Luc Ruest

Residue Gln4863 within a predicted transmembrane sequence of the Ca2+ release channel (ryanodine receptor) is critical for ryanodine interaction.

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, 4844IIFDITFFFFVIVILLAIIQGLII4867, of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca2+ in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [3H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [3H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca2+. 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 Mg2+ 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.

A Convenient Synthesis OF 2-Carbomethoxycyclohexanone

Abstract In connection with a synthetic problem of our laboratory, it became necessary to have pure 2-carbomethoxycyclohexanone (1). A search of the chemical literature revealed that the known procedures for the preparation of this compound do not give good yields.

An Anionic Ryanoid, 10-O-succinoylryanodol, Provides Insights into the Mechanisms Governing the Interaction of Ryanoids and the Subsequent Altered Function of Ryanodine-receptor Channels

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.

Quantification of the effects of a ryanodine receptor channel mutation on interaction with a ryanoid

Understanding the nature of the interaction of the plant alkaloid ryanodine with its receptor channel (RyR) is important to aid interpretation of physiological studies and provide structure-function information about RyR. We present here the first quantitative description of the relative single-channel kinetic effects of a single-point mutation in RyR2. We exploit the well-characterized ryanoid 8β-amino-9α-hydroxyryanodine that displays reversible kinetics with RyR2. We explicitly show that the effect of the Q4863A mutation is to increase the apparent dissociation constant by increasing the apparent dissociation rate of the ryanoid. The voltage-dependence of the interaction displays no change. We infer that Q4863 is not involved with the voltage-drop but is able to influence ryanoid-bound structural changes. We discuss structural mechanisms by which this mutation could affect ryanoid interaction.

Excess noise in modified conductance states following the interaction of ryanoids with cardiac ryanodine receptor channels

The interaction of ryanodine with the ryanodine receptor (RyR) produces profound changes in channel function. Open probability increases dramatically and conductance is reduced. In this report we describe differences in the properties of reduced conductance states produced by the interaction of ryanodine derivatives with RyR channels. Some reduced conductance states are considerably noisier than the normal open state of the RyR channel. Inspection and analysis of these events reveals that the excess noise arises from transitions between two conductance states. Following the interaction of certain ryanodine derivatives, RyR channels undergo transitions between two conformations with slightly different ion‐handling properties.

The Interaction of an Impermeant Cation with the Sheep Cardiac RyR Channel Alters Ryanoid Association

In previous studies, we have demonstrated that the interaction of ryanoids with the sarcoplasmic reticulum Ca2+-release channel [ryanodine receptor (RyR)] incorporated into planar lipid bilayers reduced the effectiveness of tetraethylammonium (TEA+) as a blocker of K+ translocation (J Gen Physiol 117: 385-393, 2001). In the current study, we investigated both the effect of TEA+ on [3H]ryanodine binding and the actions of this impermeant cation on the interaction of the reversible ryanoid 21-amino-9α-hydroxyryanodine with individual, voltage-clamped RyR channels. A dose-dependent inhibition of [3H]ryanodine binding was observed in the presence of TEA+, suggesting that the cation and alkaloid compete for access to a common site of interaction. Single channel studies gave further insights into the mechanism of the competition between the two classes of ligands. TEA+ decreases the association rate of 21-amino-9α-hydroxyryanodine with its receptor, whereas the dissociation rate of the ryanoid from the channel was unaffected. Our results demonstrate that TEA+ inhibits both K+ translocation through RyR, and ryanoid interaction at the high affinity ryanodine site on the channel. These actions involve binding of TEA+ to different, but weakly interacting, sites in the RyR channel.

Pharmacology of the Ryania Alkaloids: The Ester A, a Ryanodine Analog That Only Increases Sarcoplasmic Reticulum Calcium Permeability

Ryanodine, an alkaloid extracted from the wood of Ryania speciosa, is a specific ligand of the “foot” protein of the triad junction(1) and a modulator of sarcoplasmic reticulum (SR) calcium permeability in both striated and smooth muscles.(2–5) Ryanodine modulates the calcium permeability of sarcoplasmic reticulum terminal cisternae membranes in a complex manner. Depending on its concentration and the experimental conditions employed, ryanodine can either increase or decrease this variable.(2–4) It is important to note that either effect will reduce activation-dependent release of SR calcium in intact muscle cells.(6)