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The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

The cardiac Ca2+ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys3635 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys3602) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys3602 (C3602A) on store overload-induced Ca2+ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C3602A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca2+ release by reducing the termination threshold. As a result, C3602A markedly increased the fractional Ca2+ release. Furthermore, the C3602A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca2+ release, but it had no effect on the stimulatory action of 4,4′-dithiodipyridine (DTDP) on Ca2+ release. In addition, Cys3602 mutations (C3602A or C3602R) did not abolish the effect of CaM on Ca2+-release termination. Therefore, RyR2–Cys3602 is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys3602 plays an important role in the activation and termination of Ca2+ release, but it is not essential for CaM regulation of RyR2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the most lethal inherited cardiac arrhythmias mostly linked to cardiac ryanodine receptor (RyR2) mutations with high disease penetrance. Interestingly, a novel RyR2 mutation G357S discovered in a large family of more than 1400 individuals has reduced penetrance. The molecular basis for the incomplete disease penetrance in this family is unknown. To gain insights into the variable disease expression in this family, we determined the impact of the G357S mutation on RyR2 function and expression. We assessed spontaneous Ca2+ release in HEK293 cells expressing RyR2 wildtype and the G357S mutant during store Ca2+ overload, also known as store overload induced Ca2+ release (SOICR). We found that the G357S mutation reduced the percentage of RyR2-expressing cells that showed SOICR. However, in cells that displayed SOICR, G357S reduced the thresholds for the activation and termination of SOICR. Furthermore, G357S decreased the thermal stability of the N-terminal domain of RyR2, and markedly reduced the protein expression of the full-length RyR2. On the other hand, the G357S mutation did not alter the Ca2+ activation of [3H]ryanodine binding or the Ca2+ induced release of Ca2+ from the intracellular stores in HEK293 cells. These data indicate that the CPVT-associated G357S mutation enhances the arrhythmogenic SOICR and reduces RyR2 protein expression, which may be attributable to the incomplete penetrance of CPVT in this family.

Mutations in the skeletal muscle ryanodine receptor (RyR1) cause malignant hyperthermia (MH) and central core disease (CCD), whereas mutations in the cardiac ryanodine receptor (RyR2) lead to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most disease-associated RyR1 and RyR2 mutations are located in the N-terminal, central, and C-terminal regions of the corresponding ryanodine receptor (RyR) isoform. An increasing body of evidence demonstrates that CPVT-associated RyR2 mutations enhance the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process known as store overload-induced Ca2+ release (SOICR). Considering the similar locations of disease-associated RyR1 and RyR2 mutations in the RyR structure, we hypothesize that like CPVT-associated RyR2 mutations, MH/CCD-associated RyR1 mutations also enhance SOICR. To test this hypothesis, we determined the impact on SOICR of 12 MH/CCD-associated RyR1 mutations E2347-del, R2163H, G2434R, R2435L, R2435H, and R2454H located in the central region, and Y4796C, T4826I, L4838V, A4940T, G4943V, and P4973L located in the C-terminal region of the channel. We found that all these RyR1 mutations reduced the threshold for SOICR. Dantrolene, an acute treatment for MH, suppressed SOICR in HEK293 cells expressing the RyR1 mutants R164C, Y523S, R2136H, R2435H, and Y4796C. Interestingly, carvedilol, a commonly used β-blocker that suppresses RyR2-mediated SOICR, also inhibits SOICR in these RyR1 mutant HEK293 cells. Therefore, these results indicate that a reduced SOICR threshold is a common defect of MH/CCD-associated RyR1 mutations, and that carvedilol, like dantrolene, can suppress RyR1-mediated SOICR. Clinical studies of the effectiveness of carvedilol as a long-term treatment for MH/CCD or other RyR1-associated disorders may be warranted.

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 methionine), 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 to CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level, compared to 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 shows that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra reveals 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 RyR2s 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.

RyR2 (cardiac ryanodine receptor)-mediated Ca2+ release in cardiomyocytes terminates when the sarcoplasmic reticulum Ca2+ content depletes to a threshold level, known as the termination threshold. Despite its importance, little is known about the mechanism that regulates the termination threshold. CaM (calmodulin), by inhibiting RyR2, has been implicated in Ca2+-release termination, but whether CaM modulates the termination threshold is unknown. To this end, we monitored the endoplasmic reticulum Ca2+ dynamics in RyR2-expressing HEK (human embryonic kidney)-293 cells transfected with WT (wild-type) CaM or mutants. We found that WT CaM or CaM mutations which abolish Ca2+ binding to the N-lobe (N-terminal lobe) of CaM increased the termination threshold (i.e. facilitated termination), but had no effect on the activation threshold at which spontaneous Ca2+ release occurs. On the other hand, CaM mutations that diminish Ca2+ binding to both the N-lobe and C-lobe (C-terminal lobe), or the C-lobe only, decreased the termination threshold (i.e. delayed termination) with a similar activation threshold. Furthermore, deletion of residues 3583–3603 or point mutations (W3587A/L3591D/F3603A, W3587A, or L3591D) in the CaM-binding domain of RyR2 that are known to abolish or retain CaM binding all reduced the termination threshold without having a significant impact on the activation threshold. Interestingly, the RyR2-F3603A mutation affected both the activation and termination threshold. Collectively, these data indicate that CaM facilitates the termination of Ca2+ release by increasing the termination threshold, and that this action of CaM depends on Ca2+ binding to the C-lobe, but not to the N-lobe, of CaM. The results of the present study also suggest that the CaM-binding domain of RyR2 is an important determinant of Ca2+-release termination and activation.

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.

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 Ca 2+ release during SR (sarcoplasmic reticulum) Ca 2+ overload, a process also known as SOICR (store-overload-induced Ca 2+ 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 Ca 2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca 2+ 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 Ca 2+ 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 Ca 2+ activation and the Ca 2+ -dependent activation of [ 3 H]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 Ca 2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.

Global conformational changes in the three-dimensional structure of the Ca(2+) 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 (RyR2(Ser-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 RyR2(Ser-2367-CFP/Tyr-2801-YFP) induced by various ligands. We also monitored the extent of Ca(2+) release induced by the same ligands in HEK293 cells expressing RyR2(Ser-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 Ca(2+) or 4-chloro-m-cresol, although they all induced Ca(2+) 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.

The ryanodine receptor (RyR) channel pore is formed by four S6 inner helices, with its intracellular gate located at the S6 helix bundle crossing region. The cytoplasmic region of the extended S6 helix is held by the U motif of the central domain and is thought to control the opening and closing of the S6 helix bundle. However, the functional significance of the S6 cytoplasmic region in channel gating is unknown. Here we assessed the role of the S6 cytoplasmic region in the function of cardiac RyR (RyR2) via structure-guided site-directed mutagenesis. We mutated each residue in the S6 cytoplasmic region of the mouse RyR2 (4876QQEQVKEDM4884) and characterized their functional impact. We found that mutations Q4876A, V4880A, K4881A, and M4884A, located mainly on one side of the S6 helix that faces the U motif, enhanced basal channel activity and the sensitivity to Ca2+ or caffeine activation, whereas mutations Q4877A, E4878A, Q4879A, and D4883A, located largely on the opposite side of S6, suppressed channel activity. Furthermore, V4880A, a cardiac arrhythmia-associated mutation, markedly enhanced the frequency of spontaneous openings and the sensitivity to cytosolic and luminal Ca2+ activation of single RyR2 channels. V4880A also increased the propensity and reduced the threshold for arrhythmogenic spontaneous Ca2+ release in HEK293 cells. Collectively, our data suggest that interactions between the cytoplasmic region of S6 and the U motif of RyR2 are important for stabilizing the closed state of the channel. Mutations in the S6/U motif domain interface likely destabilize the closed state of RyR2, resulting in enhanced basal channel activity and sensitivity to activation and increased propensity for spontaneous Ca2+ release and cardiac arrhythmias.

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778–4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy usually caused by gain-of-function mutations ryanodine receptor type-2 (RyR2). Left ventricular non-compaction (LVNC) is an often genetic cardiomyopathy. A rare LVNC-CPVT overlap syndrome may be caused by exon 3 deletion in RyR2. We sought to characterize the phenotypic spectrum and molecular basis of a novel RyR2 mutation identified in a family with both conditions. Methods Several members of an affected family underwent clinical and genetic assessments. A homology model of the RyR2 pore-region was generated to predict the location and potential impact of their RyR2 mutation. Ca 2+ -release assays were performed to characterize the functional impact of the RyR2 mutant expressed in HEK293 cells. Results A multigenerational family presented with a history of sudden death and a phenotype of atypical CPVT and LVNC. Genetic testing revealed a RYR2 mutation (I4855M) in two affected individuals. A homology model of the RyR2 pore-region showed that the I4855M mutant reside is located in the highly conserved ‘inner vestibule', a water-filled cavity. I4855M may interfere with Ca 2+ permeation and affect interactions between RyR2 pore subunits, and is thus predicted in silico to be damaging. Expression and functional studies in HEK293 cells revealed that I4855M inhibited caffeine-induced Ca 2+ release and exerted a dominant-negative impact on wild type RyR2. Conclusions This study identifies a potentially lethal overlapping syndrome of LVNC and atypical CPVT related to a novel RYR2 variant. Structural and functional studies suggest that this is a loss-of-function mutation, which exerts a dominant-negative effect on wild type RyR2.

Beat-to-beat alternations in the amplitude of the cytosolic Ca2+ transient (Ca2+ alternans) are thought to be the primary cause of cardiac alternans that can lead to cardiac arrhythmias and sudden death. Despite its important role in arrhythmogenesis, the mechanism underlying Ca2+ alternans remains poorly understood. Here, we investigated the role of cardiac ryanodine receptor (RyR2), the major Ca2+ release channel responsible for cytosolic Ca2+ transients, in cardiac alternans. Using a unique mouse model harboring a suppression-of-function (SOF) RyR2 mutation (E4872Q), we assessed the effect of genetically suppressing RyR2 function on Ca2+ and action potential duration (APD) alternans in intact hearts, and electrocardiogram (ECG) alternans in vivo. We found that RyR2-SOF hearts displayed prolonged sarcoplasmic reticulum Ca2+ release refractoriness and enhanced propensity for Ca2+ alternans. RyR2-SOF hearts/mice also exhibited increased propensity for APD and ECG alternans. Caffeine, which enhances RyR2 activity and the propensity for catecholaminergic polymorphic ventricular tachycardia (CPVT), suppressed Ca2+ alternans in RyR2-SOF hearts, whereas carvedilol, a β-blocker that suppresses RyR2 activity and CPVT, promoted Ca2+ alternans in these hearts. Thus, RyR2 function is an important determinant of Ca2+, APD, and ECG alternans. Our data also indicate that the activity of RyR2 influences the propensity for cardiac alternans and CPVT in an opposite manner. Therefore, overly suppressing or enhancing RyR2 function is pro-arrhythmic.

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 (RyR2S437-YFP/S2367-CFP). We transfected HEK293 cells with RyR2S437-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 (RyR2S437-YFP) and single YFP (RyR2S2367-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.

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K+ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of ±50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.

Naturally occurring mutations in the skeletal muscle Ca2+ release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca2+ release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca2+ release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca2+ release during store Ca2+ overload, a process we refer to as store overload-induced Ca2+ release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca2+ activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca2+ and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca2+ activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca2+ regulation of RyR1 represents a primary causal mechanism of MH.

A region between residues 414 and 466 in the cardiac ryanodine receptor (RyR2) harbors more than half of the known NH(2)-terminal mutations associated with cardiac arrhythmias and sudden death. To gain insight into the structural basis of this NH(2)-terminal mutation hot spot, we have determined its location in the three-dimensional structure of RyR2. Green fluorescent protein (GFP), used as a structural marker, was inserted into the middle of this mutation hot spot after Ser-437 in the RyR2 sequence. The resultant GFP-RyR2 fusion protein, RyR2(S437-GFP,) was expressed in HEK293 cells and characterized using Ca(2+) release, [(3)H]ryanodine binding, and single cell Ca(2+) imaging studies. These functional analyses revealed that RyR2(S437-GFP) forms a caffeine- and ryanodine-sensitive Ca(2+) release channel that possesses Ca(2+) and caffeine dependence of activation indistinguishable from that of wild type (wt) RyR2. HEK293 cells expressing RyR2(S437-GFP) displayed a propensity for store overload-induced Ca(2+) release similar to that in cells expressing RyR2-wt. The three-dimensional structure of the purified RyR2(S437-GFP) was reconstructed using cryo-electron microscopy and single particle image processing. Subtraction of the three-dimensional reconstructions of RyR2-wt and RyR2(S437-GFP) revealed the location of the inserted GFP, and hence the NH(2)-terminal mutation hot spot, in a region between domains 5 and 9 in the clamp-shaped structure. This location is close to a previously mapped central disease-causing mutation site located in a region between domains 5 and 6. These results, together with findings from previous studies, suggest that the proposed interactions between the NH(2)-terminal and central regions of RyR2 are likely to take place between domains 5 and 6 and that the clamp-shaped structure, which shows substantial conformational differences between the closed and open states, is highly susceptible to disease-causing mutations.

Spontaneous Ca(2+) release occurs in cardiac cells during sarcoplasmic reticulum Ca(2+) overload, a process we refer to as store-overload-induced Ca(2+) release (SOICR). Unlike cardiac cells, skeletal muscle cells exhibit little SOICR activity. The molecular basis of this difference is not well defined. In this study, we investigated the SOICR properties of HEK293 cells expressing RyR1 or RyR2. We found that HEK293 cells expressing RyR2 exhibited robust SOICR activity, whereas no SOICR activity was observed in HEK293 cells expressing RyR1. However, in the presence of low concentrations of caffeine, SOICR could be triggered in these RyR1-expressing cells. At the single-channel level, we showed that RyR2 is much more sensitive to luminal Ca(2+) than RyR1. To identify the molecular determinants responsible for these differences, we constructed two chimeras between RyR1 and RyR2, N-RyR1(1-4006)/C-RyR2(3962-4968) and N-RyR2(1-3961)/C-RyR1(4007-5037). We found that replacing the C-terminal region of RyR1 with the corresponding region of RyR2 (N-RyR1/C-RyR2) dramatically enhanced the propensity for SOICR and the response to luminal Ca(2+), whereas replacing the C-terminal region of RyR2 with the corresponding region of RyR1 (N-RyR2/C-RyR1) reduced the propensity for SOICR and the luminal Ca(2+) response. These observations indicate that the C-terminal region of RyR is a critical determinant of both SOICR and the response to luminal Ca(2+). These chimeric studies also reveal that the N-terminal region of RyR plays an important role in regulating SOICR and luminal Ca(2+) response. Taken together, our results demonstrate that RyR1 differs markedly from RyR2 with respect to their responses to Ca(2+) overload and luminal Ca(2+), and suggest that the lack of spontaneous Ca(2+) release in skeletal muscle cells is, in part, attributable to the unique intrinsic properties of RyR1.

The intracellular Ca(2+) sensor calmodulin (CaM) regulates the cardiac Ca(2+) 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 Ca(2+) release. All mutations increased Ca(2+) release and rendered RyR2 more susceptible to store overload-induced Ca(2+) release (SOICR) by lowering the threshold of store Ca(2+) content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca(2+) 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 Ca(2+) 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 Ca(2+) binding to the N-domain but markedly decreased the affinity of the C-domain for Ca(2+). 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 Ca(2+) 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 Ca(2+)-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca(2+) concentration, is proposed.

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca(2+) is a central step in the process of Ca(2+)-induced Ca(2+) release, but the molecular basis of RyR2 activation by cytosolic Ca(2+) is poorly defined. It has been proposed recently that the putative Ca(2+) binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca(2+) sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca(2+) activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca(2+)-dependent activation of [(3)H]ryanodine binding or the cytosolic Ca(2+) activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca(2+) activation of RyR2 and spontaneous Ca(2+) release in HEK293 cells during store Ca(2+) overload or store overload-induced Ca(2+) release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca(2+), it plays an important role in luminal Ca(2+) activation and SOICR.

The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-induced Ca(2+) release and store overload-induced Ca(2+) 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. Ca(2+) release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca(2+) activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca(2+) activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca(2+) 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.

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.

The specific, high-affinity interaction of the plant toxin ryanodine with its molecular target the ryanodine receptor channel (RyR) has been instrumental in RyR research. Alanine scanning of putative pore regions of mouse RyR2 has highlighted the amino acid Gln4863, predicted to lie within trans-membrane helix TM10, as an important determinant of ryanodine binding. We have investigated the effects of several ryanodine derivatives, guanidinopropionylryanodine, 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine, 8beta-amino-9alpha-hydroxyryanodine, and 21-amino-9alpha-hydroxyryanodine, with the mouse Q4863A RyR2 mutant at the single-channel level. Our results demonstrate that the rate of dissociation of all ryanoids investigated is increased by the mutation. The modification of channel function after ryanoid binding is qualitatively similar for wild-type and mutant, but in several cases, single-channel conductances were increased with Q4863A. These novel findings have been interpreted within the framework of existing comparative molecular field analysis studies on ryanoids. We suggest that replacement of a glutamine by an alanine residue at position 4863 causes RyR2 to simultaneously alter interactions with both ends of the ryanoid molecule.

The voltage-gated sodium channel is the primary target site of pyrethroids, which constitute a major class of insecticides used worldwide. Pyrethroids prolong the opening of sodium channels by inhibiting deactivation and inactivation. Despite numerous attempts to characterize pyrethroid binding to sodium channels in the past several decades, the molecular determinants of the pyrethroid binding site on the sodium channel remain elusive. Here, we show that an F-to-I substitution at 1519 (F1519I) in segment 6 of domain III (IIIS6) abolished the sensitivity of the cockroach sodium channel expressed in Xenopus laevis oocytes to all eight structurally diverse pyrethroids examined, including permethrin and deltamethrin. In contrast, substitution by tyrosine or tryptophan reduced the channel sensitivity to deltamethrin only by 3- to 10-fold, indicating that an aromatic residue at this position is critical for the interaction of pyrethroids with sodium channels. The F1519I mutation, however, did not alter the action of two other classes of sodium channel toxins, batrachotoxin (a site 2 toxin) and Lqhalpha-IT (a site 3 toxin). Schild analysis using competitive interaction of pyrethroid-stereospecific isomers demonstrated that the F1519W mutation and a previously known pyrethroid-resistance mutation, L993F in IIS6, reduced the binding affinity of 1S-cis-permethrin, an inactive isomer that shares the same binding site with the active isomer 1R-cis-permethrin. Our results provide the first direct proof that Leu993 and Phe1519 are part of the pyrethroid receptor site on an insect sodium channel.

Ryanodine receptors (RyRs) are a family of calcium release channels found on intracellular calcium-handing organelles. Molecular cloning studies have identified three different RyR isoforms, which are 66–70% identical in amino acid sequence. In mammals, the three isoforms are encoded by three separate genes located on different chromosomes. The major variations among the isoforms occur in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and DR3). In the present study, a modified RyR2 (cardiac isoform) cDNA was constructed, into which was inserted a green fluorescent protein (GFP)-encoding cDNA within DR2, specifically after amino acid residue Thr1366 (RyR2 T1366-GFP ). HEK293 cells expressing RyR2 T1366-GFP cDNAs showed caffeine-sensitive and ryanodine-sensitive calcium release, demonstrating that RyR2 T1366-GFP forms functional calcium release channels. Cells expressing RyR2 T1366-GFP were identified readily by the characteristic fluorescence of GFP, indicating that the overall structure of the inserted GFP was retained. Cryo-electron microscopy (cryo-EM) of purified RyR2 T1366-GFP showed structurally intact receptors, and a three-dimensional reconstruction was obtained by single-particle image processing. The location of the inserted GFP was obtained by comparing this three-dimensional reconstruction to one obtained for wild-type RyR2. The inserted GFP and, consequently Thr1366 within DR2, was mapped on the three-dimensional structure of RyR2 to domain 6, one of the characteristic cytoplasmic domains that form part of the multi-domain “clamp” regions of RyR2. The three-dimensional location of DR2 suggests that it plays roles in the RyR conformational changes that occur during channel gating, and possibly in RyR's interaction with the dihydropyridine receptor in excitation–contraction coupling. This study further demonstrates the feasibility and reliability of the GFP insertion/cryo-EM approach for correlating RyR's amino acid sequence with its three-dimensional structure, thereby enhancing our understanding of the structural basis of RyR function.

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.

The cardiac ryanodine receptor (RyR2) is most abundantly expressed in the heart and brain. RyR2 is located in the sarco(endo)plasmic reticulum (ER/SR) membrane and functions as an intracellular calcium release channel, important for a number of fundamental processes, such as muscle contraction, secretion, neurotransmitter release, learning and memory. Proper function of RyR2 critically depends on its subcellular distribution. In ventricular myocyte of the heart, RyR2 is organized in highly-ordered arrays of clusters, which is thought to be important for synchronous, stable calcium release during excitation-contraction coupling. However, little is known about RyR2 distribution in other cardiac cells. In the brain, RyR2 is thought to play a central role in learning and memory, but the cellular/subcellular distribution of RyR2 in the brain remains largely undefined. Recently, we have generated a knock-in mouse model that expresses a green fluorescence protein (GFP)-tagged RyR2. This GFP-RyR2 mouse model allows us to directly and specifically monitor the cellular/subcellular distribution and expression of RyR2 in various cells and tissues. To improve the detection of GFP, we have also developed novel GFP-specific probes based on anti-GFP single domain antibodies (i.e. nanobodies). High-resolution confocal imaging of intact GFP-RyR2 heart sections and brain slices revealed highly ordered arrays of GFP-RyR2 in various regions of the heart, but disperse distribution of GFP-RyR2 in hippocampus and other regions of the brain. Using Alexa-Fluor 647 (AF647)-conjugated GFP-specific probes, we will perform super-resolution imaging to further define the subcellular distribution of GFP-RyR2 in the heart and brain (Supported by NSERC, CFI, CIHR, and LCIA).

Voltage-gated sodium channels are essential for the generation and propagation of action potentials in most excitable cells. They are the target sites of several classes of insecticides and acaricides. Isolation of full-length sodium channel cDNA is a critical and often difficult step toward an understanding of insecticide and acaricide resistance. We previously cloned and sequenced two overlapping cDNA clones covering segment 3 of domain II (IIS3) to segment 6 of domain IV (IVS6) of an arachnid sodium channel gene (named VmNa) from the varroa mite (Varroa destructor) (J. Apicultureal Res. 40 (2002) 5.). In this study, we isolated three more overlapping cDNA clones and revealed the entire coding region of VmNa (Genbank accession number: AY259834), thus providing the first complete cDNA sequence of an arachnid sodium channel gene. The composite VmNa cDNA contains 6645 nucleotides with an open reading frame encoding 2215 amino acids. The deduced amino acid sequence of VmNa shares a 51% overall identity with Drosophila Para and a 41% identity with the mammalian sodium channel alpha-subunit Na(v)1.2. All hallmarks of sodium channel proteins are conserved in the VmNa protein. Three optional exons and one retained intron were identified in VmNa. The precise position and size of only one exon is conserved in three insect sodium channel genes and mammalian Na(v)1.6 genes in human, mouse and fish, whereas the other three are novel. Interestingly, one of the novel exons is located in the C-terminus, where no alternative exons have been identified in any other sodium channel gene.

Three ryanodine receptor (RyR) isoforms, RyR1, RyR2, and RyR3, are expressed in mammalian tissues. It is unclear whether RyR isoforms are capable of forming heteromeric channels. To investigate their ability to form heteromeric channels, we co-expressed different RyR isoforms in HEK293 cells and examined their interactions biochemically and functionally. Immunoprecipitation studies revealed that RyR2 is able to interact physically with RyR3 and RyR1 in HEK293 cells and that RyR1 does not interact with RyR3. Co-expression of a ryanodine binding deficient mutant of RyR2, RyR2 (I4827T), with RyR3 (wt) restored [(3)H]ryanodine binding to the mutant. Interactions between RyR isoforms were further assessed by complementation analysis using mutants RyR2 (I4827T), RyR2 (E3987A), RyR3 (I4732T), RyR3 (E3885A), and RyR1 (E4032A), all of which are deficient in caffeine response. Caffeine-induced Ca(2+) release was restored in HEK293 cells co-transfected with mutants RyR2 (I4827T) and RyR3 (E3885A), RyR2 (E3987A) and RyR3 (I4732T), or RyR2 (I4827T) and RyR1 (E4032A), but not with RyR1 (E4032A) and RyR3 (I4732T), indicating that mutants of RyR2 and RyR3, or RyR2 and RyR1, but not RyR1 and RyR3, are able to complement each other. Co-expression of RyR3 (wt) and a pore mutant of RyR2, RyR2 (G4824A), produced regulatable single channels with intermediate unitary conductances. These observations demonstrate that RyR2 is capable of forming functional heteromeric channels with RyR3 and RyR1, whereas RyR1 is incapable of forming heteromeric channels with RyR3.

SUMMARYVarroa (Varroa destructor) has recently become resistant to Apistan, a pyrethroid pesticide with tau-fluvalinate as its active ingredient. In many insect pests, resistance to pyrethroid insecticides is due to reduced target-site (sodium channel) sensitivity to pyrethroids in the nervous system, a phenomenon called knockdown resistance (kdr). A number of studies showed that kdr and kdr-type resistance is a result of point mutations in the para family of sodium channel genes. To investigate the molecular mechanism of resistance to fluvalinate in varroa, we have cloned and sequenced a large cDNA fragment corresponding to segment 3 of domain II (IIS3) to segment 6 of domain IV (IVS6) of a para-homologous sodium channel gene (VmNa) from susceptible and resistant mite populations. The deduced amino acid sequence from this cDNA shares 71%, 60%, and 50% identity with the corresponding region of the para-homologous protein of the Southern cattle tick, Boophilus microplus, Drosophila melanogaster Para, and r...

Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) 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, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.

Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²⁺ ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²⁺ load and Ca²⁺ leak. Conversely, PLN-KO accelerates Ca²⁺ sequestration and aborts arrhythmogenic spontaneous Ca²⁺ waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²⁺-triggered arrhythmias.We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT.We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²⁺ imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²⁺ overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²⁺ sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²⁺ overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²⁺ ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li⁺ failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs.Our results demonstrate that despite severe sarcoplasmic reticulum Ca²⁺ leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²⁺ sequestration represents an effective approach for suppressing Ca²⁺-triggered arrhythmias.

Summary The N-terminal region of both skeletal and cardiac ryanodine receptor is a disease mutation hotspot. Recently, a crystal structure of the RyR1 fragment (residues 1–559) was solved. This N-terminal structure contains three separate domains, A, B, and C, and was docked into a central vestibule in a full-length RyR1 cryo-EM map. Here, we reconstructed three-dimensional cryo-EM structures of two GFP-tagged RyR2s with GFP inserted after residue Glu-310 and Ser-437, respectively. The structures of RyR2 E310-GFP and RyR2 S437-GFP displayed an extra mass on domain B and C, directly validating the predicted docking model. Next, we revealed domain movements in molecular dynamics flexible fitting models in both the closed and open state cryo-EM maps. To further probe the conformational changes, we generated FRET pairs by inserting CFP or YFP in two selected domains, FRET studies of three dual-insertion pairs and three co-expressed single-insertion pairs showed the dynamic structural changes within the N-terminal domains.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a life-threatening arrhythmia. Many congenital mutations in both the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) are known to be responsible for this disorder. It is now well established that CPVT is caused by delayed afterdepolarizations (DADs)-induced triggered activities, and that DADs are caused by spontaneous sarcoplasmic reticulum (SR) Ca release during Ca overload, a process also known as store overload induced Ca release (SOICR). A large body of evidence indicates that CPVT-causing RyR2 and CASQ2 mutations enhance the propensity for SOICR and DADs by increasing the response of RyR2 to SR luminal Ca. Recently, Marks and colleagues reported that a CPVT RyR2 mutation G230C increases the cytosolic Ca sensitivity (only after PKA phosphorylation) of single RyR2 channels in lipid bilayers, but has no effect on the luminal Ca sensitivity of the channel. These observations have led to the conclusion that SOICR is not involved in the disease mechanism of the RyR2-G230C mutation. However, the cellular impact of this mutation on SOICR has yet to be determined. To this end, we generated stable, inducible HEK293 cell lines expressing RyR2-WT and the RyR2-G230C mutant. We induced SOICR in these cells by elevating extracellular Ca, and found that the RyR2-G230C mutation markedly enhances the propensity for SOICR. Further, we employed single cell luminal Ca imaging to monitor the luminal Ca dynamics in RyR2-WT- and G230C-expressing cells during store Ca overload. We found that the G230C mutation significantly reduces the luminal Ca level at which spontaneous Ca release occurs (i.e. the SOICR threshold). Therefore, these results and those of previous studies demonstrate that reduced SOICR threshold is a common defect of CPVT-associated RyR2 mutations.

Rationale : Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca 2+ ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca 2+ load and Ca 2+ leak. Conversely, PLN-KO accelerates Ca 2+ sequestration and aborts arrhythmogenic spontaneous Ca 2+ waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca 2+ -triggered arrhythmias. Objective : We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. Methods and Results : We generated a PLN-deficient, RyR2-mutant mouse model (PLN −/− /RyR2-R4496C +/− ) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT–associated RyR2-R4496C mutant mice. Ca 2+ imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C +/− ventricular myocytes during sarcoplasmic reticulum Ca 2+ overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca 2+ sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca 2+ overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca 2+ ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li + failed to convert mini-waves to cell-wide SCWs in PLN −/− /RyR2-R4496C +/− ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. Conclusions : Our results demonstrate that despite severe sarcoplasmic reticulum Ca 2+ leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca 2+ sequestration represents an effective approach for suppressing Ca 2+ -triggered arrhythmias.

The type 2 ryanodine receptor (RyR2) controls the release of calcium ions from the sarcoplasmic reticulum in cardiac cells—the initiating step in cardiac muscle contraction. Mutations in RyR2 are associated with cardiac diseases. Peng et al. used single-particle electron cryomicroscopy to determine the structure of RyR2 from porcine heart at 4.4-A resolution with the calcium channel closed and at 4.2-A resolution with the calcium channel open. The structures reveal how interdomain motions result in a conformational change in the cytoplasmic region of RyR2 that is transduced by a central domain to cause motions that open or close the channel. Science , this issue p. [301][1] [1]: http://www.sciencemag.org/content/354/6310/aah5324.full

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.

Rationale: Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been associated with both cardiac arrhythmias and cardiomyopathies. It is clear that delayed afterdepolarization resulting from abnormal activation of sarcoplasmic reticulum Ca 2+ release is the primary cause of RyR2-associated cardiac arrhythmias. However, the mechanism underlying RyR2-associated cardiomyopathies is completely unknown. Objective: In the present study, we investigate the role of the NH 2 -terminal region of RyR2 in and the impact of a number of cardiomyopathy-associated RyR2 mutations on the termination of Ca 2+ release. Methods and Results: The 35-residue exon-3 region of RyR2 is associated with dilated cardiomyopathy. Single-cell luminal Ca 2+ imaging revealed that the deletion of the first 305 NH 2 -terminal residues encompassing exon-3 or the deletion of exon-3 itself markedly reduced the luminal Ca 2+ threshold at which Ca 2+ release terminates and increased the fractional Ca 2+ release. Single-cell cytosolic Ca 2+ imaging also showed that both RyR2 deletions enhanced the amplitude of store overload-induced Ca 2+ transients in HEK293 cells or HL-1 cardiac cells. Furthermore, the RyR2 NH 2 -terminal mutations, A77V, R176Q/T2504M, R420W, and L433P, which are associated with arrhythmogenic right ventricular displasia type 2, also reduced the threshold for Ca 2+ release termination and increased fractional release. The RyR2 A1107M mutation associated with hypertrophic cardiomyopathy had the opposite action (ie, increased the threshold for Ca 2+ release termination and reduced fractional release). Conclusions: These results provide the first evidence that the NH 2 -terminal region of RyR2 is an important determinant of Ca 2+ release termination, and that abnormal fractional Ca 2+ release attributable to aberrant termination of Ca 2+ release is a common defect in RyR2-associated cardiomyopathies.

Carvedilol is one of the most effective beta blockers for preventing ventricular tachyarrhythmias in heart failure, but the mechanisms underlying its favorable antiarrhythmic benefits remain unclear. Spontaneous Ca(2+) waves, also called store overload-induced Ca(2+) release (SOICR), evoke ventricular tachyarrhythmias in individuals with heart failure. Here we show that carvedilol is the only beta blocker tested that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol, combined with its beta-blocking activity, probably contributes to its favorable antiarrhythmic effect. To enable optimal titration of carvedilol's actions as a beta blocker and as a suppressor of SOICR separately, we developed a new SOICR-inhibiting, minimally beta-blocking carvedilol analog, VK-II-86. VK-II-86 prevented stress-induced ventricular tachyarrhythmias in RyR2-mutant mice and did so more effectively when combined with either of the selective beta blockers metoprolol or bisoprolol. Combining SOICR inhibition with optimal beta blockade has the potential to provide antiarrhythmic therapy that can be tailored to individual patients.

Cardiac Ryanodine Receptor (RyR2) is the major intracellular calcium release channel located in the sarcoplasmic reticulum. A large number of mutations in RyR2 have been linked to inherited cardiac arrhythmias (CPVT, catecholaminergic polymorphic ventricular tachycardia and IVF, idiopathic ventricular fibrillation) and sudden cardiac death. We previously demonstrated that in contrast to CPVT RyR2 mutations, the IVF RyR2 mutation A4860G suppresses RyR2 function. However, A4860G represents the only IVF RyR2 mutation that has been functionally characterized to date. To determine how common suppression-of-function RyR2 mutations are, we systematically characterized 27 RyR2 mutations that are associated with IVF or sudden cardiac death. Each of these mutations was generated by site-directed mutagenesis and expressed in HEK293 cells. Ca release assays revealed that 7 of these mutations suppress or abolish caffeine-induced Ca release. Single cell Ca imaging studies also indicated that these suppression- or loss-of-function RyR2 mutations reduce or abolish the propensity for store-overload induced Ca release (SOICR) in HEK293 cells. In addition, single channel analysis showed that these mutations significantly reduce the response of the channel to activation by cytosolic and luminal Ca. These observations demonstrate, for the first time, that suppressed or loss of RyR2 function may be a common mechanism underlying idiopathic ventricular fibrillation, which is opposite to the gain-of-function RyR2 mutations associated with CPVT. Thus, understanding the exact molecular defects of disease-causing RyR2 mutations will help to develop novel approaches to the diagnosis and personalized treatment of these lethal cardiac arrhythmias (Supported by CFI, CIHR, AIHS, and LCIA).

A longstanding question in the field of excitation-contraction coupling in cardiac muscle is how Ca2+ release from the sarcoplasmic reticulum (SR) is terminated. Recent studies have suggested that SR Ca2+ release terminates as a result of luminal Ca2+ dependent inactivation of the Ca2+ release channel/ryanodine receptor (RyR2). However, the molecular basis of luminal Ca2+ dependent inactivation of RyR2 is unknown. We have previously shown that the pore region of RyR2 is critical for the initiation of spontaneous Ca2+ release or store overload induced Ca2+ release (SOICR). In the present study, we determined whether the pore region of RyR2 is also important for Ca2+ release termination. To this end, we mutated each residue within the inner helix and the helix bundle crossing, and generated stable, inducible HEK293 cell lines expressing theses mutants. Using the fluorescence resonance energy transfer (FRET)-based luminal Ca2+ sensing protein, D1ER, we monitored the luminal Ca2+ dynamics in HEK293 cells expressing RyR2 wt and mutants during Ca2+ overload. Interestingly, we found that the G4871R mutation significantly lowered the critical luminal Ca2+ level at which Ca2+ release is terminated (the termination threshold), but it had no effect on the critical luminal Ca2+ level at which spontaneous Ca2+ release or SOICR occurs (the SOICR threshold), as compared with wt. In contrast, the I4862A mutation markedly lowered the SOICR threshold with little impact on the termination threshold. On the other hand, the Q4876A mutation lowered both the SOICR and termination thresholds, whereas the E4872A mutation raised both thresholds. Taken together, our data demonstrate that the pore region of RyR2 is an important determinant of both activation and termination of Ca2+ release, and suggest that the pathways for Ca2+ release activation and termination are distinct but overlap.

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Abnormal calcium release through a dysfunctional RyR2 has been implicated in certain types of sudden cardiac death and heart failure. A 12.6kDa FK506 binding protein (FKBP12.6) tightly associates with RyR2, and stabilizes the close state of RyR2 calcium channel. One proposed mechanism that underlies RyR2 channel dysfunction is the destabilization of the RyR2-FKBP12.6 interaction. In the present study, we mapped the location of green fluorescent protein inserted after residue Tyr-846, near the amino-terminal diseases-causing mutation hotspot, in the three-dimensional (3D) structure of RyR2 by cryo-electron microscopy (cryo-EM). The location of the inserted GFP was found to be close to the previously mapped FKBP12.6 binding site. Based on the structural information that we have learned from 3D cryo-EM, we designed a fluorescence resonance energy transfer (FRET) pair by inserting a yellow fluorescent protein in RyR2 after residue Tyr-846, and attaching a cyan fluorescent protein to FKBP12.6. By monitoring the FRET signals between the donor and acceptor, we are investigating the interaction dynamics between RyR2 and FKBP12.6.Supported by AHA to ZL, NIH to TW, CIHR and HSFA to SRWC.

Different forms of ventricular arrhythmias have been linked to mutations in the cardiac ryanodine receptor (RyR)2, but the molecular basis for this phenotypic heterogeneity is unknown. We have recently demonstrated that an enhanced sensitivity to luminal Ca 2+ and an increased propensity for spontaneous Ca 2+ release or store-overload-induced Ca 2+ release (SOICR) are common defects of RyR2 mutations associated with catecholaminergic polymorphic or bidirectional ventricular tachycardia. Here, we investigated the properties of a unique RyR2 mutation associated with catecholaminergic idiopathic ventricular fibrillation, A4860G. Single-channel analyses revealed that, unlike all other disease-linked RyR2 mutations characterized previously, the A4860G mutation diminished the response of RyR2 to activation by luminal Ca 2+ , but had little effect on the sensitivity of the channel to activation by cytosolic Ca 2+ . This specific impact of the A4860G mutation indicates that the luminal Ca 2+ activation of RyR2 is distinct from its cytosolic Ca 2+ activation. Stable, inducible HEK293 cells expressing the A4860G mutant showed caffeine-induced Ca 2+ release but exhibited no SOICR. Importantly, HL-1 cardiac cells transfected with the A4860G mutant displayed attenuated SOICR activity compared with cells transfected with RyR2 WT. These observations provide the first evidence that a loss of luminal Ca 2+ activation and SOICR activity can cause ventricular fibrillation and sudden death. These findings also indicate that although suppressing enhanced SOICR is a promising antiarrhythmic strategy, its oversuppression can also lead to arrhythmias.

The 12.6-kDa FK506-binding protein (FKBP12.6) is considered to be a key regulator of the cardiac ryanodine receptor (RyR2), but its precise role in RyR2 function is complex and controversial. In the present study we investigated the impact of FKBP12.6 removal on the properties of the RyR2 channel and the propensity for spontaneous Ca(2+) release and the occurrence of ventricular arrhythmias. Single channel recordings in lipid bilayers showed that FK506 treatment of recombinant RyR2 co-expressed with or without FKBP12.6 or native canine RyR2 did not induce long-lived subconductance states. [(3)H]Ryanodine binding studies revealed that coexpression with or without FKBP12.6 or treatment with or without FK506 did not alter the sensitivity of RyR2 to activation by Ca(2+) or caffeine. Furthermore, single cell Ca(2+) imaging analyses demonstrated that HEK293 cells co-expressing RyR2 and FKBP12.6 or expressing RyR2 alone displayed the same propensity for spontaneous Ca(2+) release or store overload-induced Ca(2+) release (SOICR). FK506 increased the amplitude and decreased the frequency of SOICR in HEK293 cells expressing RyR2 with or without FKBP12.6, indicating that the action of FK506 on SOICR is independent of FKBP12.6. As with recombinant RyR2, the conductance and ligand-gating properties of single RyR2 channels from FKBP12.6-null mice were indistinguishable from those of single wild type channels. Moreover, FKBP12.6-null mice did not exhibit enhanced susceptibility to stress-induced ventricular arrhythmias, in contrast to previous reports. Collectively, our results demonstrate that the loss of FKBP12.6 has no significant effect on the conduction and activation of RyR2 or the propensity for spontaneous Ca(2+) release and stress-induced ventricular arrhythmias.

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292–296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.