Diketopyridylryanodine Has Three
Concentration-dependent Effects on the Cardiac
Calcium-release Channel/Ryanodine Receptor*
Keshore R.
Bidasee
§¶,
Le
Xu§
,
Gerhard
Meissner
, and
Henry R.
Besch Jr.**
From the
Department of Pharmacology, University of
Nebraska Medical Center, Omaha, Nebraska 68198, the
Departments of Biochemistry and Biophysics and Cell and
Molecular Physiology, University of North Carolina, Chapel Hill, North
Carolina 27599-7260, and the ** Departments of Pharmacology
and Medicine and Krannert Institute of Cardiology, Indiana Center for
Vascular Biology and Medicine, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received for publication, August 16, 2002, and in revised form, January 10, 2003
 |
ABSTRACT |
By interacting with more than one site, ryanoids
induce multiple effects on calcium-release channels. To date, the
kinetics of interaction of only one of these sites has been
characterized. Using
C4,C12-diketopyridylryanodine in both
[3H]ryanodine binding and single channel experiments we
characterized another site on the cardiac ryanodine receptor (RyR2)
with which ryanoids interact. Competitive binding of this ryanoid to
RyR2 implied a minimal two-site binding model. At the single channel level, C4,C12-diketopyridylryanodine induced
three distinct effects. At nanomolar concentrations, it increased
channel open probability severalfold without inducing a subconductance.
This effect was independent of membrane holding potential. As for other
ryanoids, low micromolar concentrations of
C4,C12-diketopyridylryanodine readily induced a
subconductance state. The major subconductance had a current amplitude
of 52% of fully open, it was reversible, and its time to induction and
duration were voltage- and concentration-dependent, affording
Hill slopes of >2. At higher micromolar concentrations C4,C12-diketopyridylryanodine induced long
lasting, yet reversible shut states. Using a pharmacological strategy
we have discerned an additional ryanoid-binding site on RyR2 that
triggers an increase in channel activity. This site likely resides
outside the strict confines of the transmembrane conducting pathway.
 |
INTRODUCTION |
Ryanodine receptor calcium release channels
(RyRs)1 are present in almost
all mammalian cells. They play an integral role in releasing calcium
ions from the internal sarco(endo)plasmic reticulum to mediate
distinct cascades of events that culminate in such vital functions as
muscle contraction, neurotransmitter release, hormone secretion, and
lymphocyte activation (1-7). Three distinct isoforms from mammalian
tissues have been cloned from three genes and are designated RyR1
(predominant in skeletal muscle), RyR2 (predominant in cardiac muscle),
and RyR3 (first isolated from brain but present in several tissues).
The plant alkaloid ryanodine is the most recognized xenomodulator
of RyRs. In calcium efflux assays using junctional sarcoplasmic reticular membrane vesicles from fast skeletal muscle, low micromolar concentrations of ryanodine activate (i.e. open) RyR1,
whereas higher concentrations deactivate or close them (8-10). At the single channel level two effects of ryanodine are typically observed, namely induction of a persistent subconductance state and long-lasting channel closure (11-13). The latter is termed the shut state to distinguish it from brief closures that occur spontaneously (14). These
two effects are reminiscent of channel activation and deactivation seen
at the aggregate channel level in membrane vesicles.
In a recent study using frog twitch fibers we observed that prior to
induction of the subconductance state (seen as a steady glow in this
preparation) and channel shutting (recorded as inhibition of
depolarization-evoked calcium release), ryanodine increased the
frequency of spontaneous sparks (15). This increase in sparking frequency, similar to that reported by Gonzales et al. (16), suggests that in addition to the two well documented effects, ryanodine
may also induce an earlier effect, namely an increase in channel gating
frequency. Two prior studies suggested that nanomolar concentrations of
ryanodine may increase the probability of RyR opening
(Po) without inducing the subconductance state (17, 18). Because none of these earlier studies recorded dose dependence, ryanodine per se appears unable to discriminate
between functional sites that induce increases in channel gating and
those producing the subconductance state. Identifying ryanoids that can
discriminate between sites having different functional effects is key
to elucidating the phenomena behind ryanoid-induced changes in RyR properties.
In a previous study we found that pyridylryanodine
(C3-O-[pyridylcarbonyl]ryanodol) has
activating potency and efficacy similar to those of ryanodine, despite
its lower affinity (14). These data suggested that pyridylryanodine or
a derivative thereof might be able to discriminate between putative
subpopulations of binding sites on RyR. It is also likely that a
ryanoid belonging to this group might bind reversibly to RyR2, because
like three other reversible ryanoids reported in the literature, it too
has a substituent on the A-ring (C3 carbon) that differs
from that of ryanodine (6, 12). We also reasoned that additional
reversibility and subsite selectivity might be engineered into
pyridylryanodine by relaxing a ring constraint of its skeleton
backbone. In the present study we used binding and single channel
experiments to show that a modified congener of pyridylryanodine,
namely C4,C12-diketopyridylryanodine (C3-O-[pyridylcarbonyl]C4,C12-seco-C4,C12-dioxoryanodol)
induces three distinct and separate concentration-dependent
effects on canine RyR2. For comparison, we also detail the binding and
single channel properties of its parent ryanoid, pyridylryanodine.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The pyridylryanodine used in this study was
isolated from chipped Ryania wood supplied by Integrated
Biotechnology Corporation (Carmel, IN) and purified by chromatography
to
99% (19). [3H]Ryanodine (specific activity 87 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston,
MA). Precoated silica gel plates (with fluorescence indicator) were
obtained from Sigma. Lipids were obtained from Avanti Polar Lipids Inc.
(Alabaster, AL). All other reagents, buffers and solvents used were of
analytical grade.
Synthesis of
C4,C12-Diketopyridylryanodine--
Synthesis
of C4,C12-diketopyridylryanodine (for
structure, see Fig. 1) was carried out as described (20, 21). Briefly, 25 mg (49 µmol) of pyridylryanodine was dissolved in 2 ml of methanol and 113 mg (588 µmol) of periodic acid was added. The solution was
stirred for 2 h at room temperature, at the end of which 1 ml of
water was added. The reaction mixture was then extracted with 3 × 25 ml of methylene chloride. The pooled methylene chloride fractions
were dried over anhydrous sodium sulfate and rotary evaporated to
dryness. The residue was redissolved in 2 ml of methylene chloride and
chromatographed on a silica gel column (20 g), eluting sequentially
with 100 ml each of methylene chloride, methylene chloride/2%
methanol, and methylene chloride/4% methanol. The fractions containing
the product of interest were pooled and rotary evaporated to dryness
before being redissolved in dioxane for freeze drying. The structure of
the product was confirmed using 1H and 13C NMR
and electrospray mass spectrometry. Purity was ascertained using thin
layer chromatography (spraying with 5% ceric ammonium nitrate in 80%
phosphoric acid and charring), analytical high performance liquid
chromatography, and electrospray mass spectrometry.
Preparation of Sarcoplasmic Reticular Vesicles and
Purification of RyR2--
Animal procedures used in this study were
approved by Institutional Animal Care and Use Committees. Crude
sarcoplasmic reticular membrane vesicles (a mixture of junctional and
longitudinal) were prepared from canine heart as described previously
(22), except that aprotonin (0.5 µg/ml), pepstatin A (0.5 µg/ml),
and benzamide (80 µM) were added to isolation,
homogenization, and storage buffers. These vesicles were used for
[3H]ryanodine displacement binding affinity assays to
determine the affinity of ryanoids for RyR2. Junctional sarcoplasmic
reticular membrane vesicles were prepared in the presence of the
protease inhibitors, using discontinuous sucrose gradients as described previously (23). Junctional sarcoplasmic reticular membrane vesicles
were solubilized with CHAPS and 30 S RyR2 complexes were isolated by
rate density gradient centrifugation and reconstituted into
proteoliposomes (24).
Relative Binding Affinities of the Ryanoids--
The affinities
of ryanodine and pyridylryanodine for RyR2 were determined from their
ability to compete with [3H]ryanodine for binding sites
on the receptor (20, 22). The affinity of
C4,C12-diketopyridylryanodine for RyR2 was
determined as described previously, except that 1 µM
ryanodine was used to determine nonspecific binding to accommodate the
lower affinity and limited aqueous solubility of the diketone. The
binding displacement data were fit by nonlinear regression to one- and
two-site competition models given by equations,
|
(Eq. 1)
|
|
(Eq. 2)
|
where Y is specific binding, X is the
logarithm of the concentration of the unlabeled ligand, and log
EC50 is the competitor concentration that displaces half of
the specifically bound [3H]ryanodine. Goodness of fit was
evaluated by F tests and the simpler model was chosen unless
the more complex model provided a better fit at p < 0.05. Equilibrium dissociation constant (Kd) values
were ascertained using the Cheng-Prusoff relationship (25) given
by,
|
(Eq. 3)
|
where L is the concentration of
[3H]ryanodine used (6.7 nM) and
KL is the equilibrium dissociation constant of
[3H]ryanodine (1.2 nM for RyR2 (22)).
Single Channel Measurements and Analyses--
Phospholipid
bilayers were formed from a suspension of
phosphatidylethanolamine:phosphatidylserine:phosphatidylcholine in n-decane (in a ratio of 5:3:2 in a total of 35 mg of
phospholipid/ml of n-decane) across a 200-µm diameter
hole. Proteoliposomes containing the purified RyR2 were fused with the
bilayer. The side of the bilayer to which the proteoliposomes were
added was designated the cis side. The trans side
was defined as ground. Single channels were recorded in symmetric KCl
buffer solution (0.25 M KCl, 20 mM K-Hepes, pH
7.4) with 2 µM calcium or as specified. In this study,
free calcium concentrations were titered against EGTA. Ryanoids were
made up in 100% dimethyl sulfoxide at concentrations up to 500 times
higher than that anticipated for use in the bilayer bath. Accordingly,
after dilution in recording buffer the dimethyl sulfoxide concentration
in the bath was always less than 1%. All experiments were carried out
at room temperature (23-25 °C) and for all data shown, the drugs
were added only to the cis chamber. Electrical signals were
filtered at 2 kHz, digitized at 10 kHz, and analyzed as described (26,
27). Data acquisition and analyses were performed using commercially
available instruments and software packages (Axopatch 1D, Digidata
1200A or 1322A and pClamp 8.2, Axon Instruments, Burlingame, CA).
Normal gating mode is defined to include periods of rapid channel
transitions between the closed and full open states, and subconductance
mode is defined to include periods in which the channel exhibits a
persistent reduced conductance. At high ryanoid concentrations the
channel may transition into the shut state and this is distinguished
from the brief normal forays to the closed state in the absence of
ryanoid. The magnitude of the subconductance state was calculated by
dividing the amplitude of the subconductance induced by the ryanoid by
the conductance of the channel prior to ryanoid interaction (full
conductance). Amplitudes were monitored by manual positioning of
cursors at each level corresponding to closed, subconductance, and open
conductance states. The probability of the subconductance state
(Ps) was calculated from the dwell time in the
subconductance divided by the sum of times in normal gating and
subconductance modes.
Cumulative dwell time histograms in normal gating and in the
subconductance mode were obtained by nonlinear regression fits to
single and dual phase exponential functions given by the equations,
|
(Eq. 4)
|
|
(Eq. 5)
|
where K is the time constant and T is
time.
norm and
sub are calculated as
0.69/K. Goodness of fit was evaluated by F tests and the
simpler model was chosen unless the more complex model provided a
better fit at p < 0.05. From the single exponential fit, Kon and Koff were
obtained, according to the following defining equations,
|
(Eq. 6)
|
|
(Eq. 7)
|
|
(Eq. 8)
|
where X is the concentration of ryanoid and
V is the transmembrane holding potential. At a given ryanoid
concentration in solution and at a fixed holding voltage, the apparent
dissociation constant is defined by the following.
|
(Eq. 9)
|
Dose-response relationships to determine apparent dissociation
constants KH(V) at voltages other than 0 mV and
Hill slopes (nH) were determined by nonlinear
regression fit to the general four parameter logistic equation,
|
(Eq. 10)
|
where Ps is the probability of occurrence
of the subconductance state (at a given holding potential),
Ps(min) and Ps(max) are
minimum and maximum probability of substate occurrences, X is the log concentration of the drug, KH(V) is
the log concentration of drug required to elicit
Ps(0.5) and nH is the
Hill slope (28). For a reaction that does not exhibit cooperativity,
the Hill slope is 1.0. Goodness of fit was evaluated by F
tests and the simpler model was chosen unless the more complex model
provided a better fit at p < 0.05.
The relationships between Kon and
Koff and holding potentials were fitted using
the defining following equations.
|
(Eq. 11)
|
|
(Eq. 12)
|
The relationship between Ps and holding
potential was fit to the Boltzmann equation,
|
(Eq. 13)
|
where ztotal is the voltage dependence of
the subconductance state induced by ryanoid interacting with RyR2,
F is the Faraday constant, V is the transmembrane
voltage, and Gi/RT is constant at a holding
potential of 0 mV.
 |
RESULTS |
Synthesis of
C4,C12-Diketopyridylryanodine--
A strategy
similar to that used for the synthesis of
C4,C12-diketoryanodine and
C4,C12-diketo-9,21-didehydroryanodine (20, 21)
was used for the synthesis of
C4,C12-diketopyridylryanodine (Fig.
1). Using this method pyridylryanodine
underwent rapid and selective modification with periodic acid yielding
C4,C12-diketopyridylryanodine as the primary
product. Using gravity silica gel chromatography, the product eluted
from the column with methylene chloride/4% methanol and after rotary
evaporation and freeze-drying afforded 18 mg (72% yield). The
structure of this compound was confirmed using 1H and
13C NMR. The purity of
C4,C12-diketopyridylryanodine was determined to
be greater than 99.5% using analytical high performance liquid chromatography (methanol/water, 1:1 as mobile phase, UV detection at
260 nm). Non-UV active contaminants were sought using thin layer
chromatography (after spraying the plate with 5% ceric ammonium nitrate in 80% phosphoric acid and charring) and electrospray mass
spectrometry. None of these methods revealed contaminants.

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Fig. 1.
Preparation of
C4,C12-diketopyridylryanodine from
pyridylryanodine. Reaction conditions and reagents are described
in the text. Configurations shown represent projections of globally
minimized structures (MM2), using the algorithms of the software
packages of CS Chem 3D ProTM version 5 (Cambridge
Scientific Computing, Inc., Cambridge, MA).
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RyR2 Binding Affinities of Ryanodine, Pyridylryanodine, and
C4,C12-Diketopyridylryanodine--
We have yet
to synthesize radiolabeled forms of pyridylryanodine or its congener
C4,C12-diketopyridylryanodine. As such, the affinities of these ryanoids for RyR2 were determined from their ability to compete with 6.7 nM [3H]ryanodine
for binding to RyR2 using equilibrium displacement binding affinity
assays. The affinity data for all three ryanoids used in this study
were fit by nonlinear regression to one- and two-site competition
models given by Equations 1 and 2 under "Experimental Procedures."
The data for ryanodine and pyridylryanodine fit well to the one-site
binding model (r2 > 0.99 and
syx < 3.7 for both) yielding IC50 of
values of 7.8 ± 0.4 nM (Kd = 1.2 nM ± 0.2 nM) for unlabeled ryanodine and
716.8 ± 13.3 nM (Kd = 108.8 ± 5.4 nM) for unlabeled pyridylryanodine (Fig.
2). For
C4,C12-diketopyridylryanodine, however, the
two-site model given in Equation 2 provided a significantly better fit
(p = 0.005). The higher affinity site comprises 21% of
the total binding sites and has an IC50 of 299.5 ± 49.1 nM (Kd = 45.4 ± 10.4 nM) while the second site constitutes the remainder having
an apparent IC50 of 72,170 ± 2169 nM
(Kd = 10,962.5 ± 452.8 nM).
Extension of the two-site model to a three-site model failed to improve
the fit. Also, in preliminary experiments incubation was stopped after
1 and 3 h. Results obtained were similar to those after 2 h,
although Bmax was optimal after 2 h
incubation.

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Fig. 2.
Relative affinities of ryanodine,
pyridylryanodine, and
C4,C12-diketopyridylryanodine for RyR2.
[3H]Ryanodine displacement binding affinity assays were
used to determine the affinities of the ryanoids for RyR2. Briefly, SR
membranes (0.1 mg of protein/ml) were incubated for 2 h at
37 °C in the presence of 6.7 nM
[3H]ryanodine and varying concentrations of the
designated unlabeled ryanoids, in 250 mM KCl, 20 mM Tris-HCl, 0.1 mM EGTA, and 0.3 mM CaCl2 (pH 7.4 at 37 °C). At the end of
the incubation, the vesicles were filtered and washed.
[3H]Ryanodine bound to the receptors was determined by
liquid scintillation counting. Data for each compound represent the
mean ± S.E. from four experiments using two different SR
preparations. Curves were fitted using the binding analysis programs of
GraphPad Prism 3.0a (PrismPad Software Inc., San Diego, CA).
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|
Functional Effects of Pyridylryanodine on Single RyR2
Channels--
From previous studies we hypothesized that the biphasic
effects of ryanoids on RyRs represent interactions with two
functionally distinct binding site classes (10, 29). Pyridylryanodine
is unique among ryanoids investigated so far in that with calcium flux
assays it functionally separates activation from deactivation while
preserving the full extent of both. So separate are the dual
concentration-effect curves of pyridylryanodine that their intersection
presents a plateau, in marked contrast to the peak typical of other
ryanoids (14). The present studies on single RyR2 in bilayers provide
an explanation for the plateau: pyridylryanodine increases
Po in two concentration-dependent
steps as described below.
At the threshold concentration of 500 nM pyridylryanodine
(in a final concentration of 0.02% dimethyl sulfoxide in the
cis recording bath) channel Po was
significantly increased within 2 min (Fig.
3) from 0.05 ± 0.01 to 0.14 ± 0.02 at +60 mV, and from 0.06 ± 0.01 to 0.22 ± 0.5 at
60
mV, in four of five experiments. The Po increase
resulted from increases in both the frequency of transitions between
closed and full open states (number of events increased from 60 ± 5 to 200 ± 18 per s) and the mean dwell time in the open state
(from 0.5 ± 0.1 to 1.5 ± 0.3 ms). Po
increases were seen equally at positive and negative holding
potentials. This effect was not observed when dimethyl sulfoxide alone
(with 1.0% dimethyl sulfoxide Po increased by
126 ± 32% as of control, n = 7) or when lower
concentrations of pyridylryanodine (50-200 nM in dimethyl
sulfoxide) were added to the cis chamber, even after 15 min.

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Fig. 3.
Effects of submicromolar concentrations of
pyridylryanodine on RyR2. Single channel currents were recorded at
+60 mV (left panels, upward deflections) and 60 mV
(right panels, downward deflections) in symmetric
KCl/K-Hepes buffer, pH 7.4, as described in the text. The upper
panels represent control recordings in the presence of 2 µM cytosolic Ca2+ before addition of drug or
solvent to the cis chamber (Po = 0.05 at +60 mV and 0.07 at 60 mV). The middle panels represent
control recordings in the presence of 2 µM cytosolic
Ca2+ after addition of 1.0% dimethyl sulfoxide to the
cis chamber (Po = 0.06 at +60 mV and
0.08 at 60 mV). The lower panels show a typical increase
in Po induced by 500 nM
pyridylryanodine (Po = 0.14 at +60 mV and 0.22 at 60 mV).
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Doubling the cis concentration of pyridylryanodine to 1 µM readily induced a persistent subconductance state of
RyR2 (Fig. 4). The current amplitude of
this subconductance state was 0.32 ± 0.01 (n = 12) of fully open (764 ± 6 versus 246 ± 9 pS).
Whereas the subconductance amplitude characterizes different ryanoids, the appearance of a subconductance is common among all ryanoids. Induction of the subconductance state of pyridylryanodine was more
likely to occur and persisted for longer times at positive holding
potentials. In fact, the time to modification and probability of
occurrence of this subconductance state was about 6.5 times more likely
to occur at positive holding potentials than at negative potentials.

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Fig. 4.
The effects of low micromolar concentrations
of pyridylryanodine on RyR2. A, single channel currents
were recorded at +35 mV (left panels, upward deflections)
and 35 mV (right panels, downward deflections) in
symmetric KCl/K-Hepes buffer, pH 7.4. The upper panels
represent control recording in the presence of 2 µM
cytosolic Ca2+ before addition of pyridylryanodine to the
cis chamber. Po was 0.11 (left) and 0.06 (right). The lower
panels show typical subconductance states induced by 1 µM pyridylryanodine at +35 mV and
Po in normal gating mode was 0.19 (left
panel) and its reversal at 35 mV (right) and
Po in normal gating mode was 0.32. Panel
B shows the I/V curves for the experiment. Black
circles (behind the open circles) represent
channel full open state before pyridylryanodine was added and
open circles represent the full open state after addition of
1 µM pyridylryanodine. Open triangles reflect
the pyridyl-induced subconductance state. Pyridylryanodine did not
significantly change the conductance of full open state and the
magnitude of the subconductance state represents 32 ± 1% of full
open. Data shown is representative of four experiments.
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It should be pointed out that in most experiments (five of seven), the
Po of RyR2 in the normal gating mode immediately
prior to and after reversal of the subconductance state was higher than the channel Po prior to addition of the drug
(Fig. 4A, lower panels). As with most other
ryanoids, detailed kinetic analyses of the interaction of
pyridylryanodine with the site that induces the subconductance state
were precluded by its slow dissociation kinetics.
In one of five experiments pyridylryanodine (10 µM)
produced an alternate subconductance state, which had a current
amplitude of 60% of full open (data not shown). The dwell time in this
subconductance state lasted tens of seconds to minutes. This second
subconductance state could not have been due to an impurity because
three sensitive analytical techniques (see "Experimental
Procedures") all indicated the presence of only pyridylryanodine in solution.
At concentrations greater than 10 µM, pyridylryanodine
readily induced a reversible shut state of the channel (Fig.
5). Although induction of this shut state
occurred both at
60 and +60 mV, the dwell time was shorter at +60 mV
(1.1 ± 0.2 compared with 1.9 ± 0.3 s,
n = 6). Mechanistic interpretation of the shorter dwell
time at positive holding potentials is confounded by the voltage
dependence of induction of the subconductance state. At
60 mV,
Ps is small so that shutting can occur without
prior intervention of the subconductance state. As shown in Fig. 5, at
+60 mV the channel reverts from shut back to the subconductance state,
whereas at
60 mV it reverts from shut back to the normal gating
mode.

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Fig. 5.
The effects of high micromolar concentrations
of pyridylryanodine on RyR2. Single channel currents were recorded
at +60 mV (left traces) and 60 mV (right
traces) in symmetric KCl/K-Hepes, pH 7.4. These traces show that
pyridylryanodine (20 µM) can trigger a reversible closed
state of RyR2 at both positive and negative holding potentials.
However, at +60 mV RyR2 typically closes from the subconductance state
and returns back to the subconductance state, whereas at 60 mV, RyR2
transitions from the normal gating mode (high
Po) to the closed state and back to the normal
gating mode without entering the subconductance state. It should also
be mentioned that the dwell time in the closed state was twice as long
at 60 mV than it was at +60 mV. Data shown are representative of six
experiments.
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Taken together, these results show that pyridylryanodine is able to
induce three distinct dose-dependent effects on single RyR2
channels. At nanomolar concentrations it increases
Po without altering unitary conductance, at low
micromolar concentrations it induces a major subconductance state, and
at higher concentrations it causes a persistent but nevertheless
reversible shut state. These three functional effects were not
predicted from the binding studies with RyR2 in vesicles, perhaps
because of its weak subsite selectivity. Moreover, high affinity of
pyridylryanodine precluded detailed kinetic analyses.
Effects of C4,C12-Diketopyridylryanodine on
RyR2 Single Channels at Nanomolar Concentrations--
A
pyridylryanodine derivative with greater subsite selectivity and faster
kinetics was therefore desirable. We reasoned that both might be
achieved by reducing the rigidity of the ryanoid skeletal backbone. Two
principal ways to relax the skeletal backbone of pyridylryanodine are
through partial oxidation of either (i) the vicinyl diols on
C4 and C12 with concomitant breakage of the carbon-carbon bond that is shared by the A and B rings, or (ii) the
hemiacetal moiety of the D-ring, leading to breakage of the C1,C15 carbon-carbon bond and thereby
eliminating the E ring (20, 21). In this study we used the former,
affording C4,C12-diketopyridylryanodine (Fig.
1).
As shown in the example records of Fig.
6A, addition of 50 nM C4,C12-diketopyridylryanodine to
the cis chamber approximately doubled the channel
Po at holding potentials of +35 and
35 mV (184% at +35 mV and by 212% at
35 mV). This increased
Po was independent of the concomitant addition
of 0.02% dimethyl sulfoxide. Closer examination of the recordings
showed that the increase in Po resulted from
increases in gating frequency (the number of events/time) and mean open
time (Table I). Data from nine experiments show that the normalized Po was
significantly increased by 50 nM
C4,C12-diketopyridylryanodine equally at both
holding potentials, to mean values of 165 ± 30% at +35 mV and
183 ± 30% at
35 mV (Fig. 6B).

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Fig. 6.
The effects of submicromolar
C4,C12-diketopyridylryanodine concentrations on
gating characteristics of RyR2 in the presence of 2 µM
cis Ca2+. A, single channel currents
were recorded at +35 mV (left panels, upward deflections)
and 35 mV (right panels, downward deflections) in
symmetric 0.25 M KCl, 20 mM K-Hepes, pH 7.4, buffer containing 2 µM free Ca2+. Top
traces show control Po at +35 mV (0.19, left panel) and 35 mV (0.26, right panel).
Lower traces show increases in Po
after the addition of 50 nM
C4,C12-diketopyridylryanodine to the
cis chamber. At +35 mV Po increased
to 0.35 whereas at 35 mV Po increased to 0.55 (right panel). B, the relationship between
normalized Po and
C4,C12-diketopyridylryanodine concentration at
±35 mV are shown. The solid (-) line represents the fit of
the pooled data at ±35 mV using non-linear regression analysis (four
parameter logistic equation). Apparent dissociation constant
KH = 68 ± 16 nM (±35 mV) and
Hill slope (nH) of 1.7 ± 0.8 were
obtained. The dashed and dotted lines show the
fit at +35 mV ( ) and 35 mV ( ). Whereas the data at 35 mV was
accommodated by the logit function (Equation 10) affording
Po(max) = 386.6 ± 42.2%,
KH = 125.7 ± 13.1 nM, and Hill
slope = 1.0 ± 0.9, the data at +35 mV did not. Data are the
mean ± S.E. of 4 to 11 experiments.
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Table I
Channel parameters that are altered by 50 nM
C4,C12-diketopyridylryanodine
Control: 2 µM Ca2+, Po values
were 0.121 ± 0.043 and 0.133 ± 0.044 at 35 mV, and +35
mV, respectively. Topen and
Tclosed, mean open time and mean closed time,
respectively.
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Increasing the concentration of
C4,C12-diketopyridylryanodine incrementally
above 50 nM up to 1000 nM increased mean
channel Po, apparently in a
concentration-dependent manner. However, because of
experimental variation, no statistical differences could be shown
between mean values at +35 and
35 mV at each concentration. Thus, for
initial dose-response analyses these values were pooled. Concentration-effect relationships were assessed by nonlinear regression fit to the general four parameter logistic equation with
minimal Po = 100%. Goodness of fit was compared
between the version in which nH was constrained
to 1 and one in which regression was used to fit
nH. For the pooled data at ±35 mV (Fig.
6B, solid line, r2 = 0.73, sxy = 39.2), Po(max) was 328.3 ± 25.5%, the apparent dissociation constant KH
was 68 ± 16.0 nM and nH was 1.7 ± 0.8.
It was of interest to evaluate the possibility of concentration
dependence of the normalized Po data
independently at the two holding potentials. The interrupted lines of
Fig. 6B show this analysis. Above 50 nM
C4,C12-diketopyridylryanodine, dependence of
normalized Po on drug concentration was apparent
but only at the negative holding potential. The data at
35 mV are
well accommodated by the logit function (Equation 10) with values of
Po(max) = 386.6 ± 42.2%,
KH = 125.7 ± 13.1 nM, and Hill
slope = 1.0 ± 0.9. This site likely corresponds to the
higher affinity site detected in displacement binding assays. These
data represent the first clear example of a ryanoid that increases
channel Po in a
concentration-dependent manner without a transitioning into
a subconductance state.
Effects of C4,C12-Diketopyridylryanodine at
Micromolar Concentrations on RyR2 Single
Channels--
C4,C12-diketopyridylryanodine at
1 µM was the threshold concentration for induction of the
subconductance state, given prolonged incubation times in the
cis chamber. With advancing concentrations of
C4,C12-diketopyridylryanodine (2-20
µM) RyR2 quickly entered the reduced conductance state
with a current amplitude of 52 ± 1% of full open
(n = 9, Fig. 7). This
subconductance state was frequent at positive but only rarely seen at
negative voltages. On examination of the recordings at 5 µM, the increased channel gating previously seen at
nanomolar concentrations was apparent prior to induction and following
reversal of the subconductance state at +35 mV and also at
35 mV
where the subconductance did not occur (Fig. 7, lower
panels). Po in the normal gating mode increased from 0.11 to 0.44 at +35 mV and from 0.12 to 0.54 at
35 mV.
The subconductance amplitude was not different at the different drug
concentrations nor was the unitary conductance (data not shown).

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Fig. 7.
The effects of a low micromolar concentration
of C4,C12-diketopyridylryanodine on RyR2 in the
presence of 2 µM cis Ca2+.
Single channel currents were recorded at +35 mV (left
panels, upward deflections) and 35 mV (right panels,
downward deflections) in symmetric 0.25 M KCl, 20 mM K-Hepes, pH 7.4, buffer containing 2 µM
free Ca2+. The upper panels show the control
recording before addition of 5 µM
C4,C12-diketopyridylryanodine to the
cis chamber. Po was 0.11 at + 35 mV
(upper left panel) and 0.12 at 35 mV (upper right
panel). The lower left panel (+35 mV) shows that after
5 µM
C4,C12-diketopyridylryanodine was added to the
cis chamber, RyR2 entered into a reversible subconductance
state (52 ± 1% of full open). This subconductance state was not
observed at 35 mV (lower right panel).
Po in normal gating mode was 0.44 at +35 mV
(lower left panel) and 0.54 at 35 mV (lower right
panel).
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The ready reversibility of the
C4,C12-diketopyridylryanodine subconductance
state at positive holding potentials afforded the opportunity to
evaluate reversibility of this ryanoid under experimental conditions
designed to vitiate subtle Po effects. To
achieve a high initial Po, we used a bathing
calcium concentration of 25 µM, producing a control
Po of 0.90 ± 0.03 (n = 8, Fig. 8). Over a period of 9 s of
recording, six transitions from the normal gating mode to the
subconductance state occurred (Fig. 8B).

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Fig. 8.
Multiple subconductance states and
reversibility C4,C12-diketopyridylryanodine on
RyR2. Single channel currents were recorded at +35 mV in symmetric
0.25 M KCl, 20 mM K-Hepes, pH 7.4, buffer
containing 25 µM free Ca2+. A, the
upper two traces show a continuous control recording with
Po = 0.95. B, these continuous traces
show that after 5 µM
C4,C12-diketopyridylryanodine was added to the
cis chamber, RyR2 entered and reversed from the
subconductance state of 52 ± 1% of full open. C, this
panel shows an infrequently occurring subconductance state of 75% of
full open conductance.
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In addition to the major subconductance of 52%,
C4,C12-diketopyridylryanodine occasionally
induced a second subconductance of 75% of fully open (Fig.
8C). This subconductance is the largest yet reported for a
ryanoid. On one occasion, a third subconductance of ~25%
was observed. To evaluate whether these two additional subconductance
states might have resulted from contamination we used analytical high
performance liquid chromatography, thin layer chromatography, and
electrospray mass spectrometry. By all three of these techniques we
could find only C4,C12-diketopyridylryanodine in solution.
Kinetics of Interaction of
C4,C12-Diketopyridylryanodine with the Binding
Site That Triggers the Major Subconductance State--
The
reversibility of C4,C12-diketopyridylryanodine
permitted evaluation of its kinetics of interaction with the binding
site that induces the major channel subconductance. In the first series of these experiments, the dwell times for a channel in the normal gating and major subconductance states were monitored over 6 min at +35
mV in the presence of 10 µM
C4,C12-diketopyridylryanodine and 25 µM calcium. The distribution of dwell times in the normal and subconductance modes fit best to single exponentials
(r2
0.98, syx
2.88) and
afforded a mean dwell time in the unmodified state
(
norm) of 2450 ms, and in the modified state (
sub) of 335 ms (Fig. 9).
In the presence of 10 µM
C4,C12-diketopyridylryanodine K
= 0.0408 s
1
µM
1 and Koff = 2.98 s
1 were obtained using Equations 6-8. At 35 mV the
apparent dissociation constant KH for
C4,C12-diketopyridylryanodine is 73.0 µM (Equation 9). This value is slightly larger but in
reasonable agreement with the aggregate Kd of
10.9 ± 0.5 µM at 0 mV for the lower affinity
fraction of sites obtained using displacement binding assays at a
calcium concentration optimized for binding.

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Fig. 9.
Dwell time histograms in normal and
subconductance modes after addition of
C4,C12-diketopyridylryanodine. Cumulative
dwell time histograms in normal gating mode (left panel) and
in subconductance mode (right panel) were obtained from a
6-min recording recorded at 35 mV in symmetric 0.25 M KCl
buffer containing 25 µM cytosolic Ca2+. Both
dwell time histograms were fitted to single exponential function with
time constants of norm 2450 ms (left panel)
and sub 335 ms (right panel),
respectively.
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In the second series of experiments, the effect of
C4,C12-diketopyridylryanodine concentration on
Ps was evaluated at three increasingly positive
holding potentials. Increasing the drug concentration at a given
holding potential decreased the mean dwell time in the normal gating
mode and increased the mean dwell time in the subconductance state
(Table II). Thus as the drug concentration increased, the rate at which ryanoid molecules become bound increases and this in turn decreases the time to induction, as
well as increasing the duration of the subconductance state. Increasing
the holding potential from +20 to +50 mV at a given concentration of
C4,C12-diketopyridylryanodine did not affect the number of transitions between the subconductance and normal gating
mode (Table III). These results are
consistent with previous reports that the time to onset of the
subconductance state decreases as the holding potential increases (12,
26, 30). They are also consistent with the general notion that positive
holding potentials facilitate ryanoid interactions with RyR2.
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Table II
Channel parameters that are altered by
C4,C12-diketopyridylryanodine at +35 mV
In the absent of drug, Po = 0.90 ± 0.03 (n = 8). Derived data were obtained from single channel
recordings similar to those shown in Fig. 8B.
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Table III
Channel parameters that are altered with increasing transmembrane
holding potential at 10 µM
C4,C12-diketopyridylryanodine
In the absent of drug, Po = 0.90 ± 0.03 (n = 8). Derived data were obtained from single channel
recordings similar to those shown in Fig. 8B.
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In a third set of experiments association and dissociation rates were
investigated at 35 mV over the concentration range of 5-20
µM. Consistent with the data of Table II above, the
association rate constant of
C4,C12-diketopyridylryanodine with RyR2
increased as a function of concentration at +35 mV
(r2 = 0.99), affording an association rate
(slope of the curve) of 0.044 ± 0.004 s
1
µM
1 (Fig.
10,
). On the other hand,
dissociation from RyR2 was not statistically related to the
concentration of C4,C12-diketopyridylryanodine as its slope was not significantly different from zero
(p = 0.07), because of the large standard errors. The
rate of dissociation of
C4,C12-diketopyridylryanodine concentration
(Koff) was determined to be 2.8 s
1. Using the values obtained, KH
approximates 63.6 µM (35 mV) as calculated from Equations
11 and 12. This value is in close agreement with the
KH obtained using probability density function
curves.

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Fig. 10.
The dependence of association (open
circles) and dissociation (filled circles) rates on
concentration of
C4,C12-diketopyridylryanodine.
Kon and Koff were
obtained using Equations 6 and 8. K was
derived using Equation 7. Whereas Kon was
dependent on concentration of
C4,C12-diketopyridylryanodine,
Koff was not. Each point represents the
mean ± S.E. from three to five experiments.
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The dose-response relationships between concentration of
C4,C12-diketopyridylryanodine and
Ps at three holding potentials are summarized in
Fig. 11. The data fit well to the four
parameter logistic function (Equation 13) giving
r2 > 0.90 and syx
0.092 at all three holding potentials. Ps values at
+20 mV were doubled at +35 mV and nearly doubled again at +50 mV. The
apparent dissociation constant KH
(i.e. voltage-dependent Kd)
values were 29.0 ± 1.2 µM at 20 mV, 15.8 ± 1.2 µM at 35 mV, and 8.9 ± 1.0 µM at
50 mV and corresponding Hill slopes of 2.1 ± 0.1, 2.1 ± 0.1, and 2.3 ± 0.2. These data strongly suggests that induction
of the subconductance state results after binding of more than one
molecule of C4,C12-diketopyridylryanodine to RyR2. These results are internally consistent, in that the first molecule bound increases channel Po, whereas a
subsequent molecule induces the subconductance state.

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Fig. 11.
The relationship between
Ps and concentration of
C4,C12-diketopyridylryanodine at increasing
positive holding potentials. Ps was calculated
from recordings similar to those shown in Fig. 8A (25 µM Ca2+). Po in the
absence of the drug was 0.90 ± 0.03 (n = 8). The
lines represent the logit fits to the data using Equation 13. KH values of 29.0, 15.8, and 8.9 µM and nH values of >2 were
obtained for 20, 35, and 50 mV, respectively. Values shown represent
the mean ± S.E. of three to eight experiments.
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Because the channel dwell time in the subconductance state is
voltage-dependent, we further evaluated the relationship
between Ps and transmembrane holding potential
at 10 µM
C4,C12-diketopyridylryanodine. Fig.
12A represents the nonlinear
regression best fit of ln(Ps) as a function of
transmembrane holding potential (Equation 13). A slope of 0.055 (r2 = 0.99) was obtained and from this value, an
effective gating charge (ztotal) of 1.4 was
derived. The ztotal was also calculated from the
rates of association and dissociation of
C4,C12-diketopyridylryanodine at the three
holding potentials. As shown in Fig. 12B,
Kon increases as the holding potential is made
more positive; at the same time as Koff
decreases. The lines shown were obtained by nonlinear regression fit to the first-order polynomial equation. Values for
zon and zoff obtained
from the slopes of these lines were 0.60 and 1.1, respectively. This
afforded a total valence, ztotal of 1.7.

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Fig. 12.
Relationship between
Ps and holding potential at 10 µM
C4,C12-diketopyridylryanodine.
A, Ps was obtained from single
channel currents similar to those in Fig. 8B. Each point
represents the mean ± S.E. of three to five experiments. Points
were fitted using the Boltzmann equation (Equation 13 in text) and
afforded a ztotal of 1.4. B, the
relationship between ln(Kon) an
ln(Koff) at various positive holding potentials.
Each point represents the mean ± S.E. obtained from three to five
experiments (on open circles, S.E. values are too small to
be seen). zon of 0.6 and
zoff of 1.1 were obtained using Equations 11 and
12, respectively. ztotal = zon + zoff = 1.7. ztotal obtained from A and
B are very similar.
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Effect of a High Micromolar Concentration of
C4,C12-Diketopyridylryanodine on RyR2 Single
Channel Activity--
At a concentration of 250 µM
C4,C12-diketopyridylryanodine induced a long
lasting closed state that was nonetheless reversible (Fig.
13). Such shutting was observed in
three separate experiments. In each experiment, the dwell time in the
shut state lasted for tens of seconds. It should be pointed out that
the shutting closures induced by
C4,C12-diketopyridylryanodine at positive
holding potentials were much longer lasting (about 8 times) than that
caused by parent pyridylryanodine.

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Fig. 13.
The effects of high micromolar
concentrations of C4,C12-diketopyridylryanodine
on RyR2. These panels show continuous recordings obtained after
250 µM
C4,C12-diketopyridylryanodine was added to
the cis chamber. Three separate recording were carried out
using 25 µM Ca2+ in the cis
chamber. Note the long lasting, but reversible shut state.
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DISCUSSION |
The principal finding of the present study is that
C4,C12-diketopyridylryanodine and its parent
pyridylryanodine induce three distinct,
concentration-dependent effects on RyR2 single channels. These ryanoids differ from ryanodine in that their C3
hydroxyl carries a pyridyl substituent instead of a pyrrole. At
nanomolar concentrations the diketo derivative and pyridylryanodine
first increase channel Po (by ~3-fold) without
inducing a persistent subconductance state. The initial
Po increase consists of an increase in both
gating frequency and mean open dwell time. At higher concentrations, they induce the hallmark ryanoid subconductance state and at even higher concentrations they produce a persistent shut state that nevertheless is reversible. These effects are summarized in Scheme 1, where RyR2n represents the
normal gating of the channel, RyR2
reflects the voltage-independent increase in channel gating frequency,
RyR2
represents induction of the
subconductance state, and RyR2
represents the long lasting but reversible transition to the shut state. These three effects were more easily identified with
C4,C12-diketopyridylryanodine than they were
with pyridylryanodine. The first effect was hardly discernable when
control Po had been elevated to ~0.9 by
adjusting the calcium concentration to 25 µM.
Another major finding of the present study is that unlike its parent,
the chemically modified ryanoid
C4,C12-diketopyridylryanodine (having a
backbone-relaxed structure) is able to discriminate two populations of
ryanoid binding sites on RyR2. Analysis of the displacement binding
data revealed that of the total binding sites, 21% exhibit nanomolar
affinity for C4,C12-diketopyridylryanodine whereas the remaining 79% exhibit micromolar affinity. Because RyR2 is
the only isoform detected in canine left ventricle, either (a) the single isoform comprises two populations of binding
sites or (b) among the several energetically favored
conformations of C4,C12-diketopyridylryanodine,
there are conformers that can discriminate between multiple members of
a single class of binding sites on RyR2. Data from several laboratories
are supportive of the notion that RyR2 has more than one ryanoid
binding site. Because each effect recorded in the present study is
distinct and concentration-dependent (and one is also
voltage-dependent), it is likely that each effect is
induced by a ryanoid molecule binding to a separate site on a single
RyR2 molecule. Three concentration-dependent effects then
should result from serial binding of three ryanoid molecules. The
competition displacement studies (Fig. 2) discern only two. Because
effects on single channels in bilayers are more discriminant than
aggregate effects on multiple channels in vesicles, it is not unlikely
that within the two populations of binding sites, subpopulations may
exist that remain poorly discernable with binding affinity assays.
From here on, the discussion will focus primarily on the effects
induced by C4,C12-diketopyridylryanodine on
RyR2. References to the effects of pyridylryanodine will occasionally
be made for comparison. Within the nanomolar concentration range,
C4,C12-diketopyridylryanodine induces a
voltage-independent increase in Po (of
300%)
without a step-change to a channel subconductance. This increase in
Po first became evident with channels that were
submaximally activated at the start of the experiment but persisted
even when control Po had been elevated. The
dose-response data at
35 mV taken alone gave a Hill slope
(nH) of 1.0 suggesting that this effect requires binding of only a single molecule of
C4,C12-diketopyridylryanodine to RyR2. However,
given the data variance the alternate fit to the pooled (±35 mV) data
was equally valid statistically. The fit for the pooled data yielded a
nH of 1.7. The latter interpretation suggests
the possibility that more than one ryanoid molecule may participate in
the increased Po effect. Pyridylryanodine also increased the frequency of channel gating, but this required a concentration 10 times higher than that for
C4,C12-diketopyridylryanodine. Consistent with
recent reports (31-33) it seems reasonable to view the ryanoid-induced
increase in Po as resulting from destabilization of the closed state of the channel.
At low to intermediate micromolar concentrations
C4,C12-diketopyridylryanodine induces a
voltage-dependent major subconductance state of 52% of
fully open. Pyridylryanodine induced a smaller subconductance state.
These data are consistent with previous studies showing that the
magnitude of the subconductance states varies as a function of ryanoid
structure and that the frequency of occurrence of the substate
increases with increasing positive holding potentials (6, 12, 26). When
probability of occurrence of the major substate
(Ps) was plotted against concentration of C4,C12-diketopyridylryanodine at several
membrane holding potentials, Hill slopes of ~2 or greater were
obtained. These data suggest that substate induction occurs as a
consequence of the binding of more than one ryanoid molecule to RyR2.
These data differ from those for ryanodol (30) and
21-amino-9
-hydroxyryanodine (26), as the two latter compounds
display apparent bimolecular kinetics, consistent with there being only
a single ryanoid molecule needed for induction of the subconductance state.
Both C3-modified ryanoids used in this study induced more
than one subconductance state (32 and 60% for pyridylryanodine and 25, 52, and 75% for
C4,C12-diketopyridylryanodine). As
contamination was analytically ruled out, considerations of other means
by which a single ryanoid can induce more than one subconductance state are in order. Two explanations are likely. First, there may be alternate ryanoid-induced conformations of RyR2, depending on how many
ryanoid molecules have become bound per molecule of RyR2. Second,
multiple conformers of the ligand may each induce distinct conformations of the receptor.
Functional effects suggest at least three ryanoid binding sites per
RyR2 homotetramer. If the conductance amplitude of the subconductance
state ratchets down as each ryanoid molecule becomes bound to RyR2,
then one would expect that Ps should serially
diminish as each ryanoid molecule thereafter gets bound. With
C4,C12-diketopyridylryanodine, the observed
subconductance state of 75% could reflect binding of one ryanoid
molecule (in addition to the one bound to induce the increase in
channel Po), the subconductance of 52% could
reflect binding of a second additional molecule and the subconductance of 25% could reflect binding of a third additional ryanoid molecule. This leaves unanswered the question of why the 52% subconductance state should be the most frequent. It can be speculated that it is the
most stable. In parallel reasoning, the subconductance state of 60%
could reflect binding of one molecule of pyridylryanodine and the
subconductance of 32% could reflect binding of two such molecules. The
latter subconductance state might be more stable as it is more
frequently observed. The stepwise decrease in subconductance amplitude
with an increasing number of ryanoid molecules bound is supportive of
this hypothesis and is also consistent with the model described by
Pessah and co-workers (34).
On the other hand, multiple subconductances might reflect alternate
steric configurations of the ligand. Schliefer (35) suggested that
pyridylryanodine can exist in two alternate low energy conformers,
based on the arrangement of the pyridine nitrogen relative to that of
the carbonyl oxygen on C22 as indicated in Fig.
14A. These two conformers
might induce two distinct substates. The major substate would
presumably be the one that is induced by the predominant conformer.
However, it is indeterminate whether this may be the
E-/syn-conformer (oxygen and the nitrogen on the same sides)
or Z-/anti-conformer (oxygen and the nitrogen on opposite sides).

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Fig. 14.
Energy minimized conformations of
pyridylryanodine and
C4,C12-diketopyridylryanodine. The
illustrations depict the lowest steric energy forms for
pyridylryanodine and diketopyridylryanodine. These views are elaborated
from the studies of Jefferies et al. (21) and Schleifer
(35). The conformations were achieved by taking two-dimensional
representations of the ryanoids generated using ChemDraw Ultra software
(ChemOffice, version 5) and minimizing steric interactions using
MM2 minimization to define the given configurations of the molecules.
After minimization, the molecules were rotated to a standard
presentation of the C3 substituent. MOPAC data files were
then generated and physical parameters were obtained for
pyridylryanodine (A) and its modified congener
C4,C12-diketopyridylryanodine
(B(i)). Green lines are drawn to
reflect an arbitrary dihedral angle between the skeletal backbone and
the C3 substituent. The structures on the right
differ from those on the left only with respect to the
orientation of the nitrogen of the pyridyl ring relative to the
C22 carbonyl. Panel B (ii) shows
conformational inversion of the C3 pyridine ring (from the
-side to the -side) and of the isopropyl group on C2
(from the -side to -side) following breakage of the
C4-C12 carbon bond.
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With respect to C4,C12-diketopyridylryanodine,
relaxing the ring constraint on the skeletal backbone induces two major
structural changes to the molecule. First, the conformation of the
C-ring changes from chair to boat/half-boat and this alters the
orientation of the methyl functionality on the C9 carbon.
Second, merging of A and B rings into a single 8-membered ring can also
result in conformational inversion of the pyridine ring on the
C3 carbon (from
to
side) and the isopropyl group on
the C2 carbon (from
to
side) (21). For each of
these two major conformations, the E-/syn and
Z-/anti forms can exist. Thus, a total of four conformers
are likely (Fig. 14, B(i) and
B(ii)). If each conformation induces a single
substate, then C4,C12-diketopyridylryanodine should be capable of inducing four subconductance states. In the simplest case, the frequency of occurrence of each substate should be
proportional to the ratio of the conformers in solution. Thus far, we
have detected only three of the four subconductances suggested by this
physiochemical rationalization.
When exposed to low micromolar concentrations of
C4,C12-diketopyridylryanodine at +35 mV, RyR2
spends a minor percentage of its time in the major subconductance state
and the majority of its time in the normal gating mode. Plots of
Ps as a function of holding potentials in the
presence of 10 µM
C4,C12-diketopyridylryanodine fit well to the
logit function and afford an effective gating charge
(ztotal) of 1.4. A similar
ztotal (1.7) was obtained from plots of ln
Koff/ln Kon as a function
of holding potentials. Because C4,C12-diketopyridylryanodine carries a formal
charge of +1, and if the voltage dependence of the interaction is
derived solely from transmembrane movement of the charge, then at least
two molecules will be needed to accommodate the effect of the voltage
drop. Whereas this hypothesis is at least tangentially supported by Hill slopes of >2, it seems unlikely on physical grounds.
Both pyridylryanodine and
C4,C12-diketopyridylryanodine induced long
lasting shut states of RyR2 that were nonetheless reversible. As
previously shown for other ryanoids (6, 12, 26, 31), channels in the
normal gating mode can transition to the subconductance state and from
the latter back either to the full open or to the closed state. In
addition, we found that at negative holding potentials pyridylryanodine
caused RyR2 to directly transit from normal gating to the closed state
and back to normal gating without the intermediate intervention of a
subconductance. Thus, it is clear that induction of a subconductance
state is not a necessary prerequisite for ryanoid-induced shutting of RyR2.
Another interesting question raised by the present results is whether
reducing the rigidity of the skeletal backbone alone is sufficient to
induce subsite selectivity and reversibility among ryanoids. We tested
this by preparing C4,C12-diketo derivatives of
ryanodine and didehydroryanodine. In competition binding affinity assays neither compound was able to discern two classes of ryanoid binding sites on RyR2 (data not shown). In single channel measurements, C4,C12-diketodidehydroryanodine induced a
persistent subconductance state of 39% of full open, which was not
reversible on the time scale of our bilayer experiments (30-60 min)
(data not shown). These results suggest that subsite selectivity and
reversibility require not only a reduced rigidity of the ryanoid
skeletal backbone, but also alterations in the C3
substituent of the A-ring of ryanodine.
Photoaffinity labeling, protein degradation, and mutation studies have
shown that at least one site of ryanoid interaction is located in the
C-terminal, membrane spanning region of RyR (36-40). Zhao et
al. (40) showed that point mutations around a probable
pore-forming region, GVRAGGGIGD (amino acids 4820-4829), significantly
impair high affinity ryanodine binding. These data, as well as others
by the same group (41) led to the conclusion that a high affinity
ryanodine site resides within the pore-forming region of RyR2.
Fessenden et al. (34) found that the E4032A point mutation
on RyR1 significantly impaired high affinity ryanodine binding but the
channel remained sensitive to 500 µM ryanodine. This
observation led these workers to conclude that ryanodine may occlude
the pore on RyR1 by an allosteric mechanism. Data from the present
study support the existence of more than one ryanoid binding site on
RyR. One of these sites (or class) resides outside the strict confines
of the transmembrane voltage gradient whereas a second binding site may
be within the pore.
In summary, the present results show that
C4,C12-diketopyridylryanodine and its parent
pyridylryanodine induce three distinct concentration-dependent effects on RyR2, the first of which
is to increase Po without a step change to a
subconductance. We have also shown that
C4,C12-diketopyridylryanodine is able to
discriminate between two classes of binding sites on RyR2. This
suggests the intriguing possibility that further discrete modifications
on ryanoid molecules will produce even more discriminate, subsite selective ryanoids.
 |
ACKNOWLEDGEMENTS |
We thank Ashutosh Tripathy and Mirko Stange
for helpful suggestions and Daniel Pasek for preparation of some of the proteoliposomes.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants HL66898 (to K. R. B.) and HL27430 (to G. M.), and the Showalter Trust (to H. R. B.).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.
§
Both authors are considered first authors.
¶
To whom correspondence should be addressed: Dept. of
Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, NE 68198. Tel.: 402-559-9018; Fax: 402-559-7495; E-mail: kbidasee@unmc.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M208372200
 |
ABBREVIATIONS |
The abbreviations used are:
RyRs, ryanodine
receptor calcium-release channels;
pyridylryanodine, C3-O-[pyridylcarbonyl]ryanodol;
C4, C12-diketopyridylryanodine,
C3-O-[pyridylcarbonyl]C4,C12-seco-C4,C12-dioxoryanodol;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate.
 |
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