Luminal Ca<Superscript>2+</Superscript> activation of cardiac ryanodine receptors by luminal and cytoplasmic domains

The ryanodine receptors form the calcium release channel in the membrane of the sarcoplasmic reticulum (SR, the main intracellular Ca²⁺ store). The importance of ryanodine receptors (RyRs) to cardiac pacemaking and rhythmicity is highlighted by more than 69 mutations, RyR mutations, which underlie arrhythmias and sudden cardiac death. Although most of these mutations lie in cytoplasmic domains, they all cause increased RyR activation by Ca²⁺ in the SR lumen. Presented here is a review of the mechanisms by which cytoplasmic domains of the RyR can determine luminal activation.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptorCa²⁺ release channel (RyR1) in the presence of nanomolarCa²⁺ concentrations. However, the role of CaM activation inthe mechanisms that control Ca²⁺ release from thesarcoplasmic reticulum (SR) in skeletal muscle and in the heart remainsunclear. In media that contained 100 nM Ca²⁺, the rate of⁴⁵Ca²⁺ release from porcine skeletal muscle SRvesicles was increased approximately threefold in the presence of CaM(1 µM). In contrast, cardiac SR vesicle⁴⁵Ca²⁺ release was unaffected by CaM,suggesting that CaM activated the skeletal RyR1 but not the cardiacRyR2 channel isoform. The activation of RyR1 by CaM was associated withan approximately sixfold increase in the Ca²⁺ sensitivityof [³H]ryanodine binding to skeletal muscle SR, whereasthe Ca²⁺ sensitivity of cardiac SR[³H]ryanodine binding was similar in the absence andpresence of CaM. Cross-linking experiments identified both RyR1 andRyR2 as predominant CaM binding proteins in skeletal and cardiac SR,respectively, and [³⁵S]CaM binding determinations furtherindicated comparable CaM binding to the two isoforms in the presence ofmicromolar Ca²⁺. In nanomolar Ca²⁺, however,the affinity and stoichiometry of RyR2 [³⁵S]CaM bindingwas reduced compared with that of RyR1. Together, our results indicatethat CaM activates RyR1 by increasing the Ca²⁺ sensitivityof the channel, and further suggest differences in CaM's functionalinteractions with the RyR1 and RyR2 isoforms that may potentiallycontribute to differences in the Ca²⁺ dependence of channelactivation in skeletal and cardiac muscle.

     

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin

The mammalian ryanodine receptor Ca²⁺ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca²⁺. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2 μM Ca²⁺, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca²⁺ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1–3725 and RyR2 C-terminal aa 3692–4968 were inhibited by CaM at <1 μM Ca²⁺ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1–4301 and RyR2 4254–4968 was activated at <1 μM Ca²⁺ similar to RyR1. Replacement of RyR1 aa 3726–4298 with corresponding residues from RyR2 conferred CaM inhibition at <1 μM Ca²⁺, which suggests RyR1 aa 3726–4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca²⁺ binding motifs in RyR1 aa 4081–4092 (EF1) and aa 4116–4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca²⁺ concentrations.     

Ca-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

We have shown that physiological levels of Ca²⁺-calmodulin (Ca²⁺CaM; 50-100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca²⁺ sparks and waves in cardiac cells. In contrast, it is well known that Ca²⁺CaM inhibits [³H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [³H]ryanodine binding technique does not reflect the effects of Ca²⁺CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca²⁺CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca²⁺CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M₁ and M₂, in a voltage-dependent manner. Ca²⁺CaM significantly alters the equilibrium between these two states. The results suggest that Ca²⁺CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca²⁺CaM at the single-channel level are not mirrored by [³H]ryanodine binding studies.     

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca²⁺ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca²⁺− and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca²⁺-independent basal activity and Ca²⁺-dependent activation of [³H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca²⁺ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca²⁺ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.     

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²⁺ release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²⁴⁴²-Pro²⁴⁷⁷ of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²⁺ release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²⁺ release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²⁺ current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.     

Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine: IMPLICATIONS IN CALCIUM SIGNALING*

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca²⁺ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca²⁺ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca²⁺ signaling, we hypothesize that SPC could lead to Ca²⁺ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca²⁺ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca²⁺-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors.     

Characterization of the Calcium-release Channel/Ryanodine Receptor from Zebrafish Skeletal Muscle

Calcium (Ca²⁺)-mediated signaling is fueled by two sources for Ca²⁺: Ca²⁺ can enter through Ca²⁺ channels located in the plasma membrane and can also be released from intracellular stores. In the present study the intracellular Ca²⁺ release channel/ryanodine receptor (RyR) from zebrafish skeletal muscle was characterized. Two RyR isoforms could be identified using immunoblotting and single-channel recordings. Biophysical properties as well as the regulation by modulators of RyR, ryanodine, ruthenium red and caffeine, were measured. Comparison with other RyRs showed that the zebrafish RyRs have features observed with all RyRs described to date and thus, can serve as a model system in future genetic and physiological studies. However, some differences in the biophysical properties were observed. The slope conductance for both isoforms was higher than that of the mammalian RyR type 1 (RyR1) measured with divalent ions. Also, inhibition by millimolar Ca²⁺ concentrations of the RyR isoform that is inhibited by high Ca²⁺ concentrations (teleost α RyR isoform) was attenuated when compared to mammalian RyRs. Due to the widespread expression of RyR these findings have important implications for the interpretation of the role of the RyR in Ca²⁺ signaling when comparing zebrafish with mammalian physiology, especially when analyzing mutations underlying physiological changes in zebrafish. Received: 15 February 2001/Revised: 1 June 2001     

Investigating dual Ca2+ modulation of the ryanodine receptor 1 by molecular dynamics simulation

The ryanodine receptors (RyR) are essential to calcium signaling in striated muscles. A deep understanding of the complex Ca²⁺-activation/inhibition mechanism of RyRs requires detailed structural and dynamic information for RyRs in different functional states (eg, with Ca²⁺ bound to activating or inhibitory sites). Recently, high-resolution structures of the RyR isoform 1 (RyR1) were solved by cryo-electron microscopy, revealing the location of a Ca²⁺ binding site for activation. Toward elucidating the Ca²⁺-modulation mechanism of RyR1, we performed extensive molecular dynamics simulation of the core RyR1 structure in the presence and absence of activating and solvent Ca²⁺ (total simulation time is >5 μs). In the presence of solvent Ca²⁺, Ca²⁺ binding to the activating site enhanced dynamics of RyR1 with higher inter-subunit flexibility, asymmetric inter-subunit motions, outward domain motions and partial pore dilation, which may prime RyR1 for subsequent channel opening. In contrast, the solvent Ca²⁺ alone reduced dynamics of RyR1 and led to inward domain motions and pore contraction, which may cause inhibition. Combining our simulation with the map of disease mutation sites in RyR1, we constructed a wiring diagram of key domains coupled via specific hydrogen bonds involving the mutation sites, some of which were modulated by Ca²⁺ binding. The structural and dynamic information gained from this study will inform future mutational and functional studies of RyR1 activation and inhibition by Ca²⁺.     

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca²⁺, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca²⁺, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca²⁺, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca²⁺ stabilized the closed conformation by a factor of two. We conclude that the Ca²⁺-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca²⁺-dependent structural dynamics of bound CaM.     

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules

Highly localized Ca²⁺ release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca²⁺ syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca²⁺ pool. Traditional sources of intracellular Ca²⁺ appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca²⁺-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca²⁺ from vesicular stores. Such localized vesicular Ca²⁺ release events at the precise location of exocytosis could provide a Ca²⁺ amplification mechanism capable of modulating neuropeptide release physiologically.     

All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells

The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that is probably caused by Ca²⁺ release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca²⁺ release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to monitor changes in [Ca²⁺]_i and perfused with either cold (0°C), room temperature (RT), or RT buffer containing 40 mM caffeine. Control cells showed no significant response to cold or caffeine, whereas robust Ca²⁺ transients were recorded in response to both RC and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response and that all three isoforms respond in a similar manner. Ca²⁺ release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state. calcium release channel; sarcoplasmic reticulum; excitation-contraction coupling     

Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-mediated Ca2+ Release

The intracellular Ca²⁺ sensor calmodulin (CaM) regulates the cardiac Ca²⁺ release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome. Here, we investigated the effect of CaM mutations causing CPVT (N53I), long QT syndrome (D95V and D129G), or both (CaM N97S) on RyR2-mediated Ca²⁺ release. All mutations increased Ca²⁺ release and rendered RyR2 more susceptible to store overload-induced Ca²⁺ release (SOICR) by lowering the threshold of store Ca²⁺ content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca²⁺ binding of the N- and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM-binding domain of RyR2. The N53I mutation decreased the affinity of Ca²⁺ binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V, and D129G had little or no effect on Ca²⁺ binding to the N-domain but markedly decreased the affinity of the C-domain for Ca²⁺. These results suggest that mutations D95V, N97S, and D129G alter the interaction between CaM and the CaMBD and thus RyR2 regulation. Because the N53I mutation minimally affected Ca²⁺ binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca²⁺-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca²⁺ concentration, is proposed.     

The insecticide chlorantraniliprole is a weak activator of mammalian skeletal ryanodine receptor/Ca2+ release channel

Chlorantraniliprobe (Chlo), a potent insecticide, demolishes intracellular Ca²⁺ homeostasis of insects by inducing uncontrolled Ca²⁺ release through ryanodine receptors (RyRs). Chlo is lethal to insects but has low toxicity to mammals. In this study, we investigated the effects of Chlo on RyR1 from mammalian skeletal muscle. Ca²⁺ release assay indicated that Chlo at high concentrations promoted Ca²⁺ release from sarcoplasmic reticulum through RyR1 channels. Single channel recording of purified RyR1 showed that Chlo activated RyR1 channel, increased channel open probability P_o, reduced channel mean close time T_c, but did not change the channel mean open time T_o, suggesting that Chlo destabilized the closed RyR1 channel, rendered the channel easy to open. The dissociation constant K_d values of Chlo for RyR1 were of micromolar level, approximately 100-fold larger than that for insect RyR. The K_d values were smaller for open states than for closed/blocked states of the RyR1 channel. The maximal binding capacity B_max did not change in the presence of either channel activators or inhibitors/blockers. Our results demonstrate that the insecticide Chlo is a weak activator of mammalian RyR1. It can interact with mammalian RyR1 and activate RyR1 channel but with much lower affinity compared with insect RyR; Chlo has a binding site distinct from all known RyR channel modulators and represents a novel type of RyR channel modulator. Our data provide biochemical and pharmacological insights into its high specificity to insect RyR and high selectivity of poisoning to insects over mammals.     

Recruiting RyRs to Open in a Ca2+ Release Unit: Single-RyR Gating Properties Make RyR Group Dynamics

In cardiac myocytes, clusters of type-2 ryanodine receptors (RyR2s) release Ca²⁺ from the sarcoplasmic reticulum (SR) via a positive feedback mechanism in which fluxed Ca²⁺ activates nearby RyRs. Although the general principles of this are understood, less is known about how single-RyR gating properties define the RyR group dynamics in an array of many channels. Here, we examine this using simulations with three models of RyR gating that have identical open probabilities: the commonly used two-state Markov gating model, one that utilizes multiple exponentials to fit single-channel open time (OT) and closed time (CT) distributions, and an extension of this multiexponential model that also includes experimentally measured correlations between single-channel OTs and CTs. The simulations of RyR clusters that utilize the multiexponential gating model produce infrequent Ca²⁺ release events with relatively few open RyRs. Ca²⁺ release events become even smaller when OT/CT correlations are included. This occurs because the correlations produce a small but consistent bias against recruiting more RyRs to open during the middle of a Ca²⁺ release event, between the initiation and termination phases (which are unaltered compared to the uncorrelated simulations). In comparison, the two-state model produces frequent, large, and long Ca²⁺ release events because it had a recruitment bias in favor of opening more RyRs. This difference stems from the two-state model’s single-RyR OT and CT distributions being qualitatively different from the experimental ones. Thus, the details of single-RyR gating can profoundly affect SR Ca²⁺ release even if open probability and mean OTs and CTs are identical. We also show that Ca²⁺ release events can terminate spontaneously without any reduction in SR [Ca²⁺], luminal regulation, Ca²⁺-dependent inactivation, or physical coupling between RyRs when Ca²⁺ flux is below a threshold value. This supports and extends the pernicious attrition/induction decay hypothesis that SR Ca²⁺ release events terminate below a threshold Ca²⁺ flux.     

Altered RyR2 regulation by the calmodulin F90L mutation associated with idiopathic ventricular fibrillation and early sudden cardiac death

Calmodulin (CaM) association with the cardiac muscle ryanodine receptor (RyR2) regulates excitation–contraction coupling. Defective CaM–RyR2 interaction is associated with heart failure. A novel CaM mutation (CaM^F90L) was recently identified in a family with idiopathic ventricular fibrillation (IVF) and early onset sudden cardiac death. We report the first biochemical characterization of CaM^F90L. F90L confers a deleterious effect on protein stability. Ca²⁺-binding studies reveal reduced Ca²⁺-binding affinity and a loss of co-operativity. Moreover, CaM^F90L displays reduced RyR2 interaction and defective modulation of [³H]ryanodine binding. Hence, dysregulation of RyR2-mediated Ca²⁺ release via aberrant CaM^F90L–RyR2 interaction is a potential mechanism that underlies familial IVF.     

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca²⁺ mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca²⁺ concentrations in pancreatic islets, resulting from Ca²⁺ mobilization from RyRs as well as Ca²⁺ influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [³H]ryanodine binding. Intracellular Ca²⁺ release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.