Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaM^N98I, CaM^D132E, CaM^D134H and CaM^Q136P) and we used a CaM-RyR2 co-immunoprecipitation and a [³H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [³H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca²⁺, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.     

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

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

Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca²⁺ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca²⁺ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca²⁺ activation and abolished luminal Ca²⁺ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [³H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.     

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 μmol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca²⁺ sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca²⁺ sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca²⁺ leak, and then lethal arrhythmia.     

Inhibition of CaMKII Does Not Attenuate Cardiac Hypertrophy in Mice with Dysfunctional Ryanodine Receptor

In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca²⁺/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2^ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2^ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2^ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2^ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca²⁺ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2^ADA/ADA mice.     

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

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

     

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

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

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

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

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.     

Both N- and C-lobes of calmodulin are required for Ca-dependent regulations of CaV1.2 Ca channels

We investigated the concentration- and Ca²⁺-dependent effects of CaM mutants, CaM₁₂ and CaM₃₄, in which Ca²⁺-binding to its N- and C-lobes was eliminated, respectively, on the Ca_V1.2 Ca²⁺ channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM₁₂ and CaM₃₄ (0.7-10 μM) applied with 3 mM ATP produced channel activity after “rundown”. Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca²⁺], suggesting that both functional lobes of CaM were necessary for the Ca²⁺-dependent shift. However, channel activity induced by the CaM mutants showed Ca²⁺-dependent decrease, implying a Ca²⁺ sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca²⁺-dependent regulations of Ca_V1.2 Ca²⁺ channels.     

Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel (ryanodine receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.     

The CaMKII inhibitor KN93-calmodulin interaction and implications for calmodulin tuning of NaV1.5 and RyR2 function

Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca²⁺-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM–protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na⁺ channel (Na_V1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-Na_V1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca²⁺ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.     

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

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

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca²⁺, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca²⁺, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [³H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca²⁺-binding site to sensitize RyR1 to Ca²⁺. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca²⁺. However, enhanced RyR1 activation occurred in the presence of Ca²⁺, AMPPCP, and Caf. In the absence of Ca²⁺, Na⁺ inhibited [³H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca²⁺-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca²⁺, Na⁺ is predicted to inhibit RyR1 by interacting with the Ca²⁺-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca²⁺-binding site, and conversely, Ca²⁺ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca²⁺, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca²⁺-, ATP-, and Caf-binding sites.     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.     

Ionic displacement of Ca2+ by Pb2+ in calmodulin is affected by arrhythmia-associated mutations

Lead is a highly toxic metal that severely perturbs physiological processes even at sub-micromolar levels, often by disrupting the Ca²⁺ signaling pathways. Recently, Pb²⁺-associated cardiac toxicity has emerged, with potential involvement of both the ubiquitous Ca²⁺ sensor protein calmodulin (CaM) and ryanodine receptors. In this work, we explored the hypothesis that Pb²⁺ contributes to the pathological phenotype of CaM variants associated with congenital arrhythmias. We performed a thorough spectroscopic and computational characterization of CaM conformational switches in the co-presence of Pb²⁺ and four missense mutations associated with congenital arrhythmias, namely N53I, N97S, E104A and F141L, and analyzed their effects on the recognition of a target peptide of RyR2. When bound to any of the CaM variants, Pb²⁺ is difficult to displace even under equimolar Ca²⁺ concentrations, thus locking all CaM variants in a specific conformation, which exhibits characteristics of coiled-coil assemblies. All arrhythmia-associated variants appear to be more susceptible to Pb²⁺ than wild type (WT) CaM, as the conformational transition towards the coiled-coil conformation occurs at lower Pb²⁺, regardless of the presence of Ca²⁺, with altered cooperativity. The presence of arrhythmia-associated mutations specifically alters the cation coordination of CaM variants, in some cases involving allosteric communication between the EF-hands in the two domains. Finally, while WT CaM increases the affinity for the RyR2 target in the presence of Pb²⁺, no specific pattern could be detected for all other variants, ruling out a synergistic effect of Pb²⁺ and mutations in the recognition process.     

Personal recollections on the discovery of the ryanodine receptors of muscle

The intracellular Ca²⁺ release channels are indispensable molecular machinery in practically all eukaryotic cells of multicellular animals. They serve a key role in cell signaling by way of Ca²⁺ as a second messenger. In response to a signaling event, the channels release Ca²⁺ from intracellular stores. The resulting rise in cytoplasmic Ca²⁺ concentration triggers the cell to carry out its specialized role, after which the intracellular Ca²⁺ concentration must be reduced so that the signaling event can again be repeated. There are two types of intracellular Ca²⁺ release channels, i.e., the ryanodine receptors and the inositol triphosphate receptors. My focus in this minireview is to present a personal account, from the vantage point our laboratory, of the discovery, isolation, and characterization of the ryanodine receptors from mammalian muscle. There are three isoforms: ryanodine receptor 1 (RyR1), first isolated from rabbit fast twitch skeletal muscle; ryanodine receptor 2 (RyR2), first isolated from dog heart; and ryanodine receptor 3 (RyR3), first isolated from bovine diaphragm muscle. The ryanodine receptors are the largest channel structures known. The RyR isoforms are very similar albeit with important differences. Natural mutations in humans in these receptors have already been associated with a number of muscle diseases.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.     

Characterization and Ca2+-induced expression of calmodulin (CaM) in marine dinoflagellates

Calmodulin (CaM) is one of the major Ca²⁺-binding proteins in the cells, and it plays multiple roles in several Ca²⁺ signaling pathways and regulating the activities of other proteins. In the present study, we characterized CaM genes from the marine dinoflagellates Amphidinium carterae, Cochlodinium polykrikoides, Prorocentrum micans, and P. minimum, and examined their expression patterns upon the addition and chelation of calcium. Their cDNAs had same ORF length (450 bp) and encoded the same protein, but with few nucleotide differences in the ORF and different 3′- and 5′ untranslated regions (UTRs). The four CaM proteins consist of four EF-hand Ca²⁺-binding motifs, two N-terminal domains and two C-terminal domains, and they were highly conserved within eukaryotes. The CaM gene expressions in the tested species increased by calcium treatments; however, they were significantly down-regulated by the calcium-chelator EGTA. The CaM genes of the test species were inducible and regulated by different calcium doses, suggesting their major role in calcium regulation in dinoflagellates.