Electrophysiological effects of ryanodine derivatives on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Abstract

We have examined the effects of a number of derivatives of ryanodine on K+ conduction in the Ca2+ release channel purified from sheep cardiac sarcoplasmic reticulum (SR). In a fashion comparable to that of ryanodine, the addition of nanomolar to micromolar quantities to the cytoplasmic face (the exact amount depending on the derivative) causes the channel to enter a state of reduced conductance that has a high open probability. However, the amplitude of that reduced conductance state varies between the different derivatives. In symmetrical 210 mM K+, ryanodine leads to a conductance state with an amplitude of 56.8 +/- 0.5% of control, ryanodol leads to a level of 69.4 +/- 0.6%, ester A ryanodine modifies to one of 61.5 +/- 1.4%, 9,21-dehydroryanodine to one of 58.3 +/- 0.3%, 9 beta,21beta-epoxyryanodine to one of 56.8 +/- 0.8%, 9-hydroxy-21-azidoryanodine to one of 56.3 +/- 0.4%, 10-pyrroleryanodol to one of 52.2 +/- 1.0%, 3-epiryanodine to one of 42.9 +/- 0.7%, CBZ glycyl ryanodine to one of 29.4 +/- 1.0%, 21-p-nitrobenzoyl-amino-9-hydroxyryanodine to one of 26.1 +/- 0.5%, beta-alanyl ryanodine to one of 14.3 +/- 0.5%, and guanidino-propionyl ryanodine to one of 5.8 +/- 0.1% (chord conductance at +60 mV, +/- SEM). For the majority of the derivatives the effect is irreversible within the lifetime of a single-channel experiment (up to 1 h). However, for four of the derivatives, typified by ryanodol, the effect is reversible, with dwell times in the substate lasting tens of seconds to minutes. The effect caused by ryanodol is dependent on transmembrane voltage, with modification more likely to occur and lasting longer at +60 than at -60 mV holding potential. The addition of concentrations of ryanodol insufficient to cause modification does not lead to an increase in single-channel open probability, such as has been reported for ryanodine. At concentrations of > or = 500 mu M, ryanodine after initial rapid modification of the channel leads to irreversible closure, generally within a minute. In contrast, comparable concentrations of beta-alanyl ryanodine do not cause such a phenomenon after modification, even after prolonged periods of recording (>5 min). The implications of these results for the site(s) of interaction with the channel protein and mechanism of the action of ryanodine are discussed. Changes in the structure of ryanodine can lead to specific changes in the electrophysiological consequences of the interaction of the alkaloid with the sheep cardiac SR Ca2+ release channel.

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

“…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).…”

“…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).…”

“…5e. Values of slope conductance of the unmodified and ryanodine-modified states are 796 Ϯ 5.7 and 448 Ϯ 6.8 pico- 4] 0.6 Ϯ 0.8 Ϫ Ϫ I4848A [5] 59 Ϯ 10 ϩ ϩ T4849A [6] 0.5 Ϯ 0.8 ϩ ϩ F4850A [7] 0.1 Ϯ 0.5 Ϫ Ϫ F4851A [8] 0.1 Ϯ 0.3 Ϫ Ϫ F4852A [9] 84 Ϯ 16 ϩ ϩ F4853A [10] 99 Ϯ 28 ϩ ϩ V4854A [11] 36 Ϯ 1.2 ϩ ϩ I4855A [12] 3.8 Ϯ 1.6 ϩ ϩ V4856A [13] 1.0 Ϯ 0.9 ϩ ϩ I4857A [14] 64 Ϯ 2.4 ϩ ϩ L4858A [15] 2.5 Ϯ 1.6 Ϫ Ϫ L4859A [16] 3.8 Ϯ 1.4 Ϫ Ϫ A4860G [17] 58 Ϯ 3.5 ϩ ϩ I4861A [18] 98 Ϯ 5.7 ϩ ϩ I4862A [19] 61 Ϯ 2.7 ϩ Ϫ Q4863A [20] 0.0 Ϯ 0.0 ϩ Ϫ G4864A [21] 93 Ϯ 4.3 ϩ ϩ L4865A [22] 87 Ϯ 7.1 ϩ ϩ I4866A [23] 5.1 Ϯ 1.8 Ϫ Ϫ I4867A [24] 25 Ϯ 6. siemens (n ϭ 3) and are comparable to those of the corresponding states of wt RyR2 channels (23). While the Q4863A [20] mutation produces a profound alteration in the kinetics of ryanodine interaction it does not seem to cause gross alterations in channel conduction or the modulation of gating by other ligands.…”

“…Voltage Influence of Ryanodine Interaction with Single Q4863A [20] Mutant Channels-It has been shown previously that the effects of several reversible ryanoids on single RyR2 channels are dependent on the transmembrane holding potential (15)(16)(17)(18). However, the question of whether ryanodine action is also dependent on the transmembrane potential cannot be addressed due to the irreversibility of ryanodine action on single wt RyR2 channels.…”

“…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.…”