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

Abstract

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...

The Predicted TM10 Transmembrane Sequence of the Cardiac Ca2+ Release Channel (Ryanodine Receptor) Is Crucial for Channel Activation and Gating

“…7, we hypothesize that the side of the TM10 helix containing smaller and less hydrophobic residues is oriented toward the water-filled cavity, whereas the opposite side of the helix is involved in hydrophobic interactions with other helices of the channel. In support of this model, we have shown that TM10 residues such as Phe 4846 [3], Thr 4849 [6], Val 4856 [13], and Gln 4863 [20] are critical for interaction with ryanodine (26). These residues are located on the proposed pore-lining side of the TM10 helix, which is consistent with the notion that the high affinity ryanodine binding site is likely located in the outer vestibule of the RyR channel pore (31).…”

“…On the basis of these findings and the proposed pore model of RyR, it is anticipated that the putative inner helix of the RyR channel pore, TM10, would be involved in channel activation and gating. In line with this hypothesis, we have recently shown that the TM10 sequence contains an important determinant for ryanodine interaction (26) and that ryanodine is able to sensitize the RyR channel to Ca 2ϩ activation (27), suggesting the involvement of TM10 in Ca 2ϩ activation. To test this hypothesis directly, we have mutated each residue in the TM10 sequence and systematically characterized the effects of these mutations on channel activation by caffeine or by Ca 2ϩ .…”

“…A total of 24 TM10 mutants were generated. We have shown that all of the TM10 mutants were expressed in HEK293 cells and that their levels of expression were comparable with that of the wt (26). To screen the impact of this large number of mutations on channel activation quickly, we employed a simple and quick Ca 2ϩ release assay based on the use of the HEK293 cell expression system and using caffeine as a functional probe.…”

“…Site-directed Mutagenesis and DNA Transfection-Single point mutations within the proposed TM10 transmembrane sequence of the mouse RyR2 were generated by the overlap extension method as described previously (26). HEK293 cells grown on 100-mm tissue culture dishes in Dulbecco's modified Eagle's medium, supplemented with 0.1 mM nonessential amino acids, 4 mM L-glutamine, 100 units of penicillin/ml, 100 g of streptomycin/ml, 4.5 g of glucose/liter, and 10% fetal calf serum, for 18 -20 h after subcultures were transfected with 12 g of wt or mutant RyR2 cDNA using the method of Ca 2ϩ phosphate precipitation (28).…”

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

“…Unfortunately, no binding domains are known for caffeine, which also requires tens of millimolar concentrations in other cells, just as in Paramecium (25,48). Putative ryanodine-binding sites normally present in the last C-terminal transmembrane domain (100) are not present in the primary sequence of P. tetraurelia CRC-IV-1 (Fig. 2D), thus explaining their insensitivity.…”