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.
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.
Comparative molecular field analysis (CoMFA) was used to analyze the relationship between the structure of a group of ryanoids and the modulation of the calcium channel function of the ryanodine receptor. The conductance properties of ryanodine receptors purified from sheep heart were measured using the planar, lipid bilayer technique. The magnitude of the ryanoid-induced fractional conductance was strongly correlated to specific structural loci on the ligand. Briefly, electrostatic effects were more prominent than steric effects. The 10-position of the ryanoid had the greatest influence on fractional conductance. Different regions of the ligand have opposing effects on fractional conductance. For example, steric bulk at the 10-position is correlated with decreased fractional conductance, whereas steric bulk at the 2-position (isopropyl position) is correlated with increased fractional conductance. In contrast to fractional conductance, the 3-position (the pyrrole locus) had the greatest influence on ligand binding, whereas the 10-position had comparatively little influence on binding. Two possible models of ryanodine action, a direct (or channel plug) mechanism and an allosteric mechanism, were examined in light of the CoMFA. Taken together, the data do not appear to be consistent with direct interaction between ryanodine and the translocating ion. The data appear to be more consistent with an allosteric mechanism. It is suggested the ryanoids act by inducing or stabilizing a conformational change in the ryanodine receptor that results in the observed alterations in cation conductance.
In an earlier investigation, we demonstrated that the likelihood of interaction of a positively charged ryanoid, 21-amino-9α-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. 112:55–69) 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.
Ryanodine, a natural product, is a complex modulator of a class of intracellular Ca2+ release channels commonly called the ryanodine receptors. Ryanodine analogs can cause the channel to persist in long-lived, subconductance states or, at high ligand concentrations, in closed, nonconducting states. In this paper, we further explore the relationship between structure and ryanodine binding to striated muscle. Ryanodine, 3-epiryanodine, and 10-ryanodine are three structural isomers of ryanodine. The dissociation constants of these compounds were measured using rabbit skeletal muscle ryanodine receptors. Placing the pyrrole carbonyl group at the 3-epi- and 10-positions of ryanodol largely restores the large loss of binding energy observed when ryanodine is hydrolyzed to ryanodol. Comparative molecular field analysis successfully predicts the enhanced binding and indicates that the pyrrole group controls the orientation of ligand binding. We propose that the ryanoids are reorientated in the binding site of the ryanodine receptors such that the pyrrole always occupies the same subsite. By applying this model, the binding constants of other ryanoids are predicted.
Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, 4844 2؉ . Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg 2؉ were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction.Ryanodine receptors (RyRs) 1 are a family of intracellular Ca 2ϩ release channels located in the sarco(endo)plasmic reticulum of a variety of cells. They play an essential role in various cellular functions including excitation-contraction coupling, fertilization, and apoptosis (1-7). Three RyR isoforms, RyR1, RyR2, and RyR3, are expressed in mammalian tissues. Mutations in the RyR1 gene have been linked to two human diseases, malignant hyperthermia and central core disease (8 -10), while mutations in the RyR2 genes are associated with polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy type 2 (11, 12). To understand the impact of the disease-causing mutations and hence the molecular and cellular basis of the diseases, detailed knowledge of the structure-function relationships of RyRs is required.One of the most widely used probes for studying the structure and function of RyRs is ryanodine, a plant alkaloid. The high affinity and specificity of the interaction of ryanodine with RyRs has facilitated the identification, purification, and cloning of the channel (2-5). Ryanodine has also been used to investigate the structural changes associated with channel gating and the mechanisms of ion conduction. Large ryanodineinduced conformational changes in the three-dimensional structure of RyR, in particular in the cytoplasmic assembly, have been observed (13). By monitoring the actions of ryanodine on single RyR channels it has been established that this ligand causes profound alterations in channel function. Interactions of ryanodine with a high affinity site on the channel induce the occurrence of a reduced conductance state with increased open probability, producing an overall effect of channel activation. In the presence of high micromolar to millimolar concentrations of ryanodine the RyR channel closes (5,14).While the functional effects of ryanodine have been well characte...
; 2840 (1985). Six new metabolites (2, 5-9) were isolated from the extracts of Ryania Speciosa Vahl. These compounds are closely related to ryanodine, the known insecticidal toxic alkaloid from this plant.Luc RUEST, DAVID R. TAYLOR et PIERRE DESLONCCHAMPS. Can. J. Chem. 63, 2840 (1985). Six nouveaux mktabolites (2, 5-9) ont CtC isolCs des extraits de la plante Ryar~ia Speciosa Vahl. Ces composCs sont Ctroitement reliCs i la ryanodine, un alcaloide toxique connu provenant de cette plante.Although the insecticidal diterpene ester ryanodine (1) (1) has been isolated from Ryarzia Speciosa since 1948 (2), to our knowledge no other diterpene had been reported from this genus prior to 1984. Very recently Waterhouse, Holden, and Casida (3) reported the isolation and characterization of dehydroryanodine (2), the second most abundant toxic (3) alkaloid (besides ryanodine) from Ryania Speciosa. Ryanodine (1) is a complex, highly oxygenated diterpene in which the unrearranged geranyl-geraniol (3) unit is easily discerned as proposed by Wiesner (1). An examination of the constituents of Ryania Speciosa was undertaken to seek compounds related to ryanodine as (i) alternate synthetic targets;' (ii) possible precursors on the biogenetic pathway to ryanodine. The actual growing interestZ in the toxic constituents of Ryania Speciosa prompted us to publish some results obtained some years ago in our laboratory.This report describes the isolation and identification of six diterpene esters of a-pyrrole carboxylic acid closely related to ryanodine (1): the already known (3) dehydroryanodine (2), diterpene esters A (5), B (6), C , (7), C2 (8), and D (9).The structure of dehydroryanodine (2), the major new diterpene slightly more polar than ryanodine (see Experimental), was proven by spectral analysis and by catalytic hydrogenation to a mixture of ryanodine (1) and 9-epiryanodine (10). We will not discuss further the structure of this compound (cf. Experimental for details) since it has been at least partly described in ref. 3.Compounds A, £3, C,, CZ, and D were isolated by chromatography of the crude extracts of powdered stems of Ryania Speciosa (2). 'Their Rf values are all greater than that of ryanodine and they are Iisted in order of increasing polarity, A being the least polar (see Experimental).Spectral data of diterpene ester A (5) are in agreement in all respects with the proposed structure. The mass spectrum indicates a formula of C26H,,010N, which is equivalent to the addition of CHZO to ryanodine, suggesting that ester A has one more hydroxyl group than ryanodine and that one of the hydroxyl groups has been methylated. The structure is supported by nmr spectral analysis: it shows three multiplets from 6.2 to 7.1 6 (for the pyrrole-a-carboxylate moiety), a sharp singlet at 5.68 6 (3-H), a methoxy at 3.56 6, an AB pattern at 2.62 and 1.83 6 (for 14-H2 on the heterocyclic ring), two tertiary and three secondary methyl groups. In addition, after DzO ex-' For a total synthesis of ryanodol (4) see rcf. 4.'At submicromolar concentr...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.