Calmodulin Mutants Research Articles

Calmodulinopathy in inherited arrhythmia syndromes.

Calmodulin (CaM) is a ubiquitous intracellular calcium sensor that controls and regulates key cellular functions. In all vertebrates, three CaM genes located on separate chromosomes encode an identical 149 amino acid protein, implying an extraordinarily high level of evolutionary importance and suggesting that CaM mutations would be possibly fatal. Inherited arrhythmia syndromes comprise a spectrum of primary electrical disorders caused by mutations in genes encoding ion channels or associated proteins leading to various cardiac arrhythmias, unexplained syncope, and sudden cardiac death. CaM mutations have emerged as an independent entity among inherited arrhythmia syndromes, referred to as calmodulinopathies. The most common clinical presentation associated with calmodulinopathy is congenital long QT syndrome, followed by catecholaminergic polymorphic ventricular tachycardia, both of which significantly increase the possibility of repeated syncope, lethal arrhythmic events, and sudden cardiac death, especially in young individuals. Here, we aim to give an overview of biochemical and structural characteristics of CaM and progress toward updating current known CaM mutations and associated clinical phenotypes. We also review the possible mechanisms underlying calmodulinopathy, based on several key in vitro studies. We expect that further experimental studies are needed to explore the complexity of calmodulinopathy.

Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1–5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ∼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.

Ca2+-CaM Dependent Inactivation of RyR2 Underlies Ca2+ Alternans in Intact Heart.

Ca2+ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca2+ alternans remains undefined. Increasing evidence suggests that Ca2+ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. To determine the role of CaM (calmodulin) on Ca2+ alternans in intact working mouse hearts. We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca2+ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca2+ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca2+ alternans and prolonged Ca2+ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca2+ current but had no significant impact on sarcoplasmic reticulum Ca2+ content. Furthermore, we developed a novel numerical myocyte model of Ca2+ alternans that incorporates Ca2+-CaM-dependent regulation of RyR2 and the L-type Ca2+ channel. Remarkably, the new model recapitulates the impact on Ca2+ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca2+ elevation as a result of rapid pacing triggers Ca2+-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca2+ release, which, in turn, reduces diastolic cytosolic Ca2+, leading to alternations in diastolic cytosolic Ca2+, RyR2 inactivation, and sarcoplasmic reticulum Ca2+ release (ie, Ca2+ alternans). Our results demonstrate that inactivation of RyR2 by Ca2+-CaM is a major determinant of Ca2+ alternans, making Ca2+-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.

Met125 is essential for maintaining the structural integrity of calmodulin\u2019s C-terminal domain

We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM is a Ca2+-sensitive regulatory protein that interacts with numerous targets. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species under conditions of cell stress and age-related muscle degeneration. CaM oxidation alters regulation of a host of CaM’s protein targets, emphasizing the importance of understanding the mechanism of CaM oxidation in muscle degeneration and overall physiology. It has been shown that the M125Q CaM mutant can mimic the functional effects of methionine oxidation on CaM’s regulation of the calcium release channel, ryanodine receptor (RyR). We report here that the M125Q mutation causes a localized unfolding of the C-terminal lobe of CaM, preventing the formation of a hydrophobic cluster of residues near the EF-hand Ca2+ binding sites. NMR analysis of CaM oxidation by peroxide offers further insights into the susceptibility of CaM’s Met residues to oxidation and the resulting structural effects. These results further resolve oxidation-driven structural perturbation of CaM, with implications for RyR regulation and the decay of muscle function in aging.

Different arrhythmia-associated calmodulin mutations have distinct effects on cardiac SK channel regulation.

Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.

Preclinical proof-of-concept study: antisense-mediated knockdown of CALM as a therapeutic strategy for calmodulinopathy

Abstract Background Calmodulin (CaM) is a ubiquitous Ca2+ sensor molecule encoded by three distinct calmodulin genes, CALM1–3, and has an important role for cardiac ion channel function. Recently, heterozygous missense mutations in CALM genes were reported to cause a new category of life-threatening genetic arrhythmias such as long-QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT), which is called as “calmodulinopathy”. The patients with calmodulinopathy show poor prognosis and there is no effective treatment for them. Purpose Considering the dominant-negative effect of mutant calmodulin proteins produced by heterozygous missense mutations in CALMs, we aimed to prove the concept of antisense-based therapy to treat calmodulinopathy using human iPS cell-derived cardiomyocyte (hiPSC-CM) model. Methods We designed multiple locked nucleic acid (LNA) gapmer-antisense oligonucleotides (ASOs) targeting CALM2 and analyzed the silencing efficiency and toxicity in cultured cells to select the most potent ASO. Using CMs differentiated from hiPSCs which were generated form a 12-year-old boy with LQTS carrying a heterozygous CALM2-N98S mutation, CALM2 expression and action potentials (APs) were analyzed to evaluate the efficacy of ASOs. Results We identified several ASOs which reduced CALM2 expression without affecting cell viability in human cultured cells (HepG2) (ASO 50 nM, n=2; Figure 1A). Considering further experiments in vivo mouse model, we investigated the CALM2 silencing activity in mouse cultured cells (3T3-L1) without transfection (free-uptake) (ASO 1 μM, n=2; †ASOs have homologous sequence between human and mouse; Figure B). After free-uptake CALM2 silencing analysis in 3T3-L1 cells, we identified that ASO #2 has the most potent CALM2 silencing activity and low cytotoxicity (Figure 1B). ASO #2 effectively reduced CALM2 expression even in hiPSC-CMs (ASO(−): n=3, lipofection: n=4, free-uptake: n=3; P<0.05; Figure 1C). In action potential recordings, we demonstrated that ASO #2 ameliorated prolonged AP durations (APD90) in N98S-hiPSC-CMs at 0.5 Hz pacing (ASO(−): 666±123 ms (n=7), lipofection: 329±21 ms (n=8), free-uptake: 388±34 ms (n=12); P<0.05; Figure 1D). Conclusion Our results using patient-derived hiPSC-CM model suggest that ASO-based therapy might be a promising strategy for the treatment of calmodulinopathy. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Nissan Chemical Corporation

CaV channels reject signaling from a second CaM in eliciting Ca2+-dependent feedback regulation

Calmodulin (CaM) regulation of voltage-gated calcium (CaV1-2) channels is a powerful Ca2+-feedback mechanism to adjust channel activity in response to Ca2+ influx. Despite progress in resolving mechanisms of CaM-CaV feedback, the stoichiometry of CaM interaction with CaV channels remains ambiguous. Functional studies that tethered CaM to CaV1.2 suggested that a single CaM sufficed for Ca2+ feedback, yet biochemical, FRET, and structural studies showed that multiple CaM molecules interact with distinct interfaces within channel cytosolic segments, suggesting that functional Ca2+ regulation may be more nuanced. Resolving this ambiguity is critical as CaM is enriched in subcellular domains where CaV channels reside, such as the cardiac dyad. We here localized multiple CaMs to the CaV nanodomain by tethering either WT or mutant CaM that lack Ca2+-binding capacity to the pore-forming α-subunit of CaV1.2, CaV1.3, and CaV2.1 and/or the auxiliary β2A subunit. We observed that a single CaM tethered to either the α or β2A subunit tunes Ca2+ regulation of CaV channels. However, when multiple CaMs are localized concurrently, CaV channels preferentially respond to signaling from the α-subunit-tethered CaM. Mechanistically, the introduction of a second IQ domain to the CaV1.3 carboxyl tail switched the apparent functional stoichiometry, permitting two CaMs to mediate functional regulation. In all, Ca2+ feedback of CaV channels depends exquisitely on a single CaM preassociated with the α-subunit carboxyl tail. Additional CaMs that colocalize with the channel complex are unable to trigger Ca2+-dependent feedback of channel gating but may support alternate regulatory functions.

Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation.

Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. Mouse lines heterozygous for the Calm1N98S allele (Calm1N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca2+]i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. Two biologically independent knock-in mouse lines heterozygous for the Calm1N98S allele were generated. Calm1N98S/+ mice of either sex and line exhibited sinus bradycardia, QTc interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of ICa.L significantly more in Calm1N98S/+ versus Calm1+/+ ventricular myocytes, increasing late ICa.L in the former. Rapidly paced Calm1N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. Heterozygosity for the Calm1N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced ICa.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1N98S/+ hearts.

Calmodulin-Cork Model of Gap Junction Channel Gating-One Molecule, Two Mechanisms.

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

Effect of calmodulin and its mutants on binding to NaV1.2 IQ

To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (NaV1.2 IQ). The cDNA of NaV1.2 IQ was constructed by PCR technique, CaM mutants CaM12, CaM34 and CaM1234 were constructed with QuickchangeTM site-directed mutagenesis kit (QIAGEN). The binding of NaV1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. NaV1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and NaV1.2 IQ at [Ca2+]-free was greater than that at 100 nmol/L [Ca2+] (P < 0.05). In the absence of calcium, the binding amount of CaM wild-type to NaV1.2 IQ was greater than that of its mutant, and the binding affinity of CaM1234 to NaV1.2 IQ was the weakest among the three mutants (P < 0.05). The binding ability of CaM and CaM mutants to NaV1.2 IQ is Ca2+-dependent. This study has revealed a new mechanism of NaV1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.

The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a flickering gating mode of the CaV1.2 channel.

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.

Calmodulin mutant in central linker reduces the binding affinity with PreIQ and IQ while interacting with CaV1.2 channels

Calmodulin (CaM) was reported to interact with PreIQ and IQ of CaV1.2 channels, but to date, no explicit binding sites of CaM were illustrated. Therefore, in the present study, we firstly used MOE (Molecular Operating Environment) for protein-protein docking and we found that the most likely residues of CaM that play an important role in the interface are concentrated in central linker region. Next we examined the binding properties of CaM and its mutants to PreIQ and IQ by GST pull-down assays. Here we confirmed that CaM binds to PreIQ and IQ in a concentration-dependent and [Ca2+]-dependent manner. However, silencing the effect of N-lobe and C-lobe by mutating two Ca2+ binding sites of each lobe abolished [Ca2+]-dependence of CaM binding, but could not influence the combination. And the mutant in central linker reduced the binding of CaM/PreIQ and CaM/IQ especially at low [Ca2+]. We confirmed that N-lobe and C-lobe play vital role in sensing the change of Ca2+, and found that the central linker of CaM is involved in the binding of CaM to CaV1.2 channels in particular at low [Ca2+], not only participates in the combination with PreIQ, but also with IQ.

Calmodulin Mutations Associated with Heart Arrhythmia: A Status Report

Calmodulin (CaM) is a ubiquitous intracellular Ca2+ sensing protein that modifies gating of numerous ion channels. CaM has an extraordinarily high level of evolutionary conservation, which led to the fundamental assumption that mutation would be lethal. However, in 2012, complete exome sequencing of infants suffering from recurrent cardiac arrest revealed de novo mutations in the three human CALM genes. The correlation between mutations and pathophysiology suggests defects in CaM-dependent ion channel functions. Here, we review the current state of the field for all reported CaM mutations associated with cardiac arrhythmias, including knowledge of their biochemical and structural characteristics, and progress towards understanding how these mutations affect cardiac ion channel function.

Arrhythmia mutations in calmodulin can disrupt cooperativity of Ca2+ binding and cause misfolding.

Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel CaV 1.2. Arrhythmia mutations in CaM can lead to loss of Ca2+ binding, uncoupling of Ca2+ binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease. Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca2+ channel CaV 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the CaV 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca2+ binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca2+ -saturating conditions when unbound to the IQ domain. Ca2+ binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca2+ ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of CaV 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease.

Arrhythmogenic Calmodulin Mutations Can Disrupt the Globular Structure and Uncouple Ca2+ Binding Cooperativity

Calmodulin (CaM) is a highly conserved Ca2+ sensor that modulates hundreds of proteins including the L-type voltage-gated Ca2+ channel CaV1.2 and cardiac Ryanodine Receptor (RyR2). Mutations in CaM are associated with life-threatening arrhythmia syndromes such as early-onset long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). In the heart, CaM interacts with the C-terminal IQ domain of CaV1.2 to facilitate calcium dependent inactivation (CDI), and several CaM disease mutants were found to suppress CaV1.2 CDI and have decreased Ca2+ affinity. Here, we utilize structural biology techniques and binding assays to characterize arrhythmia-linked CaM variants and their interactions with the CaV1.2 IQ domain. From the crystal structure, we find one LQTS-associated variant (D129G) causes unfolding of the C-lobe that completely disrupts the globular structure and a loss of Ca2+ binding in EF-hand 4. Another mutation (Q135P) severely reduces CaM's affinity for the IQ domain, and shows changes in the CD spectra compared to wild-type Ca2+/CaM when unbound to the IQ domain. The N97S CaM variant displays uncoupled Ca2+ binding cooperativity between EF-hands 3 and 4, resulting in a C-lobe that can have either one or two Ca2+ ions bound. An N-lobe variant (N53I) linked to CPVT does not exhibit significant differences in complex with the IQ domain, in agreement with past studies which show N53I CaM does not affect CaV1.2 regulation. These results confirm that different CaM variants have distinct effects on ion channel regulation and act via diverse molecular mechanisms to cause disease.

Energetics of Calmodulin Recognition of a Skeletal Muscle Ryanodine Receptor Site

In skeletal muscle, the calcium-binding protein calmodulin (CaM) modulates the opening and closing of the ryanodine receptor (RyR1), the main ion channel responsible for calcium release from the sarcoplasmic reticulum during excitation-contraction coupling. By weakly activating RyR1 at low (nM) calcium and inhibiting it at high (µM) calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction. A short region on RyR1 (residues 3614-3640, RyR1p) serves as an anchor for CaM. We investigated the energetics of CaM-RyR1p recognition using Förster resonance energy transfer in auto-fluorescent biosensor constructs (YFP-RyRp-CFP). Using this system, we systematically explored the roles of individual RyR1 residues and each CaM domain at the interface, both in the presence and the absence of calcium. The interplay between the processes of calcium- and target-binding to CaM was probed by quantifying the energy of interaction between wild-type or mutated RyR1p sequences and CaM mutants having non-functional domain-specific Ca2+-binding sites. At low calcium, the interaction is weak (low µM), and mutation Trp3620Ala in the N-terminus of RyR1p (but not mutation Tyr3636Ala in the C-terminus) renders it non-specific. At high calcium, the interaction is at least three orders of magnitude stronger, and CaM C-domain binding to the N-terminal half of RyRp is the main mediator of recognition. A CaM C-domain Ca2+-binding mutant and/or Trp3620Ala RyR1p mutation significantly decrease the binding affinity, while a CaM N-domain Ca2+-binding mutant and/or Tyr3636Ala RyRp mutation have weaker effects. The primary role of CaM C-domain at the interface is maintained in the Tyr3636Ala RyRp complex, whereas Trp3620Ala mutation almost equalizes the energetic contributions of the two CaM domains. Future NMR studies of co-expressed CaM-RyR1p complexes will complement the thermodynamic data with atomic-resolution structural information.

A Calmodulin Mutation that Dysregulates NaV1.6 But Not NaV1.5

A Calmodulin Mutation that Dysregulates NaV1.6 But Not NaV1.5

Defective Interaction of Cam with RyR2 Cam-Binding Pocket Might Contribute to Arrhythmogenic Cardiac Disease

Ryanodine receptor 2 (RyR2) is a large transmembrane calcium (Ca2+) release channel that mediates Ca2 release from the sarcoplasmic reticulum to activate cardiac muscle contraction. Calmodulin (CaM) regulation of RyR2 is essential for normal cardiac function. A number of linear fragments of RyR2 have been reported as potential CaM-binding sequences. The sequence 3583-3603aa of human RyR2, which is highly conserved among mammalian isoforms, has been identified as a CaM-binding site in almost all relevant studies and therefore this region is considered as a well-established CaM-binding domain of RyRs. Besides 3583-3603aa region, other RyR2 regions have been also reported as potential CaM-binding sequences. Herein, we used recombinant wild-type CaMprotein and isothermal titration calorimetry (ITC) experiments to screen a number of RyR2-specific synthetic peptides corresponding to the region 4240-4277aa of RyR2, which has been previously proposed as a putative CaM-binding RyR2 region. From all the synthetic peptides screened, a peptide corresponding to 4255-4271aa region of human RyR2 was found to interact with significant affinity with RyR2, in the presence and absence of Ca2+ (Kd values 0.60 and 16.58 μM, respectively). Moreover, investigation of the interaction of four arrhythmogenic CaM mutants (N98I, D132E, D134H and Q136P) with this synthetic peptide, as well as the peptide corresponding to the well-established CaM-binding domain of RyR2 (3583-3603aa), revealed that all mutants show disparate binding properties to these two RyR2 peptides, which have been previously proposed to contribute to a putative intra-subunit CaM-binding pocket. Our findings extend our previous observations suggesting that CaM mutations may trigger arrhythmogenic cardiac disease by altering both intrinsic Ca2+-binding, as well as by dysregulating RyR2-mediated Ca2+ release via defective interaction of CaM with a distinct CaM-binding pocket that multiple RyR2 regions might contribute.

LQTS-Associated Mutants of Calmodulin Show Disrupted Interaction with L-Type Calcium Channels

Long QT syndrome (LQTS) is a major cause of sudden cardiac death, and is characterised by dysfunctional ion channel activity. Genetic screening of clinical populations has identified mutations in the calcium sensor calmodulin (CaM). However, little is currently known about the molecular mechanism of CaM-associated LQTS. A small but ubiquitous protein, CaM is involved in the calcium-dependent regulation of multiple targets such as Cav1.2, where it mediates calcium-dependent inactivation by interaction with CaM binding domains present on the channel, including the N-terminal spatial calcium transforming element (NSCaTE) and the C-terminal IQ domain. Ergo, CaM mutations that affect binding to Cav1.2 may affect channel modulation. We investigated the interaction of clinically relevant CaM mutants D132E, D134H, Q136P and E141G with Cav1.2. Here, we show that these mutations in the fourth EF hand of CaM differentially alter CaM's stability and structure, in addition to its interaction with Cav1.2 CaM binding domains. Circular dichroism experiments reveal alterations to thermal stability and secondary structure content. This is corroborated by HSQC NMR spectra showing differences in conformation which are exacerbated by interaction with NSCaTE or IQ domains. Furthermore, isothermal titration calorimetry demonstrates that in calcium-saturating conditions, Q136P and E141G isoforms have a significantly decreased affinity for NSCaTE (up to 6-fold), whilst D132E, D134H and E141G have an increased affinity for IQ (up to 3-fold). Therefore, mutations have clear but distinct effects on the interaction of CaM with Cav1.2, suggesting differences in the mechanisms underlying the pathophysiology of the disease. This is likely to disrupt channel function, leading to prolonged calcium influx and manifestation of the LQTS phenotype. This work is funded by the Wellcome Trust PhD Studentship (to NG) and the British Heart Foundation Intermediate Basic Science Research Fellowship FS/17/56/32925 (to NH).

Long-QT Syndrome-Associated Calmodulin Mutations and Their Interactions at the Kv7.1 Potassium Channel

Long-QT Syndrome-Associated Calmodulin Mutations and Their Interactions at the Kv7.1 Potassium Channel