RyR2 Channel Gating Research Articles

Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour

ATP is an essential constitutive regulator of cardiac ryanodine receptors (RyR2), enabling small changes in cytosolic Ca2+ to trigger large changes in channel activity. With recent landmark determinations of the full structures of RyR1 (skeletal isoform) and RyR2 using cryo-EM, and identification of the RyR1 ATP binding site, we have taken the opportunity to model the binding of fragments of ATP into RyR2 in order to investigate how the structure of the ATP site dictates the functional responses of ligands attracted there. RyR2 channel gating was assessed under voltage-clamp conditions and by [3H]ryanodine binding studies. We show that even the triphosphate (PPPi) moiety alone was capable of activating RyR2 but produced two distinct effects (activation or irreversible inactivation) that we suggest correspond to two preferred binding locations within the ATP site. Combinations of complementary fragments of ATP (Pi + ADP or PPi + AMP) could not reproduce the effects of ATP, however, the presence of adenosine prevented the inactivating PPPi effects, allowing activation similar to that of ATP. RyR2 appears to accommodate diverse types of molecules, including PPPi, deep within the ATP binding site. The most effective ligands, however, have at least three phosphate groups that are guided into place by a nucleoside.

S4-S5 Linker Regulates RyR2 Channel Gating through Multiple Interactions

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel in the sarcoplasmic reticulum of cardiac muscle and plays an important role in excitation-contraction coupling. Although recent cryo-EM studies provided near-atomic structures of type 1 ryanodine receptor (RyR1), molecular mechanism of the channel opening remains largely unknown. The S4-S5 linker (S45L) is an α-helical structure connecting the S4 and S5 transmembrane segments and mediates signal transmission in a wide variety of channels. To address the role of S45L in the RyR2 channel gating, we systematically mutated residues in S45L (Thr4751-Asn4762) and neighboring transmembrane segments (S5 and S6). The mutant RyR2 was stably expressed in HEK293 cells, and the channel activity was investigated by ER luminal Ca2+ measurements and [3H]ryanodine binding. Using high sequence identity between RyR1 and RyR2 (65% in the whole molecule and 93% in the core channel domain after S4), we constructed structural model of RyR2 based on the structures of RyR1, and the phenotypes of mutants were interpreted by the model. Plausible interactions between amino acid residues in S45L and neighboring domains that may regulate channel gating will be discussed.

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca(2+) channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (Po) are associated with Ca(2+) leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca(2+)]o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca(2+)]o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca(2+) transient was normal, despite the reduced SR Ca(2+) content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca(2+) transients because they are incapable of properly increasing the amount of Ca(2+) stored in the SR because of a larger SR Ca(2+) leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca(2+) channel current (ICa-L) and SR Ca(2+) content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 Po and fractional Ca(2+) release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca(2+) content, but cannot compensate for the inability to increase SR Ca(2+) content during inotropic challenge.

Is there something fishy about the regulation of the ryanodine receptor in the fish heart?

What is the topic of this review? Excitation-contraction coupling in fish hearts is maintained over a range of temperatures that would be cardioplegic to most mammals. Here, we review what is known about the fish cardiac ryanodine receptor, and consider how it may be regulated in a different manner from the mammalian cardiac isoforms of this channel. What advances does it highlight? We highlight how a better understanding of the basic gating and conducting properties of fish cardiac ryanodine receptors could provide considerable insight into mechanisms underlying sarcoplasmic reticulum calcium release in fish hearts and the role of the sarcoplasmic reticulum in the evolution of the heart. The fish cardiac sarcoplasmic reticulum (SR) holds large quantities of Ca(2+), but calcium-induced calcium release (CICR) is weak in these myocytes, and contraction and relaxation are largely determined by transsarcolemmal Ca(2+) flux. Many fish species live in a cold and seasonally variable thermal habitat, which could provide challenges to regulation of excitation-contraction coupling. Here, we focus on the cardiac SR Ca(2+)-release channel (RyR2) in fish and ask whether it may be regulated in a different manner from the mammalian RyR2. We review data indicating that fish cardiac RyR are present at lower density, are more spatially separated within the SR membrane and are less responsive to cytosolic Ca(2+) than mammalian RyR2 channels. All of these features would contribute to the weak CICR evident from functional studies. We also consider how CICR can be enhanced in fish myocytes following β-adrenergic stimulation and application of low levels of caffeine, and how acute and chronic temperature change may affect the gating properties of fish RyR2s. It is clear that a lack of insight into the fundamental gating and conductance properties of fish RyR2 channels is hindering our understanding of the role of the SR in fish cardiac excitation-contraction coupling. We conclude by reflecting on how studies that probe the biophysical properties of fish RyR2 channel gating in response to various ligands and temperatures would be very instructive for our understanding of the role of the SR in the evolution of the heart.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin affinity

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain–domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM–RyR2 and FK506-binding protein (FKBP12.6)–RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM–RyR2 binding, but had no effect on FKBP12.6–RyR2 binding. Dantrolene restored CaM–RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10–RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM–RyR2 affinity, has no effect on FKBP12.6–RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM–RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

The mechanism of flecainide action in CPVT does not involve a direct effect on RyR2.

Flecainide, a class 1c antiarrhythmic, has emerged as an effective therapy in preventing arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) refractory to β-adrenergic receptor blockade. It has been proposed that the clinical efficacy of flecainide in CPVT is because of the combined actions of direct blockade of ryanodine receptors (RyR2) and Na(+) channel inhibition. However, there is presently no direct evidence to support the notion that flecainide blocks RyR2 Ca(2+) flux in the physiologically relevant (luminal-to-cytoplasmic) direction. The mechanism of flecainide action remains controversial. To examine, in detail, the effect of flecainide on the human RyR2 channel and to establish whether the direct blockade of physiologically relevant RyR2 ion flow by the drug contributes to its therapeutic efficacy in the clinical management of CPVT. Using single-channel analysis, we show that, even at supraphysiological concentrations, flecainide did not inhibit the physiologically relevant, luminal-to-cytosolic flux of cations through the channel. Moreover, flecainide did not alter RyR2 channel gating and had negligible effect on the mechanisms responsible for the sarcoplasmic reticulum charge-compensating counter current. Using permeabilized cardiac myocytes to eliminate any contribution of plasmalemmal Na(+) channels to the observed actions of the drug at the cellular level, flecainide did not inhibit RyR2-dependent sarcoplasmic reticulum Ca(2+) release. The principal action of flecainide in CPVT is not via a direct interaction with RyR2. Our data support a model of flecainide action in which Na(+)-dependent modulation of intracellular Ca(2+) handling attenuates RyR2 dysfunction in CPVT.

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12E31Q/D32N/W59F) where the residues Glu31, Asp32, and Trp59 were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12E31Q/D32N/W59F mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12E31Q/D32N/W59F activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu31, Asp32, and Trp59 in FKBP12 and Gln31, Asn32, and Phe59 in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.

Gating of the Pore of the Human Ryanodine Receptor Type 2 does not require Glycine Hinges

Many potassium channels use a conserved glycine residue to allow flexibility of the inner helices and facilitate gating. A KcsA-based analogy model of the RyR2 pore-forming region (Welch et al Biophys J 87: 2335-2351) indicates that this region is composed of structural elements equivalent to those found in potassium channels and includes a potentially equivalent inner-helix hinge motif (GXXXXA).We studied the functional consequences of substitutions of the conserved glycine residue at position 4864 in recombinantly expressed human RyR2 GFP-tagged proteins. Whilst wild type (WT) RyR2 and the mutants G4864A and G4864V were expressed at equivalent levels in HEK293 cells, G4864P was less tolerated with a reduced expression. This was confirmed by western blot analysis in HEK293 membrane preparations.Caffeine-induced intracellular calcium release was observed in Fluo3-loaded HEK293 cells expressing WT, G4864A and G4864P RyR2 channels. In contrast cells expressing G4864V were not sensitive to caffeine. [3H]-ryanodine binding to HEK293 membrane preparations resulted in similar specific binding for WT and G4864A at 0.192 ± 0.005 and 0.190 ± 0.002 pmol/mg, while G4864V and G4864P showed comparable low values of 0.006 ± 0.001 and 0.002 ± 0.002 pmol/mg, respectively. Characteristic single channel current fluctuations were observed following the incorporation of purified WT and G4864A RyR2 proteins into planar phospholipid bilayers and G4864A channels displayed ion handling, and calcium-dependent gating properties comparable to WT channels. No equivalent activity was observed for G4864V and G4864P.Our investigation indicates that whilst functionally sensitive to mutagenesis, a glycine residue at 4864 is not essential for RyR2 channel gating. Rather, there is a requirement for amino acids with small side chains that allow close packing of helices during transitions from closed to open states. The BHF supported this research.

FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6.

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, ‘leaky’ RyR2 channels and Ca2+-dependent arrhythmias.

Oligomerisation of the Human Cardiac Ryanodine Receptor Amino-Terminus

The cardiac ryanodine receptor (RyR2) mediates the release of calcium from the sarcoplasmic reticulum of cardiac myocytes. The functional channel is composed of four identical subunits with the C-terminal part comprising the transmembrane domain predicted to form the Ca2+-conducting pore. The large N-terminal cytoplasmic portion of RyR2 is believed to serve as a scaffold for interaction with accessory proteins, ions and other regulatory molecules. RyR2 channel gating is regulated by a complex network of inter- and intra-subunit interactions between discrete structural domains. It has been proposed that disruption of inter-domain cross-talk results in abnormal RyR2 channel function, as observed in catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure.Here, we report that the RyR2 amino-terminus, containing one of the three CPVT-associated mutation hot spots, is capable of self-association. Chemical cross-linking of an RyR2 N-terminal fragment (BT4L; residues 1-906) indicated that it can assemble into tetramers. Moreover, BT4L expressed in mammalian HEK293 cells was found to form tetramers through endogenous disulphide bonds. We undertook a site-directed mutagenesis approach to identify the cysteines involved in BT4L disulphide bond formation, whereby cysteine residues were substituted by serine. The BT4L Cys361 Ser mutant did not form DTT-sensitive tetramers, suggesting that Cys361 participates in disulphide bond formation. When BT4L was co-expressed with full-length RyR2 in HEK293 cells it translocated from the cytosol to the microsomal fraction. The functional significance of RyR2 N-terminus self-association was studied by [3H]ryanodine binding assays. We found that the BT4L fragment activates the channel at low Ca2+ concentrations most likely by disrupting inter-subunit N-terminal self-association within the tetrameric channel. Our findings suggest that the RyR2 N-terminus regulates channel function through inter-subunit interactions. This work was supported by the British Heart Foundation.

Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor.

Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been linked to certain types of cardiac arrhythmias and sudden death. Two mutation hotspots that lie in the N-terminal and central regions of RyR2 are predicted to interact with one another and to form an important channel regulator switch. To monitor the conformational dynamics involving these regions, we generated a fluorescence resonance energy transfer (FRET) pair. A yellow fluorescent protein (YFP) was inserted into RyR2 after residue Ser437 in the N-terminal region, and a cyan fluorescent protein (CFP) was inserted after residue Ser2367 in the central region, to form a dual YFP- and CFP-labeled RyR2 (RyR2(S437-YFP/S2367-CFP)). We transfected HEK293 cells with RyR2(S437-YFP/S2367-CFP) cDNAs, and then examined them by using confocal microscopy and by measuring the FRET signal in live cells. The FRET signals are influenced by modulators of RyR2, by domain peptides that mimic the effects of disease causing RyR2 mutations, and by various drugs. Importantly, FRET signals were also readily detected in cells co-transfected with single CFP (RyR2(S437-YFP)) and single YFP (RyR2(S2367-CFP)) labeled RyR2, indicating that the interaction between the N-terminal and central mutation regions is an inter-subunit interaction. Our studies demonstrate that FRET analyses of this CFP- and YFP-labeled RyR2 can be used not only for investigating the conformational dynamics associated with RyR2 channel gating, but potentially, also for identifying drugs that are capable of stabilizing the conformations of RyR2.

Localization of a Disease-associated Mutation Site in the Three-dimensional Structure of the Cardiac Muscle Ryanodine Receptor

The cardiac muscle ryanodine receptor (RyR2) functions as a calcium release channel in the heart. Up to 40 mutations in RyR2 have been linked to genetic forms of sudden cardiac death. These mutations are largely clustered in three regions of the sequence of the polypeptide: one near the N terminus, one in the central region, and the third in the C-terminal region. The central region includes 11 mutations, and an isoleucine-proline motif (positions 2427 and 2428) in the same region is predicted to contribute to the binding of FKBP12.6 protein. We have mapped the central mutation site in the three-dimensional structure of RyR2 by green fluorescent protein insertion, cryoelectron microscopy, and single-particle image processing. The central mutation site was mapped to a "bridge" of density that connects cytoplasmic domains 5 and 6, which have been suggested to undergo conformational changes during channel gating. Moreover, the location of this central mutation site is not close to that of the FKBP12.6-binding site mapped previously by cryoelectron microscopy.

Enhanced basal activity of a cardiac Ca2+ release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death.

Mutations in the human cardiac Ca2+ release channel (ryanodine receptor, RyR2) gene have recently been shown to cause effort-induced ventricular arrhythmias. However, the consequences of these disease-causing mutations in RyR2 channel function are unknown. In the present study, we characterized the properties of mutation R4496C of mouse RyR2, which is equivalent to a disease-causing human RyR2 mutation R4497C, by heterologous expression of the mutant in HEK293 cells. [3H]ryanodine binding studies revealed that the R4496C mutation resulted in an increase in RyR2 channel activity in particular at low Ca2+ concentrations. This increased basal channel activity remained sensitive to modulation by caffeine, ATP, Mg2+, and ruthenium red. In addition, the R4496C mutation enhanced the sensitivity of RyR2 to activation by Ca2+ and by caffeine. Single-channel analysis showed that single R4496C mutant channels exhibited considerable channel openings at low Ca2+ concentrations. HEK293 cells transfected with mutant R4496C displayed spontaneous Ca2+ oscillations more frequently than cells transfected with wild-type RyR2. Substitution of a negatively charged glutamate for the positively charged R4496 (R4496E) further enhanced the basal channel activity, whereas replacement of R4496 by a positively charged lysine (R4496K) had no significant effect on the basal activity. These observations indicate that the charge and polarity at residue 4496 plays an essential role in RyR2 channel gating. Enhanced basal activity of RyR2 may underlie an arrhythmogenic mechanism for effort-induced ventricular tachycardia.