Correspondence to: Alan J. Williams, Department of Cardiac Medicine, National Heart & Lung Institute, Imperial College of Science, Technology & Medicine, Dovehouse Street, London SW3 6LY, United Kingdom. Fax:44 0 171 823 3392 E-mail:a.j.williams{at}ic.ac.uk.
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Abstract |
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In an earlier investigation, we demonstrated that the likelihood of interaction of a positively charged ryanoid, 21-amino-9-hydroxyryanodine, with the sarcoplasmic reticulum Ca2+-release channel (ryanodine receptor, RyR) is dependent on holding potential (Tanna, B., W. Welch, L. Ruest, J.L. Sutko, and A.J. Williams. 1998. J. Gen. Physiol. 112:5569) and suggested that voltage dependence could result from either the translocation of the charged ligand to a site within the voltage drop across the channel or a voltage-driven alteration in receptor affinity. We now report experiments that allow us to assess the validity of these alternate mechanisms. Ryanodol is a neutral ryanoid that binds to RyR and induces modification of channel function. By determining the influence of transmembrane potential on the probability of channel modification by ryanodol and the rate constants of ryanodol association and dissociation, we demonstrate that the influence of voltage is qualitatively the same for both the neutral and positively charged ryanoids. These experiments establish that most, if not all, of the modification of ryanoid interaction with RyR by transmembrane holding potential results from a voltage-driven alteration in receptor affinity.
Key Words: ryanodine receptor, sarcoplasmic reticulum, calcium channel, ryanodine, ryanodol
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INTRODUCTION |
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Ryanodine is a plant alkaloid that binds with high affinity and specificity to a class of intracellular membrane Ca2+-release channels (
The kinetic parameters of the ryanodineRyR interaction are traditionally determined by monitoring the binding of [3H]ryanodine to populations of receptors, either in intact membrane vesicles, or after receptor purification (-hydroxyryanodine, with the sheep cardiac isoform of the RyR channel and have identified a number of novel features (
-hydroxyryanodine binding to single channels with changing concentrations of the ryanoid indicate that modifications of RyR channel function induced by this ryanoid result from the interaction of a single molecule of the ryanoid with each channel protein. Related experiments demonstrated that 21-amino-9
-hydroxyryanodine has access to its binding site only in open conformations of the channel protein, that the ryanoid binding site can only be reached from the cytosolic side of the channel, and that the interaction of the ryanoid with its binding site is influenced strongly by transmembrane voltage (
The quantitative determination of the voltage dependence of the interaction of 21-amino-9-hydroxyryanodine with RyR indicates that approximately two positive charges move through the voltage drop across the channel during the activation of 21-amino-9
-hydroxyryanodine binding by voltage (
-hydroxyryanodine (net charge +1) to a binding site within the voltage drop across the channel. If all the voltage dependence of the interaction arises in this way, either two molecules of the ryanoid would need to be translocated across the entire voltage drop, or several ryanoid molecules could interact with sites located nearer the cytosolic entry to the voltage drop. Neither of these possibilities is consistent with our observation that other aspects of the interaction of 21-amino-9
-hydroxyryanodine with RyR can be described in terms of a bimolecular reaction (
-hydroxyryanodine with RyR is of interest in its own right, but may also provide information on the location of the ryanodine binding site in RyR. For example, translocation of 21-amino-9
-hydroxyryanodine into the voltage drop across the channel would suggest that the binding site is within the conduction pathway of the RyR channel.
In this communication we have addressed these issues by investigating the influence of transmembrane voltage on the interaction of a neutral ryanoid, ryanodol, with single RyR channels. Our experiments demonstrate that ryanodol displays the same qualitative voltage dependence as the positively charged 21-amino-9-hydroxyryanodine and are consistent with the proposal that most, if not all, of the influence of voltage on the interaction of ryanoids with the RyR channel is derived from a voltage-driven alteration in the affinity of the receptor.
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MATERIALS AND METHODS |
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Materials
Phosphatidylethanolamine was supplied by Avanti Polar Lipids, Inc. and phosphatidylcholine by Sigma-Aldrich. [3H]Ryanodine was purchased from New England Nuclear Ltd. Aqueous counting scintillant was purchased from Packard. Standard chemicals were obtained as the best available grade from BDH Ltd. or Sigma-Aldrich. Ryanodol was synthesized as described earlier (
Isolation of Sheep Cardiac Heavy Sarcoplasmic Reticulum Membrane Vesicles and Solubilization and Separation of the Ryanodine Receptor
Heavy sarcoplasmic reticulum (HSR) membrane vesicles were prepared using procedures described earlier (
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 (0.5 ml) and trans (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 currentvoltage converter (
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). Single channel current amplitudes and lifetimes were measured 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 Corp.) for annotation and printing.
Monitoring the Interaction of Ryanodol with Single Channels
Ryanodol binds to the high affinity ryanodine binding site on the SR Ca2+-release channel and induces modifications of channel function; channel conductance is reduced and Po increases (-hydroxyryanodine and the resulting modification of channel function can be described by a simple bimolecular reaction scheme (
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(1) |
and
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(2) |
Dwell times and the probability that the channel is in the ryanoid-modified state (Pmod) were determined using a pattern recognition program as described in
The Modification of the Interaction of Ryanodol with RyR by Voltage
If the transition between the normal gating state of the RyR and the modified state resulting from the interaction of ryanodol is dependent on holding potential, Pmod will be determined by the Boltzmann distribution,
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(3) |
where F is the Faraday constant, V is the transmembrane voltage, R is the gas constant, T is temperature (°K), zt is the voltage dependence of the occurrence of the ryanoid modified state, Gi is the difference in free energy of the unmodified and ryanoid-modified states, and
Gi/RT is an expression of the equilibrium of the reaction at a holding potential of 0 mV.
For such a relationship, the rate constants at a given voltage will be described as follows:
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(4) |
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(5) |
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. Plots of the natural logarithm of kon and koff against holding potential should be linear with slopes zonF/RT and -zoffF/RT and intercepts ln[kon(0)] and ln[koff(0)], respectively. The total voltage dependence (ztotal) of the reaction is then zon + zoff.
The Probability of the Channel Being Open
While the rates of dissociation of ryanodol and 21-amino-9-hydroxyryanodine from RyR are independent of Po, the rates of association of both ryanodol (data not shown) and 21-amino-9
-hydroxyryanodine (
-hydroxyryanodine (
-hydroxyryanodine have been normalized to a Po of 1.0.
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RESULTS |
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Is the Interaction of Ryanodol with the RyR Channel Influenced by Transmembrane Holding Potential?
In previous studies, we demonstrated that the interaction of 21-amino-9-hydroxyryanodine with the high affinity ryanodine binding site on the RyR channel is influenced by transmembrane voltage (
2. As outlined in the INTRODUCTION, this dependence on holding potential could arise from the translocation of the positively charged ryanoid into the voltage drop across the RyR channel. Alternatively, voltage dependence could be derived from a voltage-driven movement of charge within the RyR channel that results in a conformational change that switches the ryanoid binding site between two states of different affinity (
-hydroxyryanodine. If the site were located outside the voltage drop across the channel, all of the voltage dependence would be derived from the proposed voltage-driven conformational change. However, if the site were located within the voltage drop, a proportion of the total voltage dependence would arise from the movement of the charged ryanoid into and out of the voltage drop. These alternatives can be distinguished by monitoring the voltage dependence of a ryanoid such as ryanodol that carries no ionic groups.
Fig 1 presents the electrical potential of 21-amino-9-hydroxyryanodine (right, net charge +1) and ryanodol (left, no ionic groups) together with ball and stick models of the molecules. The top shows positive and the bottom shows negative electrostatic fields. 21-amino-9
-hydroxyryanodine is shown with the pyrrole ring at the bottom left. Ryanodol, which lacks the pyrrole ring, is in the same orientation. The wire frames in all diagrams represent the surface where the electrostatic potential equals 10 kcal/mol. Note the extensive electrical potential of 21-amino-9
-hydroxyryanodine (top right) due to the ammonium ion (located in the upper right of the ball and stick diagram). In comparison with ryanodol (top left), the far more extensive electrical interactions of 21-amino-9
-hydroxyryanodine with other molecules is clearly visible. While ryanodol is neutral (has no net charge), the presence of hydroxyl groups creates a nonuniform distribution of potential in the molecule. The hydroxyl groups on one surface mean that there will be excess negative electrostatic potential localized to a specific surface region of ryanodol. The resulting microscopic dipoles sum to yield the electrical fields shown on the left hand figures. Note that, as for the positive electrostatic field, the distribution of the negative electrostatic potential (bottom) is much different for 21-amino-9
-hydroxyryanodine and ryanodol. Fig 1 visualizes the large difference in the macroscopic dipole moments of these two molecules. The dipole (24.2 Debye) of 21-amino-9
-hydroxyryanodine runs along the long axis of the cationic ryanoid (the long axis is parallel to the plane of Fig 1). In contrast, the dipole (3.3 Debye) of the neutral ryanoid is at right angles to that of 21-amino-9
-hydroxyryanodine. Therefore, these two ryanoids will experience considerably different torsional forces within any electrical potential gradient, including the applied transmembrane voltage. The large permanent dipole will tend to orient 21-amino-9
-hydroxyryanodine with the long axis parallel to the electric field.
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Fig 2 shows current fluctuations of a single RyR channel at holding potentials ranging from -50 to 20 mV in the presence of 20 µM ryanodol. As is the case with 21-amino-9-hydroxyryanodine, ryanodol interacts reversibly with the RyR channel, inducing modifications to both channel gating and ion handling; however, the fractional conductance of the modified state (
-hydroxyryanodine under these conditions is
0.45 (
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Inspection of the traces in Fig 2 highlights the first novel finding of these studies. The probability of RyR channel modification by a neutral ryanoid, ryanodol, varies markedly with holding potential, and the effect of voltage is qualitatively the same as that observed with 21-amino-9-hydroxyryanodine: Pmod for both ryanoids rises as holding potential is shifted to more positive values. The relationship between Pmod and holding potential, in the range -60 to 20 mV, for several channels in the presence of 20 µM ryanodol is shown in Fig 3. The solid line is the best fit Boltzmann distribution (Equation 3) obtained by nonlinear regression with a value for zt of 1.54 (r = 1.0).
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Fig 4 shows the influence of holding potential on kon (a) and koff (b) of ryanodol together with equivalent data for 21-amino-9-hydroxyryanodine taken from
-hydroxyryanodine data are again included for comparison.
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The ryanodol data are summarized, together with equivalent parameters for 21-amino-9-hydroxyryanodine (
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DISCUSSION |
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Our earlier investigations of the interaction of 21-amino-9-hydroxyryanodine with individual cardiac muscle RyR channels demonstrated for the first time that both the association of a ryanoid with its receptor and dissociation of a ryanoid from its receptor could be sensitive to transmembrane voltage (
These experiments establish that transmembrane voltage influences the likelihood of a ryanoid being bound to the receptor on the RyR channel by a mechanism that is independent of the translocation of a charged moiety into the voltage drop across the channel.
A likely mechanism for the modulation of ryanoid interaction with the receptor on RyR would then involve a voltage-dependent equilibrium between receptor states of different affinity (
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While the experiments reported here provide an unequivocal demonstration that modulation of ryanoid binding by voltage is not dependent on the ryanoid carrying a positive charge, they do suggest that the net charge of the ryanoid may produce small quantitative differences in the influence of transmembrane voltage. The total voltage dependence, monitored from variations in kon and koff, with voltage, of 21-amino-9-hydroxyryanodine is slightly greater than that of ryanodol, and this produces a significant difference in the influence of voltage on the resulting Kds for the two ryanoids. How could the net charge of the ryanoid alter the influence of voltage on the interaction of the ligand with its receptor? The binding of 21-amino-9
-hydroxyryanodine may induce a different conformer of the ligandRyR complex diagrammed in Fig 6 than that induced by ryanodol. The difference in fractional conductance of the ryanodolRyR and 21-amino-9
-hydroxyryanodineRyR complexes is evidence of such a difference. The binding of the two ryanoids may induce different distributions of charge in the ryanoidRyR complex, either directly by addition of another ionic charge or indirectly through alteration of one or more of the RyR acid dissociation constants by interaction with the electric charge of 21-amino-9
-hydroxyryanodine. The small difference in voltage dependence of the neutral and cationic ryanoid indicates that movement of charge is involved in the voltage-induced transition; however, the small magnitude of the effect makes interpretation difficult. While not eliminating the possibility of allosteric interactions between the ryanodine binding site and the ion conduction pathway, a mechanism where the ryanoid binding site is placed within the voltage drop across the channel is appealing because of its simplicity. Under these circumstances, the small increase in voltage dependence arises from the movement of a charged ryanoid into and/or out of the electric field. A location of the ryanoid binding site within the voltage drop of RyR would be consistent with the proposal that the receptor site is within the conduction pathway of the channel. The conduction pathway of the RyR channel is almost certainly formed from residues within the carboxyl terminal domains of the four RyR monomers that together make up the functional homotetramer (
In addition, several features of the interaction of ryanoids with the RyR channel are consistent with the proposal that the high-affinity binding site for these ligands is located within the conduction pathway of the channel. The magnitude of equilibrium [3H]ryanodine binding is altered by interventions that modify RyR channel Po. Activating ligands such as Ca2+, caffeine, and ATP increase binding while ligands that lower Po, such as Mg2+ and ruthenium red, reduce binding (-hydroxyryanodine on RyR channel Po was demonstrated.
The dissociation constants monitored in these single-channel experiments are in the same range as those determined for RyR in intact cardiac sarcoplasmic reticulum membrane vesicles in competition binding studies with [3H]ryanodine [2 µM 21-amino-9-hydroxyryanodine (Welch, W., unpublished results) and 1 µM ryanodol (
-hydroxyryanodine and ryanodol are more likely to result from the detailed differences in electrical and steric interactions between the receptor and ligand rather than from differences in the global ryanoidRyR interaction energy. Note that compared with ryanodine (Kd = 2 nM;
-hydroxyryanodine arises from a 4 kcal/mol unfavorable interaction between the ligand and receptor (either electrostatic repulsion, poor solvation, or both). Steric interactions can be discounted since placing the bulky BODIPY derivative at the 21 position caused only a small perturbation in binding (
-hydroxyryanodine have 4 kcal/mol less binding energy; this results in one case from the omission of an important interaction, while in the other case it is due to the addition of an unfavorable interaction.
Independent of voltage-induced changes, there are very marked differences in both the rates of association and dissociation for ryanodol and 21-amino-9-hydroxyryanodine with RyR. For example, at a holding potential of 0 mV, ryanodol is
10x less likely to associate (
G = 1.4 kcal/mol) with the ryanodine binding site on RyR than 21-amino-9
-hydroxyryanodine; once bound, ryanodol leaves this site
10x slower (
G = 1.4 kcal/mol) than 21-amino-9
-hydroxyryanodine. Therefore, the difference in energy barriers limiting association with and dissociation from the ryanoid binding site is considerably greater than the difference in total binding energy of the two ryanoids.
In summary, the experiments described in this report were designed to examine the mechanisms involved in the striking influence of transmembrane holding potential on the interaction of ryanoids with the high affinity binding site on the RyR channel. Our earlier observation of a strong influence of voltage on the interaction of a positively charged ryanoid with RyR could be explained either by the movement of the charged ligand into the voltage drop of the channel to reach its binding site and/or a voltage-driven conformational alteration in RyR leading to altered affinity of the receptor. Here we demonstrate that the apparent dissociation constant for the neutral ryanoid, ryanodol, decreases as the potential at the cytosolic face of the channel is made increasingly positive, and that this results from alterations to both the rates of association of ryanodol with its receptor and dissociation of ryanodol from its receptor. This observation provides very strong evidence in support of a voltage-driven alteration in ryanoid receptor affinity as the major determining factor in the influence of transmembrane holding potential on the interactions of ryanoids with RyR (Fig 6).
While this mechanism underlies the influence of voltage on the interaction of both neutral and positively charged ryanoids, our experiments indicate that a small additional voltage-dependent effect can be observed with the positively charged 21-amino-9-hydroxyryanodine. This may reflect the movement of the charged moiety from the cytosolic bulk solution to a binding site within the voltage drop across the channel, presumably in the conduction pathway of the channel.
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Footnotes |
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1 Abbreviation used in this paper: RyR, ryanodine receptor.
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Acknowledgements |
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This work was supported by grants from the Biotechnology and Biological Sciences Research Council, The British Heart Foundation, The Wellcome Trust, the National Institutes of Health (HL53677), and the National Science Foundation (MCB-9817605).
Submitted: 20 December 1999
Revised: 12 April 2000
Accepted: 8 May 2000
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