Biol Res 37: 527-538, 2004

ARTICLE

The interaction of ryanoids with individual ryanodine receptor channels

ALAN J WILLIAMS and BHAVNA TANNA

Cardiac Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Dovehouse St., LONDON SW3 6LY, U.K.

Dirección para Correspondencia

ABSTRACT

Ryanodine binds with high affinity and specificity to a class of Ca²⁺-release channels known as ryanodine receptors (RyR). The interaction with RyR results in a dramatic alteration in function with open probability (Po) increasing markedly and rates of ion translocation modified. We have investigated the features of ryanodine that govern the interaction of the ligand with RyR and the mechanisms underlying the subsequent alterations in function by monitoring the effects of congeners and derivatives of ryanodine (ryanoids) on individual RyR2 channels. While the interaction of all tested ryanoids results in an increased Po, the amplitude of the modified conductance state depends upon the structure of the ryanoid. We propose that different rates of cation translocation observed in the various RyR-ryanoid complexes represent different conformations of the channel stabilized by specific conformers of the ligand. On the time scale of a single channel experiment ryanodine binds irreversibly to the channel. However, alterations in structure yield some ryanoids with dissociation rate constants orders of magnitude greater than ryanodine. The probability of occurrence of the RyR-ryanoid complex is sensitive to trans-membrane voltage, with the vast majority of the influence of potential arising from a voltage-driven alteration in the affinity of the ryanoid-binding site.

Key words: calcium, Ca²⁺-release channel, sarcoplasmic reticulum, ryanodine, single channel

INTRODUCTION

The regulated release of Ca²⁺ from intracellular storage organelles such as the endoplasmic reticulum (ER) and its counterpart in muscle, the sarcoplasmic reticulum (SR), plays vital roles in the control of myriad cellular signalling processes (4; see also Carafoli, this issue, ref 12). In response to an activating signal, the release of stored calcium occurs via ligand-regulated, cation-selective, intracellular membrane channels. Two structurally related, Ca²⁺-release channels exist, one regulated primarily by inositol 1,4,5-trisphosphate (IP₃) is known as the IP₃ receptor, and the other, regulated either by Ca²⁺ or by interactions with cell surface membrane voltage sensors, is known as the ryanodine receptor (RyR). Ryanodine receptors have acquired their name because each channel molecule contains a high-affinity binding site for this plant alkaloid. Ryanodine has been an indispensable tool in the investigation of the identity, structure, function and levels of expression of RyR (33;5;20). It has also been instrumental in the identification of the involvement of RyR-mediated Ca²⁺-release in a variety of cellular processes (3;18;35;42;43) and in establishing the importance of RyR-mediated Ca²⁺ release from the SR in processes such as cardiac muscle excitation-contraction coupling (6;7;17;23;27). Ryanodine disables SR Ca²⁺ handling by modifying RyR channel function. Investigations of the action of ryanodine on isolated SR membrane vesicles indicate that low concentrations (nM-low mM) render the membrane leaky to Ca²⁺ while higher concentrations (high mM and above) prevent Ca²⁺ release (34).

SINGLE CHANNEL STUDIES

In this short review we will focus on research in which the interaction of ryanodine and ryanoids with RyR and the functional consequences of these interactions have been examined by monitoring the behavior of individual RyR channel proteins. Single RyR channel activity is monitored following the incorporation of either channels in intact SR vesicles or purified channel proteins into planar phospholipid bilayers. RyR channels incorporate in a fixed orientation so that the cytosolic and luminal faces of the protein can be defined (32). Once located in the bilayer, the gating and ion handling properties of individual channels can be monitored under voltage clamp conditions. In the experiments that we will discuss in this review the solution at the luminal face of the channel is held at ground and the solution at the cytosolic face of the channel is clamped at potentials relative to ground.

Initial research on single RyR channel function revealed the mechanisms underlying the concentration-dependent actions of ryanodine on SR Ca²⁺ permeability. Under these conditions, a variable time after addition of the ligand to the solution at the cytosolic side of the channel, nM to low mM concentrations of ryanodine produce a very characteristic modification of channel function; open probability (Po) is increased dramatically to values approaching 1.0, and at the same time, single channel current amplitude is reduced (1;10;26;29). On the time scale of a single channel experiment this modification of channel function by ryanodine can be considered as irreversible; unbound ryanodine can be perfused out of the system but the channel remains in the modified state. These experiments demonstrate that the rate of dissociation of ryanodine from its receptor is very low. Consistent with the observation of reduced Ca²⁺ permeability of intact SR vesicles, high mM concentrations of ryanodine induce closing of RyR channels (26;41). These observations are consistent with the identification of high affinity and low affinity sites in [³H]-ryanodine binding studies to populations of receptors in intact SR membrane vesicles (21;45;28;44).

Subsequent single channel experiments have provided some evidence for an additional site of ryanoid interaction. Buck et al. (9) proposed that low nanomolar concentrations of ryanodine produced an increase in RyR1 single channel open probability without modifying rates of cation translocation. This activation was reversed by removal of ryanodine. In these experiments addition of higher nanomolar concentrations of ryanodine prompted the occurrence of an irreversible high Po-modified conductance state and yet higher concentrations of the ligand ( 200 mM) caused complete channel closure.

In agreement with these observations Bidasee et al. (8) have reported that C₄,C₁₂-diketopyridylryanodine has three concentration-dependent effects on the function of individual RyR2 channels incorporated into planar phospholipid bilayers. As in the experiments of Buck et al., nanomolar concentrations of this ryanoid produced a reversible increase in channel Po with no alteration in rates of cation translocation. Low micromolar concentrations of the ryanoid produced high Po-modified conductance states and high micromolar concentrations produced long lasting closings.

THE FUNCTIONAL CONSEQUENCES OF ALTERED RYANOID STRUCTURE

Of the three potential sites of ryanoid interaction with the RyR channel, by far the most information is available for that which results in the occurrence of a high Po-modified conductance state. Unless indicated otherwise, the remainder of this review will focus on processes associated with this interaction and properties of the resulting RyR-ryanoid complex.

In recent years we have used a wide range of ryanoids to investigate the features of these molecules that govern both their association with individual RyR channels to produce high Po-modified conductance states and their dissociation from these states. We have also investigated the structural features of ryanoids that determine the function of the channel following the formation of the RyR-ryanoid complex. These investigations have revealed features common to all ryanoids so far examined. Without exception, ryanoids interact with individual RyR channels to produce high Po states with reduced rates of translocation of K⁺. However, while all ryanoids appear, at least qualitatively, to have equivalent actions on RyR Po, rates of ion translocation in the RyR-ryanoid complex differ and are determined by the structure of the ryanoid.

RYANOID STRUCTURE INFLUENCES RATES OF ION TRANSLOCATION IN THE RYR-RYANOID COMPLEX

We have quantified the rate of ion translocation in the RyR-ryanoid complex as a fractional conductance (FC) that expresses the conductance of the ryanoid-modified state as a proportion of the unitary conductance in the absence of the ryanoid (41) (Fig. 1.). To date we have monitored FC values ranging from 0.06 for 10-O-guanidinopropionylryanodine (41) to 0.93 for 10-O-succinoylryanodol (40) with K⁺ as the charge carrier. CoMFA has identified loci on the ryanoid molecule showing strong correlations with rates of K⁺ translocation in the RyR-ryanoid complex (46;47). Alterations in the physical properties of the ryanoids at and around the 9- and 10- positions have significant effects on fractional conductance. Increased steric bulk or increased positive electrostatic charge at this region of the molecule correlates with low values of FC. Combinations of positive charge and steric bulk, such as are found in 10-O-guanidinopropionylryanodine and b-alanylryanodine, result in values of FC approaching zero. Of particular interest is the observation that the locations of structural features of the ryanoid molecule associated with modulation of ion translocation in the RyR-ryanoid complex differ from those identified as correlating with the affinity of interaction of the ligand with the receptor (46). Similarly, there is no correlation between the affinity of ryanoid interaction with RyR, as monitored in binding studies to populations of channels, and the value of FC of the RyR-ryanoid complex (46).

RYANOID STRUCTURE INFLUENCES RATES OF ASSOCIATION AND DISSOCIATION

As outlined above, ryanodine that binds to RyR and modifies Po and conductance dissociates extremely slowly. Alteration of ryanoid structure yields ligands that dissociate at much higher rates. As an example, dwell times of RyR2 in the 21-amino-9a-hydroxyryanodine-modified state are measured in seconds (36). The availability of ryanoids with enhanced rates of dissociation has facilitated the measurement of the kinetics of interaction of ryanoids with individual channel proteins under voltage clamp conditions. These studies have identified novel regulatory factors that could not be monitored in conventional [³H]-ryanodine binding experiments.

Figure 1. The figure shows representative interactions of A) ryanodine and B) 21-amino-9a-hydroxyryanodine with individual sheep RyR2 channels. In both cases experiments were carried out with 610 mM K+ as the permeant cation and the holding potential is +40 mV. The interaction of the ryanoids with the channel leads to the occurrence of modified conductance states. Po is high with only occasional brief closing events whilst the ryanoid is bound. The diagram demonstrates significant differences between the two ligands. As highlighted in the text, the 21-amino-9a-hydroxyryanodine-modified state is considerably noisier than that observed following the interaction of ryanodine with RyR. Interactions of ryanodine with RyR are, on the time scale of a single channel experiment, irreversible. In contrast, 21-amino-9a-hydroxyryanodine-induced modified states last for seconds and in the continued presence of the ryanoid the kinetics of interaction can be monitored. The durations of 2 ryanoid-modified events and the intervening periods of normal gating are demonstrated in panel B. The amplitude of a ryanoid-modified state is quantified as a fractional conductance defined as X/Y. FC for ryanodine in this figure is 0.55 and for 21-amino-9a-hydroxyryanodine is 0.44.

These experiments also established that this ryanoid binding site is only accessible from the cytosolic side of the channel and that the ryanoid molecule interacts with an open conformation of the channel. The rate of 21-amino-9a-hydroxyryanodine association with RyR increases in proportion with channel Po whilst rates of dissociation are unaffected by Po.

For the first time, these experiments permitted an examination of the influence of trans-membrane holding potential on the interaction of a ryanoid with its receptor in the trans-membrane RyR channel. Initial observations were made with the cationic ryanoid 21-amino-9a-hydroxyryanodine (36). In the presence of this ryanoid the probability of occurrence of the modified conductance state (P_mod), indicating the occurrence of the RyR-ryanoid complex, was sensitive to trans-membrane holding potential. P_mod was low at negative holding potentials and rose as holding potential was taken to increasingly positive potentials. An inspection of the dwell times in the ryanoid-modified and unmodified states in these experiments demonstrated that alterations in P_mod arose from voltage dependent changes in both the rate of association of the ryanoid with its receptor (k_on) and rates of ryanoid dissociation (k_off).

Two plausible mechanisms suggested themselves as possibly contributing to this observation. Sensitivity of k_on and k_off to trans-membrane potential could result from the movement of the cationic ryanoid to and from a site of interaction within the voltage drop across the RyR channel. Alternatively, the observed effects of potential could arise from a change in the affinity of the receptor resulting from a conformational rearrangement involving the movement of charged components of the channel protein.

The mechanisms underlying the influence of trans-membrane holding potential on the interaction of ryanoids with RyR have been examined by monitoring this phenomenon using ryanoids with different formal charges. In addition to 21-amino-9a-hydroxyryanodine, which has a formal charge of +1, we have monitored the influence of trans-membrane potential on P_mod for representative ryanoids with no formal charge (ryanodol) (37) and a formal charge of -1 (10-O-succinoylryanodol) (40). In all cases, P_mod is influenced by trans-membrane potential, and irrespective of the charge of the ligand, this parameter increases as holding potential is shifted from negative to positive values. Similarly, this change in potential results in an increase in k_on and a decrease in k_off and K_D for neutral, anionic and cationic ryanoids. These data are summarized in Table I.

Dissociation constants for the ryanoids in these experiments, calculated at a holding potential of 0 mV, are comparable to equivalent parameters for the high affinity interactions of these ryanoids determined in competition studies with [³H]-ryanodine binding to populations of RyR in membrane vesicle preparations (40) (Table I.) This correlation indicates that the values determined by monitoring the interactions of ryanoids with individual RyR channels are relevant to more intact systems. It also suggests that the observed dramatic increase in Po and the associated modification of cation translocation occurs as a consequence of the interaction of a single molecule of ryanoid with the high affinity binding site monitored in [³H]-ryanodine binding experiments with populations of channels in membrane vesicles.

WHERE IS THE HIGH AFFINITY RYANOID BINDING SITE?

While the location of the high affinity ryanoid binding site within the RyR channel is yet to be established, an increasing body of circumstantial evidence suggests that the site is likely to be located at some point within the conduction pathway of the channel.

Studies involving proteolytic degradation and photoaffinity labeling have demonstrated that the site is localized to a 76-kD region of the RyR1 channel stretching from Arg-4475 to the COOH terminus of the protein (11;49). This region contains several trans-membrane helices, some of which almost certainly contribute to the formation of the conduction pore of the channel (16;48). The observation that ryanoids interact with open conformations of RyR could be consistent with a high affinity binding site within the pore of the channel, however an equally plausible explanation would be that the site is located elsewhere on the cytosolic face of the protein and is exposed as a consequence of a conformational change associated with channel opening. The small differences in voltage dependence of ryanoid association and dissociation rates monitored in single channel experiments could reflect interactions of charged ligands with the voltage drop across the channel protein. The most probable site for such interactions would be within the channel pore.

Mutation of various residues thought to contribute to the formation of the pore of the RyR channel have been shown to produce alterations in the binding of [³H]-ryanodine to populations of RyR channels. Residues equivalent to those forming the selectivity filter in K⁺-selective channels have been identified in a loop linking the final two trans-membrane spanning helices of RyR (2). In addition to reducing rates of Ca²⁺ and K⁺ translocation, mutation of residues in this region leads, in some cases, to the abolition of ryanodine binding and in some cases to an increase or decrease in K_D (13;15;19;50).

Following the identification of a sequence of residues in RyR analogous to the K⁺ channel selectivity sequence and the demonstration that these residues are involved in ion handling in the channel, it has been proposed that the pore of the RyR channel might be formed by structural elements equivalent to those found in K⁺-selective channels (31;48). The tetrameric pore in these putative structures is formed by identical elements of each RyR monomer. Using the topology defined recently by Du et al. (16) for RyR1, these elements correspond broadly to the trans-membrane spanning helices M8 and M10 together with the luminal loop linking these helices. This loop contains a short helical element (M9) and a sequence of residues analogous, respectively, to the pore helix and selectivity sequence of K⁺-selective channels. Residues of M10 would form an inner helix lining the pore from its cytosolic entrance to the putative selectivity filter located towards the luminal side of the membrane.

Recent research indicates that the mutation of residues in M10 also results in alterations in the interaction of ryanodine and other ryanoids with mouse RyR2 (45). Of particular interest is the substitution of an alanine for the glutamine residue at position 4863. This mutation abolished high affinity binding of [³H]-ryanodine to populations of receptors in isolated membrane vesicles. At the single channel level, the mutation produced no discernable effect on ion handling or the regulation of gating by physiologically relevant ligands. However an explanation for the absence of detectable [³H]-ryanodine binding became apparent following the addition of ryanodine to single Q4863A channels; the mutation produced a profound increase in the rate of dissociation of bound ryanodine and prevented modification of channel function by ryanodol. In the continued presence of ryanodine, dwell times in the unmodified and ryanodine-modified states were monitored and the probability of modification was found to have the same qualitative dependence on trans-membrane potential as that determined for 21-amino-9a-hydroxyryanodine, ryanodol and 10-O-succinoylryanodol with wild type sheep RyR2.

While the mechanisms underlying the effects on ryanoid interaction of the mutations of residues in the selectivity sequence and inner helix of the putative pore remain to be determined, a plausible interpretation is that these regions contribute in some way to the high affinity ryanoid binding site in RyR.

When assessing the probability of a location within the RyR pore for the high affinity binding site, it is important to remember that the site is unlikely to be located deep within the voltage drop across the channel. Positioning of a ligand with the dimensions of ryanodine within the voltage drop would undoubtedly produce physical occlusion of the pore. Our experimental evidence argues against this. Reduction of the rate of cation translocation following the formation of an RyR-ryanoid complex does not result from channel block; in fact with some cations unitary conductance in RyR is increased when ryanodine binds (22). Similarly, CoMFA provides no evidence for a direct steric interaction between bound ryanoids and cations during translocation through the RyR-ryanoid complex (46).

Consistent with observations reported here, Bidasee et al. (8) report that the probability of occurrence of a modified conductance state produced by C₄,C₁₂-diketopyridylryanodine (formal charge +1) is influenced by trans-membrane holding potential. In contrast, increases in channel Po produced by nanomolar concentrations of this ryanoid in their experiments were not influenced by trans-membrane potential, and these authors suggest that the site responsible for this action is likely to reside outside the conduction pathway of the channel.

HOW DOES THE INTERACTION OF A RYANOID BRING ABOUT ALTERED CHANNEL FUNCTION?

Increased Po: A dramatic increase in Po associated with modified rates of cation translocation is a characteristic common to all ryanoids so far examined at the single channel level. Tanna et al. (36) noted that while the interaction of 21-amino-9a-hydroxyryanodine with RyR2 invariably led to the occurrence of a modified conductance state with increased Po, the Po of the modified state reflected the Po of the channel during periods when the ryanoid was not bound. In other words the interaction of a ryanoid with RyR did not "lock" the channel open, rather it produced an incremental increase in Po with Po in the modified state determined by the normal gating mechanisms of the channel. Subsequently Masumiya et al. (24) and Du et al. (14) both demonstrated that increases in Po in ryanodine-modified states of individual RyR channels reflect an increased sensitivity of the channel to activating Ca²⁺ following the formation of the RyR-ryanodine complex. In addition, Du et al. (14) suggested that ryanodine interaction with RyR reduced the probability of Ca²⁺-induced inactivation and proposed that modulation of both Ca²⁺-dependent processes occurred through allosteric interactions.

Altered rates of ion translocation: Comparisons of the ion-handling characteristics of ryanodine-modified RyR2 channels with those of the channel in the absence of ryanodine have revealed that altered rates of cation translocation in the RyR-ryanodine complex reflect changes in more than one parameter (22). The affinity of the RyR pore for representative group 1a monovalent cations is increased following the interaction of ryanodine. Under these conditions the permeability of these cations relative to K⁺ is unaltered. Ryanodine modification also increases the affinity of RyR for representative alkaline earth divalent cations; however, the permeability of these cations relative to K⁺ is reduced when compared to unmodified channels. Unitary conductance of organic monovalent cations in the RyR-ryanodine complex is governed by both the size and nature of the cation. Alterations in handling parameters of ammonia and similar small organic cations follow the pattern seen with the group 1a monovalent cations. The relative permeability of larger organic cations such as diethylamine is increased in the RyR-ryanodine complex, and the conductance of this class of cations is additionally influenced by interactions with hydrophilic and hydrophobic sites of the pore. These observations have given rise to the conclusion that the interaction of ryanodine and other ryanoids with RyR results in conformational changes within the channel pore that lead to modification of the interactions of cations with this structure and produce alterations in rates of cation translocation.

Independent evidence for a conformational change in the RyR pore comes from experiments in which parameters of tetraethylammonium (TEA⁺) block of K⁺ translocation were compared for unmodified RyR2 and modified states induced by several ryanoids (38). These investigations demonstrated that the effective valence (zd), and in some cases, the affinity of TEA⁺ is altered following the interaction of ryanoids. In addition, there was a loose proportionality between the values of zd for TEA⁺ and FC for a range of ryanoids.

Our interpretation of these findings is that the interaction of ryanoids with RyR leads to a relocation of the site of interaction of TEA⁺ within the voltage drop across the channel and that this reflects a structural reorganization of the channel. It is logical to assume that this reorganization also gives rise to alterations in ion handling observed following the formation of a RyR-ryanoid complex and that the structure of the specific ryanoid in the RyR-ryanoid complex determines the degree of structural reorganization and hence the rate of cation translocation.

WHAT DETERMINES THE RATE OF CATION TRANSLOCATION FOLLOWING THE FORMATION OF THE RYR-RYANOID COMPLEX?

The preceding sections of this review demonstrate that the interaction of a ryanoid molecule with the RyR channel results in profound changes in channel function that are likely to be the result of structural alterations in the channel. The interaction of a ryanoid with RyR either induces a rearrangement of channel structure or stabilizes a conformation of the channel that is not normally resolved in the absence of the ryanoid. If we assume that different values of FC reflect different conformations of the channel pore, initial observations indicated that the conformation of the pore in the complex would be determined by the covalent structure of the component ryanoid (41;46).

However, while the interaction of the majority of ryanoids so far examined results in the occurrence of a single modified conductance state, some ryanoids give rise to several modified states of different FC.21-p-nitrobenzoylamino-9a-hydroxyryanodine is a ryanoid that dissociates very slowly from RyR and as a consequence interacts irreversibly on the time scale of a single channel experiment. In the presence of this ryanoid we have seen three distinct modified conductance states with FC values of 0.17, 0.27, and 0.59 that occurred, respectively, in 20%, 73%, and 7% of experiments (38). Even more dramatic is the observation of 33 different modified conductance states of RyR, with FC values ranging from 0.20 to 0.93, in the presence of 10-O-succinoylryanodol, a ryanoid that interacts reversibly with individual channels (40).

The mechanism underlying the observation of multiple FC states of RyR in the presence of some ryanoids emerged from molecular dynamics simulations of the motion of these ligands. These studies demonstrated the occurrence of several conformers of 21-p-nitrobenzoylamino-9a-hydroxyryanodine arising from different locations of the large p-nitrobenzoyl group. These conformers have different probabilities of occurrence with the three most common conformers found in the ratio 73:24:1% (the remaining 2% being made up of multiple conformers with very low probabilities of occurrence) (38). The very obvious correlation between the occurrence of the conformers of this ryanoid and the probability of occurrence of the 3 different FC states of the RyR-21-p-nitrobenzoylamino-9a-hydroxyryanodine complex indicates that different FC states arise from the interaction of different conformers of the ryanoid.

Similarly, molecular dynamics simulations of 10-O-succinoylryanodol reveal this to be a very flexible ligand with numerous possible locations of the succinoyl group; unlike the case with 21-p-nitrobenzoylamino-9a-hydroxyryanodine, the various conformers of 10-O-succinoylryanodol are roughly isoenergetic (40). The observation of numerous FC states of RyR in the presence of 10-O-succinoylryanodol is entirely consistent with the mechanism proposed for 21-p-nitrobenzoylamino-9a-hydroxyryanodine, with individual conductance states occurring as the consequence of the interaction of a different conformation of the ryanoid.

The observations with flexible ryanoids such as 21-p-nitrobenzoylamino-9a-hydroxyryanodine and 10-O-succinoylryanodol complement and extend the initial observations made with ryanoids that induce single modified conductance states in RyR. Clearly the ryanoid-binding site on RyR should not be thought of as a rigid template; it can accept chemically distinct ryanoids or different conformers of a single ryanoid. The formation of each individual RyR-ryanoid complex results in the occurrence of a conformation of the channel pore with its own characteristic ion handling properties. Irrespective of the potential flexibility of a ryanoid in solution, it would appear that once bound, the structure of the ligand does not change. We have observed no evidence for fluctuations between different fractional conductance states with 21-p-nitrobenzoylamino-9a-hydroxyryanodine or 10-O-succinoylryanodol (38;40). In the latter case, where association and dissociation of the ryanoid is seen in the continued presence of the ryanoid, the ligand must dissociate and rebind in a different conformation to induce a different modified state.

In contrast, fluctuations between different FC states in a single RyR-ryanoid complex are seen with some ryanoids. With ryanoids such as 21-amino-9a-hydroxyryanodine and 8b-amino-9a-hydroxyryanodine bound to the channel modified-conductance states appear "noisy" (Fig. 1.). Inspection of these events at improved time resolution reveals clear transitions between two FC states (39) and measurements of permeant cation conduction and block by TEA⁺ have established that the two states observed in the RyR-8b-amino-9a-hydroxyryanodine complex are manifestations of different conformations of the channel pore with slightly different ion-handling characteristics. Therefore, while the interactions of many ryanoids result in the stabilization of single pore conformations of RyR, fluctuations between different conformations are possible in other RyR-ryanoid complexes. The factors governing the transition between such conformations are currently under investigation.

SUMMARY

_ Measurement of single RyR Ca²⁺-release channel function suggests the possibility of three potential sites of interaction for the plant alkaloid ryanodine. This proposal is based on the observation of concentration-dependent alterations in channel function. These are i) an increase in channel Po, ii) the occurrence of states in which rates of cation translocation are modified and Po approaches 1.0 and iii) closure of the channel.

_ In this review we have discussed the mechanisms underlying the occurrence of the high Po-modified conductance state using information revealed by research in which the interaction of ryanoids of differing structure with RyR2 channels has been monitored under voltage clamp conditions.

_ The site of ryanoid interaction is only accessible from the solution at the cytosolic face of the channel and interaction occurs with an open conformation of the channel.

_ The rate of association and dissociation of the ligand, and hence the probability of occurrence of the ryanoid-modified state, are influenced by the structure of the ryanoid. These parameters are additionally sensitive to trans-membrane holding potential via a potential driven alteration in the affinity of the receptor.

_ Ryanoid structure, or the conformation of a single ryanoid, also determines the rate of cation translocation in the modified state following the formation of the RyR-ryanoid complex.

_ The interaction of a ryanoid with RyR stabilises the RyR pore in a conformation that alters the way in which the pore interacts with permeant cations during translocation.

ACKNOWLEDGEMENTS

We are grateful to the British Heart Foundation and Wellcome Trust for financial support and to our supportive and stimulating collaborators Bill Welch, Luc Ruest, Wayne Chen and John Sutko.

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Corresponding Author: AJ Williams, Cardiac Medicine, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Dovehouse St., LONDON SW3 6LY, U.K., Phone: +44 (0) 20 7351 8137, Fax: +44 (0) 20 7823 3392, E-mail: a.j.williams@imperial.ac.uk

Received: June 21, 2004. Accepted: March 12, 2004.