RESUMEN
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 8beta-amino-9alpha-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.
Asunto(s)
Activación del Canal Iónico , Proteínas Mutantes/metabolismo , Mutación Puntual , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Rianodina/análogos & derivados , Rianodina/metabolismo , Alanina/química , Animales , Sitios de Unión , Calcio/metabolismo , Clonación Molecular , Glutamina/química , Cinética , Ligandos , Ratones , Modelos Químicos , Mutagénesis Sitio-Dirigida , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/química , Retículo Sarcoplasmático/metabolismoRESUMEN
In previous studies, we have demonstrated that the interaction of ryanoids with the sarcoplasmic reticulum Ca(2+)-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 [(3)H]ryanodine binding and the actions of this impermeant cation on the interaction of the reversible ryanoid 21-amino-9alpha-hydroxyryanodine with individual, voltage-clamped RyR channels. A dose-dependent inhibition of [(3)H]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-9alpha-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.
Asunto(s)
Bloqueadores de los Canales de Potasio/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Rianodina/metabolismo , Tetraetilamonio/farmacología , Animales , Cationes Monovalentes/metabolismo , Transporte Iónico/efectos de los fármacos , Ligandos , Potasio/metabolismo , Rianodina/análogos & derivados , OvinosRESUMEN
The specific, high-affinity interaction of the plant toxin ryanodine with its molecular target the ryanodine receptor channel (RyR) has been instrumental in RyR research. Alanine scanning of putative pore regions of mouse RyR2 has highlighted the amino acid Gln4863, predicted to lie within trans-membrane helix TM10, as an important determinant of ryanodine binding. We have investigated the effects of several ryanodine derivatives, guanidinopropionylryanodine, 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine, 8beta-amino-9alpha-hydroxyryanodine, and 21-amino-9alpha-hydroxyryanodine, with the mouse Q4863A RyR2 mutant at the single-channel level. Our results demonstrate that the rate of dissociation of all ryanoids investigated is increased by the mutation. The modification of channel function after ryanoid binding is qualitatively similar for wild-type and mutant, but in several cases, single-channel conductances were increased with Q4863A. These novel findings have been interpreted within the framework of existing comparative molecular field analysis studies on ryanoids. We suggest that replacement of a glutamine by an alanine residue at position 4863 causes RyR2 to simultaneously alter interactions with both ends of the ryanoid molecule.
Asunto(s)
Alanina/genética , Glicina/genética , Miocardio/metabolismo , Mutación Puntual , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Rianodina/metabolismo , Animales , Cinética , Potenciales de la Membrana , Ratones , Mutagénesis Sitio-Dirigida , Rianodina/análogos & derivadosRESUMEN
We have investigated the influence of transmembrane holding potential on the kinetics of interaction of a cationic ryanoid, 8beta-amino-9alpha-hydroxyryanodine, with individual ryanodine receptor (RyR) channels and on the functional consequences of this interaction. In agreement with previous studies involving cationic, neutral, and anionic ryanoids, both rates of association and dissociation of the ligand are sensitive to transmembrane potential. A voltage-sensitive equilibrium between high- and low-affinity forms of the receptor underlies alterations in rates of association and dissociation of the ryanoid. The interaction of 8beta-amino-9alpha-hydroxyryanodine with RyR influences the rate of cation translocation through the channel. With this ryanoid bound, the channel fluctuates between two clearly resolved subconductance states (alpha and beta). We interpret this observation as indicating that with 8beta-amino-9alpha-hydroxyryanodine bound, the pore of the RyR channel exists in two essentially isoenergetic conformations with differing ion-handling properties. The equilibrium between the alpha- and beta-states of the RyR-8beta-amino-9alpha-hydroxyryanodine complex is sensitive to transmembrane potential. However, the mechanisms determining this equilibrium differ from those responsible for the voltage-sensitive equilibrium between high- and low-affinity forms of the receptor.
Asunto(s)
Miocardio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Rianodina/análogos & derivados , Rianodina/farmacología , Retículo Sarcoplasmático/metabolismo , Animales , Sitios de Unión , Transporte Biológico , Calcio/metabolismo , Cationes , Citosol/metabolismo , Electrofisiología , Iones , Cinética , Ligandos , Membrana Dobles de Lípidos , Potenciales de la Membrana , Modelos Estadísticos , Fosfolípidos/química , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Canal Liberador de Calcio Receptor de Rianodina/aislamiento & purificación , Ovinos , Factores de TiempoRESUMEN
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.
Asunto(s)
Glutamina , Proteínas de la Membrana/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Rianodina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Clonación Molecular , Electrofisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Unión Proteica , Canal Liberador de Calcio Receptor de Rianodina/química , TransfecciónRESUMEN
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.
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina/fisiología , Rianodina/farmacología , Succinatos/farmacología , Animales , Electrofisiología , Ligandos , Miocardio , Técnicas de Placa-Clamp , Rianodina/análogos & derivados , OvinosRESUMEN
The regulatory effect of amino acids on the production of thaxtomin A, a phytotoxin produced by Streptomyces scabies, was investigated. Tryptophan had an important inhibitory effect on the toxin biosynthesis in all five strains of S. scabies tested. Two other aromatic amino acids (tyrosine and phenylalanine) also inhibited thaxtomin A biosynthesis, while aliphatic amino acids did not cause an important decline in thaxtomin A production. Methylation of tryptophan prevented or reduced the inhibitory effect on thaxtomin A biosynthesis. In spite of the inhibitory action of tryptophan and phenylalanine on thaxtomin A production, incorporation of these radiolabeled molecules into thaxtomin A confirmed that they are metabolic precursors for the biosynthesis of the phytotoxin.
Asunto(s)
Indoles/metabolismo , Piperazinas/metabolismo , Streptomyces/metabolismo , Toxinas Bacterianas/biosíntesis , Fenilalanina/fisiología , Enfermedades de las Plantas/microbiología , Triptófano/fisiologíaRESUMEN
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.