Yingjie Liu

The ryanodine receptor store-sensing gate controls Ca2+ waves and Ca2+-triggered arrhythmias

Spontaneous Ca2+ release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload–induced Ca2+ release (SOICR) can result in Ca2+ waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca2+ activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni2+-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca2+-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca2+, explaining the regulation of RyR2 by luminal Ca2+, the initiation of Ca2+ waves and Ca2+-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.

Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum

Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.

S4153R Is a Gain-of-Function Mutation in the Cardiac Ca2+ Release Channel Ryanodine Receptor Associated With Catecholaminergic Polymorphic Ventricular Tachycardia and Paroxysmal Atrial Fibrillation

Mutations in ryanodine receptor 2 (RYR2) gene can cause catecholaminergic polymorphic ventricular tachycardia (CPVT). The novel RYR2-S4153R mutation has been implicated as a cause of CPVT and atrial fibrillation. The mutation has been functionally characterized via store-overload-induced Ca(2+) release (SOICR) and tritium-labelled ryanodine ([(3)H]ryanodine) binding assays. The S4153R mutation enhanced propensity for spontaneous Ca(2+) release and reduced SOICR threshold but did not alter Ca(2+) activation of [(3)H]ryanodine binding, a common feature of other CPVT gain-of-function RYR2 mutations. We conclude that the S4153R mutation is a gain-of-function RYR2 mutation associated with a clinical phenotype characterized by both CPVT and atrial fibrillation.

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

Background: Mutations in the Ca2+ sensing protein calmodulin (CaM) cause lethal cardiac arrhythmias. Results: CaM mutations impair the activation and termination of store overload-induced Ca2+ release via the cardiac ryanodine receptor (RyR2). Conclusion: CaM mutations alter RyR2-CaM interaction, thereby affecting RyR2-mediated Ca2+ release. Significance: Aberrant regulation of RyR2 store Ca2+ sensing is a potential component of calmodulin-mediated cardiac arrhythmias. 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.

Ligand-dependent Conformational Changes in the Clamp Region of the Cardiac Ryanodine Receptor

Background: Global structural changes occur in the ryanodine receptor (RyR) upon ligand activation. Results: Different ligands induce different conformational changes in the clamp region of RyR. Conclusion: Conformational changes in the clamp region of RyR are ligand-dependent. Significance: RyR possesses multiple, ligand-dependent gating mechanisms associated with distinct structural changes. Global conformational changes in the three-dimensional structure of the Ca2+ release channel/ryanodine receptor (RyR) occur upon ligand activation. A number of ligands are able to activate the RyR channel, but whether these structurally diverse ligands induce the same or different conformational changes in the channel is largely unknown. Here we constructed a fluorescence resonance energy transfer (FRET)-based probe by inserting a CFP after residue Ser-2367 and a YFP after residue Tyr-2801 in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (RyR2Ser-2367-CFP/Tyr-2801-YFP). Both of these insertion sites have previously been mapped to the “clamp” region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2. Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2Ser-2367-CFP/Tyr-2801-YFP induced by various ligands. We also monitored the extent of Ca2+ release induced by the same ligands in HEK293 cells expressing RyR2Ser-2367-CFP/Tyr-2801-YFP. We detected conformational changes in the clamp region for the ligands caffeine, aminophylline, theophylline, ATP, and ryanodine but not for Ca2+ or 4-chloro-m-cresol, although they all induced Ca2+ release. Interestingly, caffeine is able to induce further conformational changes in the clamp region of the ryanodine-modified channel, suggesting that ryanodine does not lock RyR in a fixed conformation. Our data demonstrate that conformational changes in the clamp region of RyR are ligand-dependent and suggest the existence of multiple ligand dependent RyR activation mechanisms associated with distinct conformational changes.

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain

Summary Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating.

The CPVT-associated RyR2 mutation G230C enhances store overloadinduced Ca2+ release and destabilizes the N-terminal domains

CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca2+ release during SR (sarcoplasmic reticulum) Ca2+ overload, a process also known as SOICR (store-overload-induced Ca2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca2+ activation and the Ca2+-dependent activation of [3H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1-547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

Background: The NH2-terminal region of cardiac ryanodine receptor (RyR2) contains three domains (A, B, and C) that harbor many disease-causing mutations. Results: Domains A, B, and C distinctively regulate the activation and termination of Ca2+ release. Conclusion: Individual NH2-terminal domains play distinct roles in RyR2 channel function. Significance: These data shed new insights into the actions of RyR2 NH2-terminal disease mutations. The NH2-terminal region (residues 1–543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH2-terminal region is involved in the activation and termination of Ca2+ release. The three-dimensional structure of the NH2-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca2+ release activation and termination are largely unknown. To understand the functional significance of each of these NH2-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca2+-induced Ca2+ release and store overload-induced Ca2+ release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH2-terminal region is not essential for channel gating. Ca2+ release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca2+ activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca2+ activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca2+ activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH2-terminal domains of RyR2 and the action of NH2-terminal disease mutations.

Generation and Characterization of a Mouse Model Harboring the Exon-3 Deletion in the Cardiac Ryanodine Receptor

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/− mice are not susceptible to CPVT. Ex3-del+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction.