave like a hinge for
this blocker/activator exchange operation.