From * Cardiac Medicine, National Heart & Lung Institute, Imperial College of Science, Technology & Medicine, London SW3 6LY,
United Kingdom; Department of Biochemistry, and
Department of Pharmacology, University of Nevada School of Medicine, Reno,
Nevada 89557; and § Department of Chemistry, University of Sherbrooke, Sherbrooke, Quebec JK1 2R1, Canada
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ABSTRACT |
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The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel
results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release
channel. The interaction of 21-amino-9
-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of
this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp
conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of
channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is
open. (c) The interaction of 21-amino-9
-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.
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INTRODUCTION |
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Ryanodine is an alkaloid found in the wood of members of the genus Ryania. Ryanodine disrupts muscle
function by binding to, and modifying the function of,
an intracellular membrane Ca2+-release channel commonly referred to as the ryanodine receptor (RyR)1
(Sutko and Airey, 1996; Sutko et al., 1997
).
The interaction of ryanodine with its receptor has been investigated using two experimental approaches: (a) directly, by monitoring the binding of [3H]ryanodine to isolated populations of membrane vesicles, or (b) indirectly, by monitoring the functional consequences of the interaction of the alkaloid with its receptor, either by determining the Ca2+ handling properties of isolated membrane vesicles or by monitoring the function of single RyR channels reconstituted into planar phospholipid bilayers.
The binding of [3H]ryanodine to isolated membrane
vesicles has provided detailed information on the distribution and density of receptors in membrane populations, the affinity of the sites on the receptor, the rates
of association and dissociation of ryanodine, and the
number of sites on the receptor (Lai et al., 1989; Pessah
and Zimanyi, 1991
; McGrew et al., 1989
; Wang et al., 1993
; Coronado et al., 1994
). Variations in the quantity
of [3H]ryanodine bound to populations of receptor in
the presence of ligands known to modify Ca2+-release
channel function suggest that the interaction of the alkaloid with its receptor may be influenced by channel
gating (Holmberg and Williams, 1990a
; Chu et al.,
1990
; Hawkes et al., 1992
; Meissner and El-Hashem,
1992
). Investigations in which the interaction with the
receptor of various analogues and derivatives of ryanodine have been determined have provided information
on the structural features of these ryanoids required
for high affinity binding (Waterhouse et al., 1987
; Jefferies et al., 1993
; Gerzon et al., 1993
; Welch et al.,
1994
, 1996
; Sutko et al., 1997
).
The interaction of ryanodine with the Ca2+-release
channel modifies its function. At low concentrations
(nano- to micromolar), ryanodine increases the Ca2+
permeability of isolated membrane vesicles; at high
concentrations (high micro- to millimolar) ryanodine
decreases Ca2+ permeability (Fairhurst and Hasselbach,
1970; Meissner, 1986
; Fleischer et al., 1985
; Lattanzio et
al., 1987
). The mechanisms underlying these concentration-dependent actions of ryanodine became apparent when the influence of ryanodine was examined on single Ca2+-release channels. Concentrations of the alkaloid that increase vesicle Ca2+ permeability modify
single channel gating and ion handling; the channel
enters a high open probability (Po), reduced-conductance state (Rousseau et al., 1987
; Lindsay et al., 1994
;
Tinker et al., 1996
). At higher concentrations of ryanodine, the Ca2+-release channel closes (Meissner, 1994
;
Tinker et al., 1996
). Consistent with the extremely slow
rates of association and dissociation of [3H]ryanodine
determined in binding assays (Anderson et al., 1989
; McGrew et al., 1989
; Needleman and Hamilton, 1997
;
DiJulio et al., 1997
), the onset of the modification of
channel function by ryanodine is slow and, on the time
scale of a single channel experiment, irreversible.
A study in which the influence, on single channel
function, of a number of analogues and derivatives of
ryanodine was examined has demonstrated that all of
these ryanoids produce modifications of channel gating and ion handling. In all cases, the interaction of the
ryanoid with the channel resulted in a dramatic increase in Po and a reduction in single channel conductance; however, the amplitude of the ryanoid-induced
reduced conductance state is influenced by the structure of the ryanoid (Tinker et al., 1996; Welch et al.,
1997
). In addition, these investigations revealed that ryanoid structure also influenced the kinetics of the interaction of the alkaloid with its receptor. In particular,
the time of residence of the channel in the ryanoid-
induced modified conductance state varied. While the
majority of the ryanoids examined, like ryanodine,
modified conductance and gating irreversibly, some were reversible. With these ryanoids, under steady state
conditions, channels alternated between periods of modified conductance and gating and periods of normal
conductance and gating. Dwell times in the modified
state lasted from tens of seconds to minutes (Tinker et
al., 1996
).
In this report, we provide a detailed description of
the interaction of one particular reversible ryanoid, 21-amino-9-hydroxy-ryanodine, with single sheep cardiac
RyR channels. With this ryanoid, dwell times in the
modified state last, in general, just a few seconds. This
property makes it possible, for the first time, to obtain kinetic information on the interaction of a ryanoid with
its receptor on a single channel.
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MATERIALS AND METHODS |
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Materials
Phosphatidylethanolamine was purchased from Avanti Polar Lipids (Alabaster, AL) and phosphatidylcholine from Sigma Ltd.,
(Poole, UK). [3H]Ryanodine was obtained from New England
Nuclear Ltd. (Stevenage, UK). Aqueous counting scintillant was
purchased from Packard (Groningen, The Netherlands). Standard chemicals were obtained as the best available grade from
BDH Ltd. (Dagenham, UK) or Sigma Ltd. 21-amino-9-hydroxy-ryanodine was synthesized as described earlier (Welch et al.,
1997
) and stored as a stock solution in 50% ethanol at
20°C.
Preparation of Sheep Cardiac Heavy Sarcoplasmic Reticulum Membrane Vesicles and Solubilization and Separation of the Ryanodine Receptor
Isolation of heavy sarcoplasmic reticulum (HSR) membrane vesicles was carried out using procedures described previously (Sitsapesan and Williams, 1990). Sheep hearts were collected from a
local abattoir in ice-cold cardioplegic solution (Sitsapesan and
Williams, 1990
). A mixed membrane fraction was obtained by differential centrifugation after homogenization of the ventricular
septum and left ventricle free wall. The mixed membrane vesicles
were further fractionated by sucrose density gradient centrifugation and the HSR fraction collected at the 30/40% (wt/vol) interface. The HSR fraction was resuspended in 0.4 M KCl before
sedimentation at 100,000 g. The resulting pellet was resuspended
in 0.4 M sucrose, 5 mM HEPES, titrated to pH 7.2 with hydroxymethyl methylamine (Tris).
HSR membrane vesicles were solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) and
RyR isolated and reconstituted into unilamellar liposomes for incorporation into planar phospholipid bilayers as described previously (Lindsay and Williams, 1991).
Planar Phospholipid Bilayers
Phospholipid bilayers were formed from suspensions of phosphatidylethanolamine in n-decane (35 mg/ml) across a 200-µm
diameter hole in a polystyrene copolymer partition that separated two chambers referred to as cis (volume 0.5 ml) and trans
(volume 1.0 ml). The trans chamber was held at virtual ground
while the cis chamber could be clamped at holding potentials relative to ground. Current flow across the bilayer was monitored
using an operational amplifier as a current-voltage converter
(Miller, 1982). Bilayers were formed with solutions containing
600 mM KCl, 20 mM HEPES, titrated to pH 7.4 with KOH, resulting in a solution containing 610 mM K+ in both chambers. An osmotic gradient was created by the addition of an aliquot (50-100
µl) of 3 M KCl to the cis chamber. Proteoliposomes were added
to the cis chamber and stirred. Under these conditions, channels
usually incorporated into the bilayer within 2-3 min. If channels
did not incorporate, a second aliquot of 3 M KCl could be added
to the cis chamber. After channel incorporation, further fusion
was prevented by perfusion of the cis chamber with 610 mM K+.
Channel proteins incorporate into the bilayer in a fixed orientation so that the cytosolic face of the channel is exposed to the solution in the cis chamber and the luminal face of the channel to
the solution in the trans chamber. The 610 mM K+ solution contained 10 µM Ca2+ as contaminant and, at this level of cytosolic
Ca2+, Po is low. Unless stated otherwise, Po was increased by the addition of 100 µM EMD 41000 (McGarry and Williams, 1994
) to
the cytosolic face of the channel. With this combination of
ligands, single channel Po was increased to ~0.7 (see RESULTS),
and under these conditions it was immediately apparent if more
than one channel was present in the bilayer. Only bilayers containing a single channel were used in the experiments described
in this communication. Experiments were carried out at room
temperature (21 ± 2°C). Initially, the influence of 21-amino-9
-hydroxy-ryanodine on channel function was investigated by
adding it to the solution bathing the cytosolic face of the channel.
Single Channel Data Acquisition
Single channel current fluctuations were displayed on an oscilloscope and stored on digital audio tape. For analysis, data were replayed, filtered at 1 kHz with an eight-pole Bessel filter and digitized at 4 kHz using Satori V3.2 (Intracel, Cambridge, UK). Single channel current amplitudes were monitored from digitized data. The representative traces shown in the figures were obtained from digitized data acquired with Satori V3.2 and transferred as an HPGL graphics file to a graphics software package (CorelDraw; Corel Systems Corporation, Ottawa, Ontario, Canada) for annotation and printing.
Analysis of Single Channel Data in the Presence
of 21-Amino-9-Hydroxy-Ryanodine
The basic observation reported in this communication is that 21-amino-9-hydroxy-ryanodine induces the occurrence of subconductance events in the sheep cardiac muscle RyR. This is shown
schematically in Fig. 1. Under steady state conditions, in the presence of 21-amino-9
-hydroxy-ryanodine, the channel oscillates
between periods of normal gating and periods in a noisy subconductance state characterized by a high Po. We assume that the occurrence of the subconductance state results from the interaction
of 21-amino-9
-hydroxy-ryanodine with the channel and that when
the ryanoid is bound the channel can close but cannot enter the
normal open state. To uncover the mechanisms underlying this
behavior we have monitored the following parameters (Fig. 1).
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Fractional conductance.
Fractional conductance is the amplitude of the subconductance state expressed as a proportion of
the normal full amplitude of the channel (Lindsay et al., 1994;
Tinker et al., 1996
). Amplitudes were monitored by placing cursors at levels corresponding to the center of the noise for each of
the closed, modified, and open conductance states.
Dwell times in the unmodified and modified conductance states, and
the probability of the channel occurring in the modified state (Pmod).
These parameters were determined by using Satori V3.2 to assign
digitized events to one of the three possible states (open, closed,
and modified). Sections of data were then defined as unmodified (periods in which the channel displayed transitions only between the open and closed levels) or modified (periods in which the channel displayed transitions only between the modified and
closed levels) using a pattern recognition program with the minimum duration of an unmodified event set at 2 ms and that of a
modified event set at 15 ms. The accuracy of this procedure was
verified by inspecting the data and counting the number of modified events. Pmod was calculated from the dwell times in the unmodified and modified events as Pmod = total time in modified
state (total time in modified state + total time in unmodified
state). In all experiments reported here, Pmod was determined
from steady state runs lasting at least 6 min.
The probability of the channel being open (Po).
The open probability of single channels in the presence of 21-amino-9-hydroxy-
ryanodine was determined by monitoring this parameter in the
sections of the recorded data during which the channel was unmodified; i.e., displaying transitions only between the open and
closed levels (Fig. 1). Po in these periods was determined by 50%
threshold analysis (Sitsapesan and Williams, 1994
).
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RESULTS |
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Initial Observations
Ryanodine alters both the gating and ion handling
properties of single RyR channels, inducing modified
conductance states of high Po (Rousseau et al., 1987;
Lindsay et al., 1994
; Tinker et al., 1996
). The same basic pattern of modification of channel function is seen
with a wide range of ryanodine analogs; however, specific features such as the amplitude of the modified
conductance state and the dwell time in the modified
conductance state have been shown to be dependent
upon the structure of the ryanoid (Tinker et al., 1996
;
Welch et al., 1997
).
Consistent with this, in the presence of 21-amino-9-hydroxy-ryanodine, the RyR channel enters a modified conductance state as shown in Fig. 2. Reduced conductance states induced by ryanoids are often referred
to as "noisy." In other words, the modified conductance
state displays excess noise when compared with the other gating states of the channel. This can be seen
clearly in Fig. 2 where the amplitude of noise in the
modified state is approximately three times larger than
that of the closed state. If current amplitude is monitored by placing cursors at the center of the noise associated with each state, the modified conductance induced by 21-amino-9
-hydroxy-ryanodine is 354 ± 6 pS
(SEM, n = 14), while the conductance of the unmodified state is 813 ± 10 pS (SEM, n = 14); a fractional
conductance of 0.44.
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Unlike ryanodine, the modification of channel function induced by this ryanoid is reversible. Under steady
state conditions, we observe spontaneous transitions
between unmodified and modified conductance states.
In this example, dwell times in the modified state vary
from <1 to >10 s. In contrast to the situation with ryanodine, washing 21-amino-9-hydroxy-ryanodine out of
the solution bathing the channel rapidly restores normal, unmodified channel function (not shown).
The simplest mechanism that could account for
these observations is one in which the interaction of a
single molecule of 21-amino-9-hydroxy-ryanodine with
its binding site on the channel protein results in the occurrence of the modified state (i.e., Scheme I).
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If a bimolecular scheme of this kind is to provide a valid
description of the interaction of 21-amino-9-hydroxy-ryanodine with its receptor, certain testable criteria
should be fulfilled. The distribution of dwell times in
both the unmodified and modified states should be described by single exponentials. The relationship between 21-amino-9
-hydroxy-ryanodine concentration and
Pmod should be described by Michaelis-Menten kinetics
(i.e., Pmod should be saturable with respect to ryanoid
concentration). The rate of association should be dependent upon the first power of the concentration of
21-amino-9
-hydroxy-ryanodine, while the rate of dissociation should be independent of 21-amino-9
-hydroxy-ryanodine concentration.
Dwell Times in the Unmodified and Modified Conductance States
A bimolecular reaction scheme (Scheme I) predicts
that, in the presence of 21-amino-9-hydroxy-ryanodine, the dwell times in both the unmodified and modified states should be distributed exponentially. Fig. 3
shows noncumulative histograms of dwell times in the
unmodified and modified states of a channel monitored over a period of 20 min at +40 mV with 500 nM
21-amino-9
-hydroxy-ryanodine in the solution at the
cytosolic face of the channel. In both plots, the solid
line is a probability density function with a single exponential term determined by maximum likelihood fitting. The mean dwell time in the unmodified state (
unmod) is
0.95 s, while that of the modified state (
mod) is 4.21 s.
As both sets of dwell times can be described by single
exponentials, the apparent rate constants for the association (kon) and dissociation (koff) of 21-amino-9
-hydroxy-ryanodine can be determined from the mean dwell
times in the unmodified and modified conductance
states as:
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(1) |
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(2) |
The Influence of 21-Amino-9-Hydroxy-Ryanodine
Concentration on Pmod
Fig. 4 shows an experiment in which a single RyR channel was exposed to increasing concentrations of 21-amino-9-hydroxy-ryanodine at the cytosolic face of the
channel. The holding potential was +40 mV. As the
concentration of the ryanoid is increased from 50 to
900 nM, the likelihood of the channel being in the
modified state increases. The relationship between 21-amino-9
-hydroxy-ryanodine concentration and Pmod
for a number of channels is shown in Fig. 5. Pmod was
determined at a holding potential of +40 mV by monitoring dwell times in the unmodified and modified
conductance states as described in MATERIALS AND
METHODS. Each point is the mean ± SEM of at least
four experiments. The curve is drawn according to a
single-site binding scheme of the form:
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(3) |
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with values of Pmax = 0.97 and Kd = 105 nM determined
by nonlinear regression. The Hill slope for this data is
0.99. These observations are consistent with a simple bimolecular scheme in which modification of channel
function results from the interaction of a single molecule of 21-amino-9-hydroxy-ryanodine with a channel.
Variations of Association and Dissociation Rate with
21-Amino-9-Hydroxy-Ryanodine Concentration
Consistent with a bimolecular reaction scheme, the apparent rate constant for the association of 21-amino-9-hydroxy-ryanodine increases linearly as its concentration is raised with a slope of 2.16 ± 0.25 × 106 s
1 M
1,
while the apparent rate constant for dissociation is independent of concentration with a mean value of 0.24 ± 0.01 s
1 (Fig. 6). The apparent Kd derived from the rate
constants is 111 nM, in excellent agreement with the
apparent Kd measured directly from ligand-induced
changes in Pmod (Fig. 5) and indicates self-consistency of the measurements. A plot of ln(rate constant) vs.
ln([21-amino-9
-hydroxy-ryanodine]) is linear with a
slope of 0.975 (not shown), supporting the contention that the rate of association of the ryanoid with its binding site is first order with respect to ligand concentration. A plot of ln(Ka) vs. ln([21-amino-9
-hydroxy-ryanodine]) is linear with a slope of
0.006 (not shown),
supporting the implicit contention that the association
constant is independent of the ryanoid concentration.
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The experiments outlined above indicate that we can
describe the modification of RyR channel function by
21-amino-9-hydroxy-ryanodine by a simple bimolecular association. The observed alterations in gating and
ion handling are the result of the interaction of a single
molecule of ryanoid with a binding site on the channel protein. We can now use this basic model to investigate
other factors that might influence the interaction of 21-amino-9
-hydroxy-ryanodine with its receptor.
The Influence of Po on the Interaction
of 21-Amino-9-Hydroxy-Ryanodine
It is well established that the interaction of [3H]ryanodine with its high affinity receptor site on the SR Ca2+-release channel is influenced by factors that are known
to alter single channel Po. Ligands such as caffeine and
ATP, which increase Po, increase binding, while ligands
that decrease Po, such as ruthenium red or Mg2+, reduce [3H]ryanodine binding (Holmberg and Williams,
1990a; Chu et al., 1990
; Hawkes et al., 1992
; Meissner
and El-Hashem, 1992
). As a consequence, the level of
binding of [3H]ryanodine is often used as an indicator
of channel function.
The availability of a ryanoid that interacts reversibly with its receptor provides us with the opportunity to monitor ryanoid binding at the single channel level. Variations in Pmod are equivalent to variations in the quantity of ryanoid bound to a population of receptors in an SR membrane vesicle preparation; i.e., Pmod is equivalent to the fractional saturation of the high affinity site. In addition, the apparent rates of association and dissociation can be determined from dwell times in the unmodified and modified conductance states as described above. Therefore, we can now make a direct determination of the influence of channel Po on the binding of a ryanoid to its receptor on the SR Ca2+-release channel.
We have investigated this by monitoring the interaction of 21-amino-9-hydroxy-ryanodine (700 nM in the
solution at the cytosolic face of the channel) at +40 mV
under conditions in which Po was varied by the addition
of increasing concentrations of EMD 41000 (McGarry
and Williams, 1994
). An example of such an experiment is given in Fig. 7. The trace in Fig. 7 (top) shows
spontaneous channel activity in the absence of any
added EMD 41000. Under these conditions, Po is very
low (0.003) and we observe only very occasional modifications by the ryanoid. As channel Po is increased by
the addition of EMD 41000 to the solution bathing the
cytosolic face of the channel (Fig. 7, center : 50 µM EMD
41000, giving a Po of 0.11; and bottom: 75 µM EMD
41000, giving a Po of 0.46), the likelihood of the channel
residing in the modified conductance state increases. It
is also noticeable that the structure of the modified conductance state changes as Po is increased; the excess noise
of this state is considerably greater when channel Po is low.
An inspection of modified conductance events at enhanced temporal resolution reveals that the excess noise
observed at low Po results from the occurrence of closing events. This is demonstrated in Fig. 8, in which typical modified conductance events are shown for a single
channel in the presence of 700 nM 21-amino-9
-hydroxy-ryanodine at a Po of 0.01 and, after the addition of
EMD 41000, 0.97. At low Po, the modified conductance
event contains numerous, well resolved, closing events;
these are not seen at high Po.
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The relationship between channel Po and Pmod is
shown in Fig. 9. Values of Pmod were monitored for 18 six-min recordings obtained from six single channels at
Po varying from 0.003 to 0.97. Under these conditions,
Pmod rises steeply as Po increases with a Pmod of 0.5 occurring at a Po of ~0.05. At this concentration of 21-amino-9-hydroxy-ryanodine, Pmod tends to saturate at
Po values in excess of 0.5.
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An inspection of the apparent rates of association
and dissociation over this range of Po reveals that the
increase in Pmod associated with an increase in Po results
from an increase in the rate of association (Fig. 10). kon
is linearly dependent on Po with a slope of 0.97 ± 0.05 s1 over the range Po = 0-1. The intercept of the regression line is effectively zero (0.02 ± 0.02 s
1). koff is
independent of Po with a mean value of 0.09 ± 0.01 s
1.
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Clearly, the Po of the RyR channel has a marked effect on the probability of 21-amino-9-hydroxy-ryanodine interacting with its binding site on the channel
and inducing a modification of ion handling and gating. These findings indicate that the ryanoid binding
site is only accessible when the channel is open.
Given this observation, it is important to monitor
and, if possible, control channel Po when investigating
the effects of other factors on the probability of channel modification by 21-amino-9-hydroxy-ryanodine. For example, in the studies reported in an earlier section of this communication, in which we determined the
influence of 21-amino-9
-hydroxy-ryanodine concentration on Pmod and the rates of association and dissociation, Po values were as follows (21-amino-9
-hydroxy-ryanodine [nM], mean Po ± SEM [n]): 50, 0.62 ± 0.15 (5);
100, 0.71 ± 0.16 (5); 300, 0.70 ± 0.16 (5); 500, 0.56 ± 0.16 (5); 700, 0.73 ± 0.15 (4); 900, 0.73 ± 0.15 (4).
Therefore, variations in Po will not have made a major
contribution to the relationships determined in these experiments.
The Influence of Transmembrane Voltage on the Interaction
of 21-Amino-9-Hydroxy-Ryanodine
In a previous study, we have demonstrated that the interaction of ryanoids with the sheep cardiac SR Ca2+-
release channel can be influenced by the transmembrane voltage (Tinker et al., 1996). The interaction of
ryanodol with the channel results in the occurrence of a
reduced conductance state with an amplitude of ~70%
of the normal unmodified conductance. As is the case
with 21-amino-9
-hydroxy-ryanodine, the interaction of
ryanodol with the channel is reversible under steady
state conditions; however, the dwell times in the modified state are very much longer than those observed
with 21-amino-9
-hydroxy-ryanodine and make it impractical to obtain quantitative estimates of the kinetics
of the interaction. However, we were able to make a
qualitative statistical demonstration of the voltage dependence of the interaction of ryanodol by comparing
the likelihood of the transition from the unmodified to
the modified state and the transition from the modified to the unmodified state at two extreme voltages
(+60 and
60 mV). These studies demonstrated that
modifications of channel function by ryanodol are
more likely to occur and are longer lasting at a holding
potential of +60 than
60 mV.
We have been able to carry out a more detailed investigation of the influence of transmembrane voltage on
the interaction of 21-amino-9-hydroxy-ryanodine with
the sheep cardiac SR Ca2+-release channel. Transmembrane voltage has a dramatic influence on the probability of the channel occurring in the modified conductance state. Fig. 11 shows traces obtained from a single channel in the presence of 500 nM 21-amino-9
-hydroxy-
ryanodine in the solution at the cytosolic side of the
channel. At
60 mV, we see no evidence of interaction
of the ryanoid with the channel; there are no discernible modified-conductance events. As the transmembrane potential is made more positive, Pmod increases.
As we have demonstrated previously with ryanodine
(Lindsay et al., 1994
), the fractional conductance of the
modified state induced by 21-amino-9
-hydroxy-ryanodine does not vary as transmembrane potential is altered (holding potential [mV], mean fractional conductance ± SEM [n]): 60, 0.462 ± 0.003 (10); 40, 0.440 ± 0.003 (10); 20, 0.438 ± 0.004 (10);
20, 0.437 ± 0.005 (10);
40, 0.442 ± 0.002 (6).
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The relationship between Pmod and transmembrane holding potential is shown in Fig. 12. The solid line is the best fit Boltzmann distribution obtained by nonlinear regression:
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(4) |
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where F is the Faraday constant, V is the transmembrane voltage, z is the voltage dependence of the occurrence of the modified conductance state and Gi/RT is an expression of the equilibrium of the reaction at a holding potential of 0 mV. The value of z derived from this plot is 2.16.
If the association and dissociation rates of 21-amino-9-hydroxy-ryanodine are described by the Boltzmann
relationship, then the rate constants at a given voltage
will be described by
![]() |
(5) |
![]() |
(6) |
where K(V) and K(0) are the rate constants at a particular voltage and at 0 mV, respectively, and z is the valence of the appropriate reaction. z may then be determined as the slope of the plot of the natural logarithm of the rate constant against holding potential and K(0) may be determined from the intercept. The total voltage dependence of the reaction is then given by zon + zoff.
A plot of this form is shown in Fig. 13. The rates of association and dissociation of 21-amino-9-hydroxy-ryanodine both vary with the applied holding potential. Kon
increases as the holding potential is made more positive;
at the same time, Koff decreases. The solid lines drawn
through the points in Fig. 13 were obtained by linear regression and the values of zon and zoff obtained from the
slopes of these lines are 1.11 and 0.87, respectively, giving a total valency of 1.98. Fig. 13 (inset) shows the relationship of the Kd of 21-amino-9
-hydroxy-ryanodine (calculated from the data in Fig. 13 as Kd = Koff (s
1)/Kon
(µM
1 · s
1) to holding potential. This plot gives a value
of Kd at +40 mV of 137 nM, which is in good agreement
with the values determined from the ligand-induced
changes in Pmod (Fig. 5) and from the rate constants monitored with varying 21-amino-9
-hydroxy-ryanodine concentration (Fig. 6) at this holding potential. The extrapolated value of Kd at 0 mV is 4.41 µM.
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Is the voltage dependence determined in these experiments real or does it reflect a voltage-dependent shift in
channel Po? We have tested this hypothesis by monitoring
Po values for these channels during the experiments that
provided the data shown in Figs. 12 and 13. The results of
this study are as follows (holding potential [mV], mean
Po ± SEM [n]): 60, 0.78 ± 0.17 (5);
40, 0.73 ± 0.13 (5);
20, 0.62 ± 0.15 (6); +20, 0.65 ± 0.09 (7); +40, 0.69 ± 0.09 (7); +60, 0.45 ± 0.06 (7). These data indicate that, under these experimental conditions, there is no marked
variation in Po with holding potential. Therefore, the variations in the parameters that we have reported in Figs. 12
and 13 reflect true voltage-dependent changes in the interaction of 21-amino-9
-hydroxy-ryanodine with its receptor on the cardiac SR Ca2+-release channel.
Where Is the Site of Interaction
of 21-Amino-9-Hydroxy-Ryanodine?
Ryanodine is membrane permeant and will induce modification of channel gating and conductance when added
to the solution on either the cytosolic or luminal sides of
the SR Ca2+-release channel. 21-amino-9-hydroxy-ryanodine carries a net charge of +1 and is therefore likely to
be considerably less membrane permeant. Fig. 14 shows
records from a representative experiment (n = 6) in
which a single Ca2+-release channel was incorporated
into the bilayer. The traces in Fig. 14 (left) were obtained
after the addition of 500 nM 21-amino-9
-hydroxy- ryanodine to the solution at the luminal face of the
channel. Under these conditions, we saw no modification of gating or conductance at either +60 or
60 mV.
Subsequent addition of the same concentration of 21-amino-9
-hydroxy-ryanodine to the solution at the cytosolic face of the channel (Fig. 14, right) led to the occurrence of modification events at +60 mV. In agreement with the voltage dependence reported above,
modification of channel function was not seen at
60
mV. These experiments indicate that the site of interaction of 21-amino-9
-hydroxy-ryanodine on the SR Ca2+-release channel is only accessible from the cytosolic
side of the channel.
|
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DISCUSSION |
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Before this study, the interaction of ryanoids, or more
precisely ryanodine, with the receptors on the sarcoplasmic reticulum Ca2+-release channel has been monitored by measuring the binding of [3H]ryanodine to
populations of receptors, either in native SR membrane vesicles or after purification of the channel protein. Such studies have revealed considerable information on the nature and properties of the binding sites.
Equilibrium and kinetic analysis has established the existence of two classes of binding site on the release
channel, high (Kd in nanomolar range) and low (Kd in
micromolar range) affinity. Each functional channel
molecule (homotetramer) has a single high affinity site
and one or more low affinity sites (Lai et al., 1988;
McGrew et al., 1989
).
Kd values determined for binding to the high affinity
site vary over a wide range (~1-200 nM) depending
upon the experimental conditions, and it has been proposed that this value represents a weighted average of
the Kd values for the binding of [3H]ryanodine to different, interconvertible conformations of the receptor
(Hawkes et al., 1992). It is generally assumed that the transition between these conformations involves the
normal gating of the channel protein, with the binding
site available when the channel is open. Consistent with
this model, ligands that raise channel Po produce an increase in the affinity of the site for [3H]ryanodine as
the result of an increase in the rate of association; ligands that reduce Po generally decrease the rate of association of [3H]ryanodine with the receptor (Chu et
al., 1990
). It should be noted that both the rate of association with the high affinity receptor and the rate of
dissociation from this site are extremely slow; the association rate being below that predicted for a reaction limited by diffusion (Chu et al., 1990
).
Interactions of ryanodine with the high affinity binding site result in increased permeability of SR vesicles to
Ca2+, while interactions with the low affinity site correlate with decreased permeability to Ca2+ (Meissner,
1986; Humerickhouse et al., 1993
). At the level of single channel resolution, these interactions appear to be
equivalent, respectively, to modification of channel gating and conductance (the induction of a subconductance state with high Po) by nano- to micromolar concentrations of ryanodine, and the closure of the channel by high micro- to millimolar concentrations (Chu
et al., 1990
; Tinker et al., 1996
).
The addition of ryanodine to the solutions bathing a
single Ca2+-release channel reconstituted into a planar
bilayer does not bring about an immediate alteration in
function. Consistent with the extremely slow rates of association determined in binding studies, periods of
tens of seconds or minutes elapse before normal channel function ceases and the channel enters a high Po,
modified conductance state. It is assumed that this
modification of function results from the interaction of
ryanodine with the high affinity binding site of the
channel (Tinker et al., 1996). At first sight, these may
appear to be contradictory statements
how can an extremely slow rate of association correlate with a high affinity interaction? Rate constants considerably below
the diffusion limit are commonly observed with tight
binding ligands (Schloss, 1988
). Two general mechanisms are offered for this observation. One postulates
that the receptor must isomerize to an active conformation before binding can occur. Binding is slow because
the concentration of the active conformer is low (the
equilibrium favors the inactive conformation). This
phenomenon is illustrated in Fig. 10, where increasing
the amount of open conformer of the channel directly
enhances the association rate constant (kon). In contrast, the other mechanism postulates that the receptor
isomerizes at a slow rate after ligand binding occurs.
Binding is slow because the rate of isomerization is
slow. The overall affinity of an interaction is dependent
upon both the rate of association and the rate of dissociation, and in the case of ryanodine the rate of dissociation dominates the equilibrium. On the time scale of a
single channel experiment, ryanodine binds irreversibly to its receptor. After modification of channel function, ryanodine can be removed from the bulk solution and the channel will remain in the modified state for
the duration of the experiment; often in excess of an
hour. With these rates of association and dissociation, it
is clearly not feasible to investigate the kinetics of the
interaction of ryanodine with its receptor on a single
channel. However, as described earlier (Tinker et al.,
1996
), alterations to the structure of ryanodine yields
ryanoids that modify the gating and conductance of the Ca2+-release channel in a characteristic fashion but interact, on the time scale of a single channel experiment, reversibly with the receptor; one such ryanoid is
21-amino-9
-hydroxy-ryanodine.
The availability of a reversible ryanoid has allowed us
to carry out the first characterization of the interaction
of a ryanoid with its receptor on a single Ca2+-release
channel. As is the case with all ryanoids that we have examined (Tinker et al., 1996), 21-amino-9
-hydroxy-ryanodine induces the occurrence of a modified conductance state and we assume that modified function results from the binding of the ryanoid to a ryanodine
binding site on the channel protein. This is supported
by the observation that the binding isotherm of 21-amino-9
-hydroxy-ryanodine demonstrates direct competition
between ryanodine and 21-amino-9
-hydroxy-ryanodine with a 1:1 stoichiometry (Welch, unpublished observations). Furthermore, the Kd of 21-amino-9
-hydroxy-ryanodine binding derived from the rate constants interpolated to 0 mV applied potential (~4 µM; Fig. 13,
inset) is in good agreement with the value of Kd obtained from binding isotherms (1 µM).
How Many Molecules of 21-Amino-9-Hydroxy-Ryanodine
Interact with a Channel to Modify Function?
[3H]Ryanodine binding data indicates that each functional RyR channel contains a single high affinity binding site and in the past we have argued that it is probable that the binding of a ryanoid to this site results in
the occurrence of the modified conductance state
(Tinker et al., 1996). If this is the case, the spontaneous
transitions between the unmodified and modified conductance states of a single channel in the presence of
21-amino-9
-hydroxy-ryanodine will be described by a
simple bimolecular scheme (Scheme I). Consistent with such a scheme, our data demonstrate that (a) the
distributions of dwell times in both the unmodified and
modified conductance states can be described by single
exponentials; (b) as the concentration of 21-amino-
9
-hydroxy-ryanodine is increased, the probability of
the channel being in the modified state increases and
saturates (this relationship can be described in terms of
Michaelis-Menten kinetics); and (c) the rate of association of 21-amino-9
-hydroxy-ryanodine with its binding
site varies linearly with ryanoid concentration, while its
rate of dissociation is unaffected by the concentration of the ryanoid. Therefore, it would appear that ryanoid-induced modifications of channel gating and conductance result from the interaction of a single molecule of
ryanoid per channel. Given the stoichiometry of binding
of [3H]ryanodine to the high affinity site, it is most probable that the interaction of a single ryanoid molecule
with this site results in the modification of function.
The Relationship between Channel Gating and the Binding
of 21-Amino-9-Hydroxy-Ryanodine
In these experiments, we have been able to make the
first direct investigation of the influence of channel Po
on the interaction of a ryanoid with its receptor on
RyR. Our data demonstrate that the gating state of the
channel has a marked influence on the ability of 21-amino-9-hydroxy-ryanodine to bind to the channel and modify function; the ryanoid will only bind to the
open channel. As channel Po is increased, the probability of a channel existing in the modified state increases
as a direct result of a linear increase in the rate of association of the ryanoid with the channel. These findings
are in agreement with the proposal that Kd values determined by equilibrium [3H]ryanodine binding represent
weighted average values for binding to different, inter-convertible conformations of the receptor (Hawkes et
al., 1992
) and confirm that these conformations are open and closed states of the channel.
Our observation that the level of noise of the 21-amino-9-hydroxy-ryanodine-modified conductance state
varies with channel Po is of interest. It would appear
that on interaction with its binding site the ryanoid
does not simply "lock" the channel into an open state;
rather, the open probability of the modified conductance state reflects the Po of the channel in the absence
of the ryanoid. If Po is high, closings of the modified
channel are rare; if Po is low, closings of the modified
channel are common.
[3H]Ryanodine binding is used routinely to monitor
the effects of ligands on the Po of populations of RyR
channels. While the data presented here indicate that
this is valid, it should be noted that [3H]ryanodine
binding will not be directly equivalent to Po over the
full range of this parameter (0-1). In our experiments,
Pmod effectively saturates at values of Po in excess of 0.5;
interventions that raise Po from this value to, say, 0.9 would produce only a very small increase in Pmod. This
observation is consistent with earlier studies in which
we demonstrated that the stimulation of [3H]ryanodine
binding by various ligands is not directly related to
channel Po. For example, the addition of doxorubicin
increases channel Po in the presence of 10 µM Ca2+,
where the initial Po is low, and at 100 µM Ca2+, where
initial Po is considerably higher. However, doxorubicin only produced a significant increase in [3H]ryanodine
binding at the lower concentration of Ca2+ (Holmberg
and Williams, 1990b).
Where Is the Ryanoid Binding Site?
The channel has to be open for 21-amino-9-hydroxy-ryanodine to have access to its binding site. This could
mean that the site is within the conduction pathway; alternatively, the site could be located elsewhere on the
protein but will only be available when the channel is in
an open conformation. Do we have any other lines of
evidence identifying the location of ryanoid binding sites on the channel protein?
The high affinity binding site for ryanodine has been
localized to a 76-kD region of RyR extending from Arg-4475 to the COOH terminus of the protein in studies
using proteolytic degradation and photoaffinity labeling (Callaway et al., 1994; Witcher et al., 1994
); this region may be involved in pore formation (Callaway et
al., 1994
). Some indications on the location of the ryanoid binding site are also available from functional
studies. As the interaction of a ryanoid with its receptor
on the RyR channel produces a reduction in single
channel conductance, it might seem reasonable to assume that this results from a partial block of the conduction pathway by the ryanoid. However, studies in
which the ion handling properties of ryanodine-modified channels have been investigated indicate that reduced channel conductance cannot be explained in
terms of a simple blocking reaction; many aspects of
ion handling are modified by ryanodine binding (Lindsay et al., 1994
). The amplitude of the ryanoid-induced
modified conductance state is dependent upon the
structure of the ryanoid (Tinker et al., 1996
), and comparative molecular field analysis has revealed specific
structural loci on the ryanoid that determine the conductance properties of the modified channel; however,
these studies provide no evidence of a direct steric interaction between the bound ryanoid and the translocated ion (Welch et al., 1997
). Taken together, these
functional studies support a scheme in which modification of ion handling results from the binding of a ryanoid to a site located outside the strict confines of the
conduction pathway, for example in the cytosolic vestibule of the channel (Tinker et al., 1996
).
21-amino-9-hydroxy-ryanodine has a net charge of
+1; therefore, if its site of interaction is within the
channel's conduction pathway, it would be expected
that the interaction would be influenced by voltage.
This is the case; the probability of the channel being in
the modified conductance state is high at high positive
holding potentials and low at high negative potential,
and both association and dissociation rates are sensitive
to voltage. Analysis of the voltage dependence of the interaction of 21-amino-9
-hydroxy-ryanodine with the
receptor indicates that the reaction has a total voltage dependence of 2. In other words, if the voltage dependence of the interaction is derived from the movement
of a charged ryanoid molecule into the applied electric
field within the conduction pathway, an absolute minimum of two molecules of 21-amino-9
-hydroxy-ryanodine would need to be translocated across the entire
voltage drop. As the binding site is only accessible from
the cytosolic face of the channel, this would place the
site at the luminal extreme of the voltage drop. Alternatively, several ryanoid molecules could interact with
sites nearer the cytosolic mouth of the channel.
Neither of these alternatives is consistent with our
earlier suggestion that the occurrence of the modified
conductance state of the channel results from the interaction of a single molecule of 21-amino-9-hydroxy-ryanodine with a high affinity site on the protein, nor
with the observation that each channel (homotetramer) possesses a single high affinity binding site for [3H]ryanodine. Similarly, it is difficult to envisage a situation in
which several ryanoid molecules could occupy sites
within the conduction pathway of the channel and yet
have no direct steric interaction with the translocated
cation. How can we resolve this apparent anomaly?
One possibility is that the measured voltage dependence of the interaction of 21-amino-9
-hydroxy-ryanodine with the channel does not result from the movement of the charged ryanoid into an electric field;
rather, we could envisage a voltage-dependent conformational change in the channel protein that switches
the ryanoid binding site between two states with different affinities. A similar mechanism has been proposed
for the voltage-dependent block of Na+ channels by
guanidinium toxins (Moczydlowski et al., 1984
).
In Scheme II, modification of channel function results
from the binding of one molecule of 21-amino-9-hydroxy-ryanodine to the channel. Fig. 13 demonstrates that the
apparent dissociation constant of 21-amino-9
-hydroxy-
ryanodine decreases with increasingly positive applied
potential due to a decreasing dissociation rate constant
and an increasing association rate constant. In the simplest terms, this can be expressed as an equilibrium between two states of RyR and is similar to the familiar T to
R transition in allosteric proteins.
|
In the case of RyR, we propose that the applied potential is the effector, shifting the equilibrium toward RyR*(unmod) with increasingly positive potential. The rates of association and dissociation of ligand are unaffected by the applied potential and the ryanoid binds preferentially to RyR*(unmod). This model deals with binding to what is generally termed the high affinity ryanodine binding site. Other experiments are required to determine what effect, if any, applied potential may exert on the low affinity sites.
Is this model anything more than a convenient explanation of a larger than expected voltage dependence?
In support of the scheme, we have demonstrated previously that the interaction of a ryanoid that carries no
net charge, ryanodol, with its receptor on the channel,
shows the same qualitative dependence on voltage as
21-amino-9-hydroxy-ryanodine. With ryanodol, entry
into the modified conductance state is more likely to
occur and dwell times in the modified state last longer
at +60 than at
60 mV (Tinker et al., 1996
). From this
it would appear that the net charge on the ryanoid
does not influence the basic voltage dependence of the
interaction; this is consistent with Scheme II. It should
be noted that voltage-dependent influences on the interaction of the ryanoids with the RyR channel will not
be detected in [3H]ryanodine binding assays to populations of receptors. Under these conditions, transmembrane potential will be 0 mV and will not change during the experiment.
Summary
In this report, we have described the first investigation
of factors regulating the interaction of a ryanoid with
the high affinity binding site on single RyR channels.
These studies have identified a number of novel features.
The modification of RyR channel function (residence
in a high Po, reduced conductance state) results from the
binding of one molecule of 21-amino-9-hydroxy-ryanodine to a site on the channel. The binding site for this
ryanoid is only accessible from the cytosolic side of the
channel and the site is only available when the channel
is open. The conformation of the open form of the channel is sensitive to the applied electrical field (Scheme
II). We propose a minimum of two forms of the open RyR channel (designated openRyR and openRyR*). Increasing positive potential favors openRyR* by increasing the value of the equilibrium constants K3 and K4. The
openRyR* form has a higher affinity for ryanoid than
the openRyR form (K1 > K2); therefore, increasing positive potential favors formation of the modified channel
(RyR* · 21-amino). Thermodynamics requires that the
equilibrium between openRyR(unmod) and RyR* · 21-amino must be independent of the path between the
two forms. That is, the equilibrium constants connecting
the various open forms are linked functions and K1 × K3 = K2 × K4. Since K1 > K2 and K4 > K3. In other words,
the binding of 21-amino-9
-hydroxy-ryanodine to the
low affinity, open form of the RyR channel favors the
conversion of the low affinity form to the high affinity
form just as the voltage-induced conversion of openRyR to openRyR* favors binding of the ryanoid.
![]() |
FOOTNOTES |
---|
Address correspondence to Prof. Alan J. Williams, Cardiac Medicine, National Heart & Lung Institute, Imperial College of Science, Technology & Medicine, Dovehouse St., London SW3 6LY, UK. Fax: +44 (0)171 823 3392; E-mail: a.j.williams{at}ic.ac.uk
Received for publication 18 March 1998 and accepted in revised form 29 April 1998.
We are grateful to Andrew Griffin for preparing RyR channels, and to Dr. Rebecca Sitsapesan for many helpful discussions.
Supported by the Biotechnology and Biological Sciences Research Council, Wellcome Trust, British Heart Foundation, American Heart Association (93012790), and National Science Foundation (MCB-9317684).
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Abbreviations used in this paper |
---|
HSR, heavy sarcoplasmic reticulum; RyR, ryanodine receptor.
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