Intracellular Ca2+ ([Ca2+]i) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca2+ release from intracellular Ca2+ stores produces [Ca2+]i oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca2+]i oscillations in ICCs are preserved in the presence of dihydropyridine Ca2+ antagonists, which suppress Ca2+ activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca2+]i oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca2+]i oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP3Rs. The reconstituted [Ca2+]i oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

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, 4844 IIFDITFFFFVIVILLAIIQGLII 4867 , has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca 2؉ . Intracellular Ca 2؉ release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca 2؉ dependence of [ 3 H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca 2؉ and enhanced the basal activity of [ 3 H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca 2؉ . Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore.

Ca+-induced Ca2+ release (CICR) in the heart involves local Ca2+ signaling between sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) α1-subunit (α1CYM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing α1CYM and RyR2 were vesiculated and exhibited spontaneous Ca2+ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with α1CYM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca2+ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca2+ transients that required extracellular Ca2+. Depolarization-induced Ca2+ release in α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing α1CYM alone (maximal fluorescence change was 2.10 ± 0.39 and 0.54 ± 0.07, respectively), despite similar Ca2+ current densities. In addition, the gain of CICR in α1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local α1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca2+ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca2+ release and the incidence of spontaneous Ca2+ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K+ channels. This study predicts that, as is the case in K+ channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K+ channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K+ channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.