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Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site.

Tanna B, Welch W, Ruest L, Sutko JL, Williams AJ - J. Gen. Physiol. (2001)

Bottom Line: In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced.It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid.The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London SW3 6LY, United Kingdom.

ABSTRACT
The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.

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Single-channel traces showing the influence of 20 mM TEA+ on the amplitude of ryanoid-modified conductance states at +40 mV. Both ryanodol and 21-azido-9α-hydroxyryanodine interact rapidly with RyR. For this reason, both unmodified and ryanoid-modified conductance states are shown in the presence of TEA+ for these ryanoids. In contrast, 21-p-nitrobenzoylamino-9α-hydroxyryanodine is a slow-binding modifier of RyR channel function and, as a consequence, traces in the presence of TEA+ show only the ryanoid-modified conductance state. C, closed conductance level; M, modified conductance level; O, fully open conductance level.
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Figure 4: Single-channel traces showing the influence of 20 mM TEA+ on the amplitude of ryanoid-modified conductance states at +40 mV. Both ryanodol and 21-azido-9α-hydroxyryanodine interact rapidly with RyR. For this reason, both unmodified and ryanoid-modified conductance states are shown in the presence of TEA+ for these ryanoids. In contrast, 21-p-nitrobenzoylamino-9α-hydroxyryanodine is a slow-binding modifier of RyR channel function and, as a consequence, traces in the presence of TEA+ show only the ryanoid-modified conductance state. C, closed conductance level; M, modified conductance level; O, fully open conductance level.

Mentions: What mechanisms are involved in the alteration in effectiveness of TEA+ as a blocker of RyR after the interaction of 21-amino-9α-hydroxyryanodine with the channel? In previous investigations, we have demonstrated that ryanoid-induced modification of permeant ion handling in RyR is strongly correlated to specific structural loci on the ryanoid molecule with contributions from both electrostatic and steric effects (Welch et al. 1997). To investigate the possibility that similar structural features underlie the alteration of the effectiveness of TEA+ as a blocker of K+ current in RyR, we have examined the influence of three additional members of the ryanoid group of ligands: ryanodol, 21-azido-9α-hydroxyryanodine, and 21-p-nitrobenzoylamino-9α-hydroxyryanodine. These ryanoids vary in both steric bulk and charge and, after interaction with the channel, yield values of FC ranging from 0.67 to 0.17. Single-channel current amplitude of both unmodified and ryanoid-modified conductance states were monitored at holding potentials within the range ±80 mV in the absence and presence of 20 mM TEA+. Examples of current fluctuations of representative channels at a holding potential of +40 mV are given in Fig. 4. The left panel of Fig. 4 demonstrates modification of the RyR channel that results from the interaction of the indicated ryanoid. In all cases, the interaction of the ryanoid with the RyR channel results in the occurrence of a modified conductance state with high Po. As outlined above, the amplitude of the modified conductance state is dependent on the structure of the ryanoid bound to the channel (Tinker et al. 1996; Welch et al. 1997). The values of FC induced by the ryanoids shown in Fig. 4 are 0.67 for ryanodol, 0.55 for 21-azido-9α-hydroxyryanodine, and 0.27 for 21-p-nitrobenzoylamino-9α-hydroxyryanodine. The influence of 20 mM TEA+ on the current amplitude of these various ryanoid-modified states is shown in the right panel of Fig. 4. In all cases, the addition of 20 mM TEA+ leads to a reduction in current amplitude. However, a comparison of the current amplitudes of the ryanoid-modified conductance states in the absence and presence of the blocking cation demonstrates that the effectiveness of TEA+ differs in the various ryanoid-modified conductance states. At this holding potential, the magnitude of block produced by TEA+ of the ryanoid-modified conductance state is in order


Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site.

Tanna B, Welch W, Ruest L, Sutko JL, Williams AJ - J. Gen. Physiol. (2001)

Single-channel traces showing the influence of 20 mM TEA+ on the amplitude of ryanoid-modified conductance states at +40 mV. Both ryanodol and 21-azido-9α-hydroxyryanodine interact rapidly with RyR. For this reason, both unmodified and ryanoid-modified conductance states are shown in the presence of TEA+ for these ryanoids. In contrast, 21-p-nitrobenzoylamino-9α-hydroxyryanodine is a slow-binding modifier of RyR channel function and, as a consequence, traces in the presence of TEA+ show only the ryanoid-modified conductance state. C, closed conductance level; M, modified conductance level; O, fully open conductance level.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233661&req=5

Figure 4: Single-channel traces showing the influence of 20 mM TEA+ on the amplitude of ryanoid-modified conductance states at +40 mV. Both ryanodol and 21-azido-9α-hydroxyryanodine interact rapidly with RyR. For this reason, both unmodified and ryanoid-modified conductance states are shown in the presence of TEA+ for these ryanoids. In contrast, 21-p-nitrobenzoylamino-9α-hydroxyryanodine is a slow-binding modifier of RyR channel function and, as a consequence, traces in the presence of TEA+ show only the ryanoid-modified conductance state. C, closed conductance level; M, modified conductance level; O, fully open conductance level.
Mentions: What mechanisms are involved in the alteration in effectiveness of TEA+ as a blocker of RyR after the interaction of 21-amino-9α-hydroxyryanodine with the channel? In previous investigations, we have demonstrated that ryanoid-induced modification of permeant ion handling in RyR is strongly correlated to specific structural loci on the ryanoid molecule with contributions from both electrostatic and steric effects (Welch et al. 1997). To investigate the possibility that similar structural features underlie the alteration of the effectiveness of TEA+ as a blocker of K+ current in RyR, we have examined the influence of three additional members of the ryanoid group of ligands: ryanodol, 21-azido-9α-hydroxyryanodine, and 21-p-nitrobenzoylamino-9α-hydroxyryanodine. These ryanoids vary in both steric bulk and charge and, after interaction with the channel, yield values of FC ranging from 0.67 to 0.17. Single-channel current amplitude of both unmodified and ryanoid-modified conductance states were monitored at holding potentials within the range ±80 mV in the absence and presence of 20 mM TEA+. Examples of current fluctuations of representative channels at a holding potential of +40 mV are given in Fig. 4. The left panel of Fig. 4 demonstrates modification of the RyR channel that results from the interaction of the indicated ryanoid. In all cases, the interaction of the ryanoid with the RyR channel results in the occurrence of a modified conductance state with high Po. As outlined above, the amplitude of the modified conductance state is dependent on the structure of the ryanoid bound to the channel (Tinker et al. 1996; Welch et al. 1997). The values of FC induced by the ryanoids shown in Fig. 4 are 0.67 for ryanodol, 0.55 for 21-azido-9α-hydroxyryanodine, and 0.27 for 21-p-nitrobenzoylamino-9α-hydroxyryanodine. The influence of 20 mM TEA+ on the current amplitude of these various ryanoid-modified states is shown in the right panel of Fig. 4. In all cases, the addition of 20 mM TEA+ leads to a reduction in current amplitude. However, a comparison of the current amplitudes of the ryanoid-modified conductance states in the absence and presence of the blocking cation demonstrates that the effectiveness of TEA+ differs in the various ryanoid-modified conductance states. At this holding potential, the magnitude of block produced by TEA+ of the ryanoid-modified conductance state is in order

Bottom Line: In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced.It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid.The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiac Medicine, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, London SW3 6LY, United Kingdom.

ABSTRACT
The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.

Show MeSH
Related in: MedlinePlus