Interactions of a Reversible Ryanoid (21-Amino-9α-Hydroxy-Ryanodine) with Single Sheep Cardiac Ryanodine Receptor Channels

Abstract

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-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9α-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-9α-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.

Residue Gln4863 within a Predicted Transmembrane Sequence of the Ca2+ Release Channel (Ryanodine Receptor) Is Critical for Ryanodine Interaction

“…4B). Consistent with our previous observation that the probability of ryanoid modification is dependent upon channel P o (16), raising the level of activating Ca 2ϩ from 0.22 to 0.49 M increased both P o and the occurrence of ryanodine modification (Fig. 4C).…”

“…I4861A [18], I4862A [19], G4864A [21], L4865A [22], and I4867A [24] all had binding capacities in excess of 25% of the wt RyR2. In contrast, mutants F4846A [3], D4847A [4], T4849A [6], F4850A [7], F4851A [8], I4855A [12], V4856A [13], L4858A [15], L4859A [16], Q4863A [20], and I4866A [23] displayed binding capacities of 5% or less of wt RyR2 ( Fig. 2 and Table I).…”

“…Caffeine and Ryanodine Response of HEK293 Cells Expressing the wt and Mutant RyR2-Since the high affinity ryanodine binding site is only available when the RyR channel is in an open conformation (16,(33)(34)(35), the profound reduction in [ 3 H]ryanodine binding to some TM10 mutants could indicate that these mutations yield channels that do not open under the conditions used in our binding experiments. In other words, the mutant channel, although readily expressed, is not a functional Ca 2ϩ release channel.…”

“…3A, b-e), indicating that these mutant channels remain sensitive to ryanodine modulation in HEK293 cells despite their lack of [ 3 H]ryanodine binding in cell lysates. On the other hand, no measurable caffeine-or ryanodine-induced intracellular Ca 2ϩ release was detected in HEK293 cells expressing mutants D4847A [4], F4850A [7], F4851A [8], L4858A [15], L4859A [16], or I4866A [23] (Fig. 3B).…”

“…Unlike ryanodine, which modifies RyR in an irreversible manner on the time scale of single channel experiments, some ryanoids exhibit a reversible effect on single RyR channel function. These reversible ryanoids have provided useful probes for studying the mechanisms of ryanoidRyR interaction and have been used to demonstrate that ryanoid-RyR interaction is influenced by transmembrane voltage (15)(16)(17)(18). Comprehensive analyses of the binding properties of various ryanoids have revealed that the pyrrole locus at the 3-position of the ryanodine molecule is absolutely required for high affinity ryanodine binding to RyR.…”