(Received for publication, June 9, 1995; and in revised form, September 18, 1995)
From the
We have used [H]ryanodine binding
experiments and single channel recordings to provide convergent
descriptions of the effect of imperatoxin A (IpTx
), a
5-kDa peptide from the venom of the scorpion Pandinus
imperator (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and
Coronado, R.(1992) Proc. Ntl. Acad. Sc. U.S.A. 89,
12185-12189) on Ca
release channels/ryanodine
receptors (RyR) of sarcoplasmic reticulum (SR). At nanomolar
concentrations, IpTx
increased the binding of
[
H]ryanodine to skeletal SR and, to a lesser
extent, to cerebellum microsomes. The activating effect of IpTx
on skeletal SR was Ca
-dependent, synergized by
caffeine, and independent of other modulators of RyRs. However,
IpTx
had negligible effects on tissues where the expression
of skeletal-type RyR isoforms (RyR1) is small or altogether absent, i.e. cardiac, cerebrum, and liver microsomes. Thus, IpTx
may be used as a ligand capable of discriminating between RyR
isoforms with nanomolar affinity. IpTx
increased the open
probability (P
) of rabbit skeletal muscle RyRs by
increasing the frequency of open events and decreasing the duration of
the closed lifetimes. This activating effect was dose-dependent
(ED
= 10 nM), had a fast onset, and was
fully reversible. Purified RyR from solubilized skeletal SR displayed
high affinity for [
H]ryanodine with a K
of 6.1 nM and B
of
30 pmol/mg of protein. IpTx
increased [
H]ryanodine binding
noncompetitively by increasing B
to
60
pmol/mg of protein. These results suggested the presence of an
IpTx
-binding site on the RyR or a closely associated
regulatory protein. This site appears to be distinct from the caffeine-
and adenine nucleotide-regulatory sites. IpTx
may prove a
useful tool to identify regulatory domains critical for channel gating
and to dissect the contribution of skeletal-type RyRs to intracellular
Ca
waveforms generated by stimulation of different
RyR isoforms.
In cardiac and skeletal muscle, the calcium release
channel/ryanodine receptor (RyR) ()constitutes the major
pathway for Ca
release from the sarcoplasmic
reticulum (SR) during excitation-contraction coupling(1) . RyRs
are also found in neurons(2, 3) , exocrine cells (4) , smooth muscle cells(5, 6) , epithelial
cells(7) , lymphocytes(8) , and sea urchin
eggs(9) . In all of these cells, RyRs play a central role in
the regulation of the intracellular free Ca
concentration, whose elevation triggers a cascade of events that
culminates in muscle contraction, hormone secretion, lymphocyte
activation, egg fertilization, etc. To gain the functional flexibility
necessary to respond to different triggering signals, at least three
tissue-specific isoforms of RyR are expressed (10) . In
mammals, RyR1 is expressed predominantly in fast- and slow-twitch
skeletal muscle, while RyR2 is expressed predominantly in cardiac
muscle. RyR3 appears to be localized to brain, smooth muscle, and
epithelial cells, although low levels of expression of RyR1 and RyR2
are also found in some of these tissues(11, 12) .
Several structural and functional characteristics confer to RyRs a
distinctive earmark. They are homotetramers of large molecular size
(2 million Da(13) ); they form Ca
-gated
Ca
-selective channels of large conductance (14) , and they are distinctively affected by the plant
alkaloid ryanodine(15) .
The effects of ryanodine on single
RyRs are complex and highly dependent on the concentration of the
alkaloid. At low concentrations (5-50 nM), ryanodine
increases the mean open time of the channel without modifying its
unitary conductance(16, 17) . At intermediate
concentrations (50 nM to 10 µM), ryanodine
``locks'' the channel in an open subconductance state that
corresponds to 50% of the full conductance level. Higher
concentrations (>100 µM) of the alkaloid fully and
irreversibly close the channel(18, 19) . Thus,
ryanodine may act as an agonist and a blocker of the RyR.
In
addition to its dual effect, ryanodine displays a slow association rate
of binding (19, 20) that renders the onset of
activation incompatible with the time frame of electrophysiological
experiments. To accelerate the onset of effect, micromolar, instead of
nanomolar levels of ryanodine are used. During the initial phase of
incubation, ryanodine may open RyRs and deplete the SR
Ca content. At equilibrium, higher ryanodine
concentrations may close RyRs. Therefore, although the effect
of depleting the SR Ca
content or blocking its
release may be macroscopically the same (paralysis), the dual action of
ryanodine makes it difficult to assess the availability of a releasable
Ca
pool at any experimental point. In addition, the
recovery of SR function after treatment with ryanodine is also
difficult to assess because the slow dissociation rate of ryanodine (20) makes the pharmacological effect essentially irreversible.
Despite these drawbacks, ryanodine has been an invaluable tool in
defining the pharmacological profile of RyRs. The binding of
[H]ryanodine is enhanced by activators of
Ca
release (µM Ca
,
ATP, caffeine) and decreased by inhibitors of Ca
release (Mg
, ruthenium red), suggesting that
the alkaloid binds to a conformationally-sensitive domain on the RyR
protein(20, 21) . Therefore,
[
H]ryanodine has been used as a probe of the
functional state of the channel. This approach has contributed to the
isolation of the RyR itself (22, 23, 24) and
to the identification of novel
ligands(25, 26, 27) , endogenous
modulators(28, 29) , and regulatory
proteins(30, 31) . Still, the detailed mechanism by
which RyRs generate primary or amplified secondary Ca
signals remains elusive, especially in cells where the
permeability of intracellular Ca
stores is also
controlled by another Ca
channel, the inositol
triphosphate receptor (IP
R), or in cells where several RyR
isoforms act simultaneously. Clearly, the elucidation of the mechanism
of Ca
release from intracellular stores will depend
critically on the specificity of ligands that selectively alter a
single intracellular Ca
channel type.
From the
venom of the African scorpion Pandinus imperator we isolated
imperatoxin A (IpTx), a
5-kDa peptide that
specifically and with high affinity increased
[
H]ryanodine binding and enhanced the activity of
single RyRs reconstituted in lipid bilayers(27) . At
concentrations well above the half-maximal effective concentration
(ED
) exhibited for RyRs, IpTx
did not affect
other Ca
channels or ion transporters of muscle and
brain. The binding of a radiolabeled derivative of IpTx
to
skeletal SR was displaced by ruthenium red with a K
of
1 µM, (
)a concentration
similar to that required for the displacement of
[
H]ryanodine from purified receptors when assays
are performed under high salt concentration(23) . All these
effects could be seen only on skeletal RyRs, suggesting that IpTx
preferentially affects the RyR1 isoform.
In this study, we
carried out an in-depth analysis of the action of IpTx on
rabbit skeletal RyRs. We show that IpTx
binds directly to
RyRs, or a closely associated regulatory protein, to produce a change
in channel kinetics that results in an increased probability of the
channel being open. By increasing the RyR sensitivity to
Ca
, IpTx
relieves the inhibition of RyRs
caused by physiological concentrations of Mg
.
Furthermore, the effect of IpTx
is specific, rapid, and
reversible, thereby overcoming some of the disadvantages of ryanodine.
IpTx
thus provides the field with alternatives to ryanodine
and may contribute unique information about modulatory domains of RyRs.
Figure 1:
Effect of IpTx on the
binding of [
H]ryanodine to several tissues. A, [
H]ryanodine (7 nM) was
incubated for 90 min at 36 °C with rabbit skeletal SR (0.3 mg/ml),
pig cardiac SR (0.4 mg/ml), rat cerebrum (0.45 mg/ml), and cerebellum
microsomes (0.5 mg/ml), and pig liver microsomes (0.35 mg/ml). In all
cases, the incubation medium was 0.2 M KCl, 10 mM Na-HEPES pH 7.2, 1 mM EGTA, and 0.997 mM CaCl
(10 µM free Ca
).
Nonspecific binding was defined as the binding of
[
H]ryanodine in the presence of 10 µM ryanodine and has been subtracted in this and subsequent figures.
The binding of [
H]ryanodine in the absence of
IpTx
(control, 100% specific binding) was 1.04 ±
0.16 (skeletal), 0.35 ± 0.08 (heart), 0.048 ± 0.013
(cerebrum), 0.061 ± 0.012 (cerebellum), and 0.046 ± 0.015
(liver) pmol/mg of protein. Bars indicate the mean
(±S.E., n = 3) of the percentage of binding
increment induced by IpTx
. B, kinetics of
association of [
H]ryanodine to skeletal RyR in
the absence and presence of 50 nM IpTx
. Data
points are the mean ± S.E. of three independent determinations.
Smooth line is a fit to data points using the formula B = B
t/(t
+ t), where B is the specific binding at
time t, B
is the amount of binding at
equilibrium, and t
is the half-time to reach
equilibrium.
Figure 5:
Dose-response relation of P as a function of IpTx
concentration. A,
single channel traces of a skeletal RyR activated by 10 µM Ca
in the absence (control) and the
presence of indicated the concentrations of IpTx
. Recording
conditions were the same as those described in the legend to Fig. 3and under ``Experimental Procedures.'' Note
different time scale. B, normalized closed-time histograms for
30-s long files obtained during control and following addition of 100
nM IpTx
. Total number of events was 1,293 and
5,938 for control and + IpTx
, respectively. The bin
width was 0.2 ms. C, logarithmic plot of P
as a function of [IpTx
]. Data points
represent the mean ± S.E. for an n = 8, 3, 5, 3,
4 and 6 for 0, 3, 10, 30, 50, and 100 nM IpTx
,
respectively.
Figure 3:
IpTx activates RyR channels
reconstituted in lipid bilayers. A, continuous recordings of a
single skeletal RyR in the absence of IpTx
. Channel
openings are presented as downward deflections in this and subsequent
figures. B, the same channel
1 min after addition of 10
nM IpTx
to the cis side. All records were low-pass
filtered at 2 KHz using an 8-pole Bessel filter and digitized at 5 KHz.
Holding potential = -30 mV. Current flows from trans
(intraluminal) to cis (cytosolic) side. Right panels, current
histograms constructed from a 100-s period of recording under each
condition. The single-channel current amplitude was -18 ±
2.7 pA before and -18.3 ± 2.4 pA after addition of
IpTx
.
Fig. 1B shows the time course of the effect of
IpTx (50 nM) on rabbit skeletal RyRs. The
stimulation of [
H]ryanodine binding by IpTx
was evident after short periods of incubation and approached
steady-state at
100 min. k
, the association
rate constants, were 0.0005 ± 0.0001 (control) and 0.0007
± 0.00015 nM
min
(+IpTx
). At equilibrium, the number of receptors
occupied by [
H]ryanodine was 1.13 (control) and
3.18 pmol/mg (+IpTx
). Thus, although IpTx
did not accelerate significantly the rate of binding, it did
increase the number of receptor sites available for binding. The
intrinsically slow association kinetics of
[
H]ryanodine precluded the accurate estimation of
IpTx
effects at early times, but the substantial increase
of binding detected at 3 min suggested that IpTx
had a fast
onset of effect.
Figure 2:
[H]Ryanodine binding
to purified skeletal RyR and activation by IpTx
. A, silver-stained SDS-PAGE gel of 50 µg of SR microsomal
protein (lane 1), and
1 µg of protein of the sucrose
gradient fraction No. 11 (
15.0% sucrose (lane 2). Arrows indicate the migration distance of M
markers. The
106-kDa protein labeled with an asterisk was identified as the Ca
-ATPase of SR by the
colorimetric method of Lanzetta (50) that determines P
derived from Ca
-dependent ATP hydrolysis. Lane 2, corresponding to
15% sucrose in panel B,
yielded a specific ATPase activity of 139 nmol of P
min
mg of protein
. In
contrast, fraction 3 (corresponding to
7.5% sucrose in panel
B, not shown) which contained no
106-kDa band, had no
measurable ATPase activity. B, protein concentration (open
circles) and [
H]ryanodine binding profile (filled circles) of the sucrose gradient fractions of
CHAPS-solubilized SR. 2 ml of solubilized SR were applied on top of a
32-ml 5-20% sucrose gradient and centrifuged at 80,000
g for 15 h. 1-ml fractions were collected and assayed for
[
H]ryanodine binding in 150 mM NaCl, 20
mM Tris maleate (pH 7.2), 50 µM CaCl
,
320 mM sucrose, and 0.1% CHAPS. Protein concentration was
determined by the Bradford assay. C, specific binding of
[
H]ryanodine to the purified receptor. Fractions
pooled from the 14.6 to 17.1% portion of the sucrose gradient were
incubated for 90 min at 36 °C with the indicated concentrations of
[
H]ryanodine in the absence (open
circles) or presence of 50 nM IpTx
(filled circles). Receptor concentration was 4
µg/ml. K
= 6.1 nM and B
= 29.3 pmol/mg (control) and K
= 5.7 nM and B
= 57.8 pmol/mg
(+IpTx
).
Figure 4:
Onset and reversibility of IpTx effect. A, representative single channel recordings of a
skeletal RyR activated by 10 µM Ca
before (control) and following addition of 10 nM IpTx
to the cis chamber. Traces labeled
``wash'' were taken from the same channel after
extensive perfusion of the cis chamber with a peptide-free solution. B, 6 min of continuous records spanning the whole experiment
were divided into intervals of 6 s; P
in each
interval is plotted as a bar of length 0-1. Empty spaces
are gaps in recording due to addition of IpTx
or perfusion.
Average P
for the channel shown was 0.14
(control), 0.36 (+ IpTx
), and 0.12
(washing).
Fig. 5C shows the log dose-response
relation of P as a function of IpTx
concentration. The mean, S.E., and number of observations at each
concentration (n) are summarized in the figure legend. In the
absence of IpTx
, with Ca
as the only
agonist of RyRs, the steady-state P
was
0.20.
IpTx
increased P
consistently and in a
dose-response manner. However, P
did not reach
1.0 even at saturating concentrations of the peptide. Data were
fitted with the equation:
On-line formulae not verified for accuracy
where (P max) is the P
observed at saturating concentrations of IpTx
(0.75),
ED
is the concentration of IpTx
that produces
half-maximal stimulation of activity (
10 nM), and nH is the Hill coefficient (1.15). Since nH was not
different from 1, this indicated that the binding of IpTx
to the RyR was not cooperative over the range of IpTx
concentrations assayed. The removal of long closings and the
increase of frequency of open events are consistent with the notion
that the stimulation of [
H]ryanodine binding
induced by IpTx
results from an overall increase in P
, which in turn favors the binding of the
alkaloid. From these results we concluded that our
[
H]ryanodine binding protocol was appropriate to
characterize the effect of IpTx
on a large population of
RyRs.
Figure 6:
Effect of IpTx on the
Ca
dependence of [
H]ryanodine
binding. The binding of [
H]ryanodine to rabbit
skeletal (A) and porcine cardiac (B) SR was performed
as described in the legend to Fig. 1, with the exception that
the incubation medium was supplemented with 1 mM EGTA and
several CaCl
concentrations to yield the indicated
[free Ca
]. Ca
:EGTA ratios
were calculated with a computer program using the stability constants
given by Fabiato(34) . IpTx
(100 nM) was
present throughout the incubation period. Data points are the mean
(±S.E.) of four independent determinations. Smooth lines linking data points have no theoretical
meaning.
In the absence of IpTx, the Ca
dependence of [
H]ryanodine binding to cardiac RyRs (Fig. 6B) also had a threshold
for detection at
pCa 7 and a maximum at pCa 5.
The EC
for activation of binding by Ca
was also
3 µM. However, a fundamental
difference with skeletal receptors was the lack of inactivation
produced by 1 mM [Ca
]. Binding
decreased only by
20% with respect to maximum in cardiac RyRs
while it decreased by
90% in skeletal RyRs. These results are
consistent with those of Chu et al.(37) , who failed
to detect inactivation of cardiac RyRs at 1 mM [Ca
]. In contrast to the dramatic
effect on skeletal RyRs, the effect of IpTx
on cardiac RyRs
was erratic, showing no effect at pCa 8 and 7, a modest
stimulation at pCa 6 (from 0.093 ± 0.015 to 0.23
± 0.08 pmol/mg protein, n = 3; values
significantly different at p = 0.05 by one-way analysis
of variance), and a modest, but statistically insignificant inhibition
at pCa 5 to 3. Therefore, the activation induced by IpTx
on cardiac RyRs occurred at one [Ca
]
and constituted a small fraction of that observed on skeletal RyRs.
Furthermore, in the presence of a low [Ca
]
(100 nM), a radiolabeled derivative of IpTx
failed
to bind to cardiac SR, but it did bind to skeletal SR(38) .
Taken together, the results indicate that the activating effect of
IpTx
on cardiac RyRs is restricted to a well defined range
of [Ca
] and is weak in absolute quantity.
Given these restrictions and the fact that intracellular
[Ca
] is a dynamic parameter of many cells
that express more than one RyR isoform, we propose that cardiac RyRs
are poor substrates for IpTx
. By contrast, the broad
Ca
dependence and conspicuous response of skeletal
RyRs make these receptor isoforms the preferred target of
IpTx
.
Figure 7:
Interaction of IpTx with other
modulators of RyRs. [
H]Ryanodine binding to
skeletal SR was performed in incubation medium containing 10 µM free Ca
. Light-shaded bars represent
the mean (±S.E., n = three independent
preparations) of binding obtained in the absence (control) and
the presence of 300 µM doxorubicin
(``DXR''), 3 mM AMP-PCP, 1 µM ruthenium red (Ru Red), 3 µM calmodulin (CaM), and 1 mM MgCl
. The specific
binding for control was 1.05 ± 0.11 pmol/mg of protein. The dark-shaded bars represent the mean (±S.E.) of
percentage of binding increment induced by 50 nM IpTx
in the absence (control), or the presence of the other
modulators.
When added individually, ruthenium red (3 µM),
calmodulin (3 µM), and free Mg (1
mM) decreased binding to 8 ± 2%, 49 ± 11%, and 6
± 3%, respectively. When IpTx
was added in tandem
with each of these inhibitors, not only was binding restored but
increased above control, to 180 ± 20%, 230 ± 28%, and 110
± 20%, respectively. Therefore, the interaction of IpTx
with RyRs relieves the inhibitory effect of ruthenium red, CaM,
and Mg
, and evokes a new level of RyR activity that
is higher than that observed in the absence of the inhibitors.
Figure 8:
Combined effect of IpTx with
caffeine or AMP-PCP. A, potentiation of IpTx
effect by caffeine. The binding of
[
H]ryanodine to skeletal SR was determined in
incubation medium containing 100 nM free Ca
(1 mM EGTA and 385 µM CaCl
).
Caffeine was added at the beginning of the incubation as 10-µl
aliquots from 100-fold stocks for concentrations up to 10 mM,
and as powder form to reach 30 mM. Open circles,
binding in the absence of IpTx
. Filled circles,
binding in the presence of 100 nM IpTx
. B, lack of synergism between IpTx
and AMP-PCP.
Binding of [
H]ryanodine to skeletal SR was
carried out in incubation medium containing 10 µM free
Ca
. Data points were obtained in the absence (Control, open circles), or the presence (+IpTx
) of 50 nM IpTx
plus the specified [AMP-PCP]. The datum labeled
``+ caffeine & IpTx
''
was obtained in the combined presence of 10 mM caffeine and 30
nM IpTx
in the absence of
AMP-PCP.
In contrast, the nucleotide-binding site appeared to be
independent of the IpTx-binding site. Fig. 8B shows the effect of incremental concentrations of AMP-PCP, a
non-hydrolyzable analog of ATP, on the binding of
[
H]ryanodine to skeletal SR in the absence and
presence of IpTx
. In these experiments, AMP-PCP was
preferred over ATP to avoid activation of both the
Ca
-ATPase of SR (which may alter the
[Ca
] of the incubation medium) and
endogenous protein kinases (which may alter the activity of the
RyR(42) ). In the absence of IpTx
, AMP-PCP
increased the binding of [
H]ryanodine from 0.87
± 0.09 to 2.20 ± 0.31 pmol/mg, i.e. a net gain
of 1.33 pmol/mg protein. Remarkably, in the presence of
IpTx
, binding increased by about the same amount, from 2.85
± 0.45 to 4.06 ± 0.38 pmol/mg, i.e. a gain of
1.21 pmol/mg protein. When IpTx
was combined with 30 mM caffeine, the binding increased to 5.1 ± 0.31 pmol/mg (filled circled labeled ``caffeine & IpTx
''). This result indicated that the combined
effect of IpTx
and AMP-PCP was not limited by the number of
receptor sites available for binding. Instead, the combined effect of
AMP-PCP and IpTx
was simply the sum of the effect of each
agonist acting separately.
Figure 9:
Inhibition of
[H]ryanodine binding by Mg
and
reversal of Mg
inhibition by IpTx
.
[
H]Ryanodine binding at the specified [free
Ca
] was determined as described under
``Experimental Procedures.'' The control data (open circles) were obtained in the absence of Mg
and IpTx
; filled circles are the data
obtained in the presence of 1 mM [free
Mg
], and filled squares are data
obtained in the combined presence of 1 mM [free
Mg
] and 100 nM IpTx
.
We have used two different parallel indicators of channel
function (i.e. [H]ryanodine binding and
direct single-channel recordings), to provide complementary lines of
evidence for a selective, fast and reversible activation of skeletal
RyRs by IpTx
. The near identity of dose-response curves for
the IpTx
effect on [
H]ryanodine
binding (27) and single-channel recordings (Fig. 5)
lends credence to the assumption that
[
H]ryanodine binding is proportional to P
, since it is difficult to have a systematic
error in both assays and maintain the similarity shown.
The
mechanism involved in the activation of skeletal RyRs by IpTx is qualitatively similar to that produced by caffeine (39) and the eudostomin-derivative MBDE(25) . The
latter compounds increase the affinity of the RyR for Ca
(``Ca
sensitization''), an effect
that is reflected as a selective shift of the ascending limb of the
Ca
dependence of P
curve to the
left. The descending limb of the curve, presumably reflecting binding
of Ca
to an inactivation site, is unaffected by these
compounds. The inactivation of RyRs by high Ca
is
only observed in skeletal RyR(37) , and its physiological
significance has been questioned(45) . At nanomolar
concentrations, IpTx
increased the amplitude and displaced
the midpoint of the ascending limb of the curve from 3 µM to 0.8 µM Ca
. The midpoint of the
inactivation curve remained unchanged (Fig. 6A).
Therefore, given the resemblance between the effect of caffeine and
IpTx
, it may be suggested that they both activate RyRs by
binding to a common receptor site. However, two lines of evidence
indicated that this is probably not the case. First, in the presence of
a saturating concentration of caffeine, IpTx
produced an
additional 6-fold increase in [
H]ryanodine
binding (Fig. 8), an effect that would not be expected if
occupation of the IpTx
site by caffeine had occurred.
Second, caffeine activates both the cardiac and skeletal RyR by a
mechanism similar to activation of skeletal RyRs by IpTx
.
If IpTx
occupied the same site as caffeine, IpTx
would also be expected to activate cardiac and skeletal RyRs
alike.
Following the same argument, the nucleotide-binding site
appeared to be different from and non-cooperative with the
IpTx site. This is evident in binding experiments where the
combined effect of the two agonists is simply the sum of each agonist
acting separately (Fig. 8). Furthermore, the nucleotide effect
spans the ascending and descending limbs of the Ca
dependence of [
H]ryanodine binding
curve(10) , as opposed to the selective effect of IpTx
on the ascending limb of the curve (Fig. 6). In addition,
single-channel experiments reveal that the kinetic parameters affected
by each agonist are different. While nucleotides activate RyRs by
increasing the frequency and duration of open events(46) ,
IpTx
does it by decreasing the duration and proportion of
the closed lifetimes (Fig. 5) without affecting the duration of
the open lifetimes. This different mechanism of action highlights a
distinctive functional output from each agonist site.
Since
IpTx fails to substantially activate cardiac RyRs (as well
as cerebrum and liver RyRs) (Fig. 1A and 6B,
see also (27) ), this in all likelihood reflects the existence
of a distinct modulatory site for IpTx
on RyR1. Thus, we
conclude that IpTx
may be used as a selective activator of
skeletal-type RyRs (RyR1). The significant stimulation obtained with
cerebellum microsomes (as opposed to cerebrum microsomes) is also
consistent with this notion, since there seems to be a relatively large
proportion of RyR1 in cerebellar Purkinje cells. But even so, we cannot
rule out that the IpTx
-binding site is altogether absent in
cardiac (RyR2) or brain (RyR3) isoforms. The Ca
dependence of [
H]ryanodine binding to
cardiac RyRs (Fig. 6B) was, albeit modestly, altered by
IpTx
. This suggests that IpTx
may interact,
directly or indirectly, with cardiac RyRs; however, the functional
consequence of such interaction is almost null and occurs only over a
narrow range of [Ca
]. By contrast,
IpTx
was highly effective in activating skeletal RyRs over
a wide range of [Ca
] (Fig. 6A). Thus, in systems where intracellular
[Ca
] fluctuates rapidly due to the
concurrent action of several RyR isoforms, as it does in non-mammalian
skeletal muscle, IpTx
is likely to target skeletal-type
RyRs and produce an exclusive and substantial modification of the
parameters directly controlled by these isoforms.
In both cardiac
and skeletal RyRs, binding was maximum when
[Ca] was
10-50 µM.
At this optimum [Ca
], the P
of single RyR channels was far from being 1.0 (P
0.2, Fig. 3Fig. 4Fig. 5), suggesting
that Ca
does not fully activate the channel when it
is present as the only activating ligand. However, it has been
established that if a fast and sustained
[Ca
] step is applied, channel activity
increases to
1.0 and then spontaneously decays to a new
steady-state level(47, 44) . The spontaneous decay of
RyR activity in the presence of a sustained
[Ca
] has been termed
``adaptation'' (47) by analogy to other receptors
that decrease their level of response despite persistent occupancy by
the agonist that initiated the response (48) . In the case of
RyRs, adaptation may be explained by the rapid association of
Ca
to a binding site (O-domain) that leads to channel
opening, followed by the slower binding of Ca
to
another binding site (A-domain) responsible for channel closing or
adaptation(49) . Thus, it is conceivable that the low P
of RyRs observed under our experimental
conditions is due to the absence of the transient peak of activity
evoked by a fast increment of [Ca
]. Since
[Ca
] remained constant during the course of
our experiments, we effectively tracked the activity elicited by the
binding of Ca
to the A-domain, which is indeed
proportional to the stationary Ca
concentration (44) but saturates at a P
<
1.0(49) . Within this scheme, the IpTx
-induced
increase of channel activity (Fig. 5) and
[
H]ryanodine binding (Fig. 6) at constant
[Ca
] is compatible with Ca
removal from the A-domain, either through a direct displacement
by IpTx
, or through a decrease of affinity resultant from
negative cooperativity between the IpTx
site and the
A-domain. An inadequate binding of Ca
to the A-site
would result in higher P
over the range of
[Ca
] in which the A-site causes adaptation.
For both cardiac and skeletal RyRs, this range is between 0.1 and
10-100 µM [Ca
](49) . However, this
conclusion awaits further experiments involving fast application of
Ca
to measure directly the parameters of these
Ca
-bindings domains and the effect of IpTx
on the A-site.
The intrinsic properties of IpTx and ryanodine confer to each ligand a unique set of attributes
that may both restrict and promote their use as probes of RyRs. The
activating effect of IpTx
on skeletal RyRs is fast and
fully reversible (Fig. 4). In this respect, IpTx
is
more advantageous to physiologists than ryanodine, which is too slow to
bind and dissociate from its effector site to attain equilibrium during
the course of experiments with intact cells. On the other hand, it was
precisely the slow dissociation of ryanodine that facilitated the
purification of the receptor(24) . As a natural peptide,
IpTx
is suitable for iodination, and for attachment of
cross-linkers for covalent binding to the receptor protein, thus
offering the prospect of receptor tagging and mapping. The preferential
effect of IpTx
for the skeletal RyR may be extremely useful
to dissect the role of RyR1 in systems where complex Ca
waveforms represent the functional output of several RyR isoforms
acting in concert. This very feature, however, may limit the use of
IpTx
in a wide variety of cells where the levels of
expression of RyR1 is low or totally absent. Finally, the results
provided here do not allow us to safely ascertain if the binding of
IpTx
, like that of ryanodine, is dependent on the
conformational state of the receptor. Although the effect of IpTx
was potentiated by ligands that open RyRs such as Ca
(Fig. 6) and caffeine (Fig. 8), an unequivocal
demonstration that IpTx
binds to the open receptor must be
provided by experiments where IpTx
is directly used as the
radiolabeled ligand.
Amino acid analysis indicates that IpTx is a positively-charged
5-kDa peptide rich in basic
residues. (
)Thus, it is important to note that although
IpTx
may contribute considerably to the understanding of
the structure-function relationship of the RyR in isolated SR vesicles
or with the purified receptor, its usefulness in intact cells may be
limited by its incapacity to permeate the external membrane. In this
respect, the lipophylic nature of ryanodine is clearly advantageous.
However, the possibility that IpTx
cannot reach its
receptor raises several questions. Is the skeletal RyR its intended
target? And if it is not, how can IpTx
display such an
exquisite selectivity and high affinity? Is IpTx
, like Escherichia coli enterotoxin, allowed into the cell by another
molecular component of the venom? Or are there instances yet unknown
where the RyR (or a structurally related protein) is expressed in the
external membrane? In all cases the possibilities are intriguing.