Calmodulin Mutants Research Articles

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12', a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12' N-lobe does not interact with the IQ motif. The CaM12' C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.

Disease-associated calmodulin mutations disrupt L-type Ca2+ channel (Cav1.2) activity and CaMKIIdelta phosphorylation in long QT syndrome

Abstract Introduction Long QT Syndrome (LQTS) is a major inherited arrhythmia syndrome that can cause sudden cardiac death. Using genome sequencing in human patients, mutations in the ubiquitous calcium (Ca2+) sensor protein calmodulin (CaM) have been associated to LQTS. CaM is an ion channel regulator and can modulate the activity of the voltage-gated calcium channel (Cav1.2) and Ca2+/CaM-dependent protein kinase II (CaMKIIδ), involved in cardiac muscle contraction. However the molecular mechanism by which CaM mutations contribute to irregular heartbeats remains unclear. Methods Interaction of CaM proteins with Cav1.2 and CaMKIId synthetic peptides (Cav1.2-NSCaTE51–68, Cav1.2-IQ1665–1685, Cav1.2-C1627–1652, CaMKIIδ294–315,) was investigated using Isothermal Titration Calorimetry (ITC) and X-ray crystallography. Whole-cell patch clamp electrophysiology was used to determine the effect of CaM mutations on L-type Ca2+ currents and Ca2+-dependent inactivation (CDI). CaMKIIδ phosphorylation activity was determined by western blot and fluorescence kinase assay. Results Binding affinity of CaMKIId and Cav1.2 peptides to the LQTS-associated CaM variants was significantly reduced, up to 7-fold. Interestingly, the Cav1.2-IQ1665–1685 peptide showed a stronger binding, up to 2-fold, towards LQTS-CaM mutants. Crystal structures of Ca2+-CaM:CaMKIId294–315 showed structural alterations induced by LQTS associated mutations. In addition, we demonstrated that CaMKIIδ autophosphorylation and kinase activity can be significantly reduced by LQTS-associated CaM mutants. Electrophysiological examination of Cav1.2 function revealed that CaM mutations significantly impaired channel CDI, without affecting the voltage dependence of activation and inactivation. Conclusions These data demonstrate a strong correlation between LQTS-associated CaM mutations and Cav1.2 activity. We provide molecular insights into the diverse factors contributing to CaM-mediated arrhythmias. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship

Long QT syndrome-associated calmodulin mutations and their interactions with the Kv7.1 voltage-gated potassium channel

Abstract Introduction Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+) dependent signalling. Its flexible structure allows CaM to bind and modulate many targets, including cardiac ion channels. Genotyping has revealed several CaM mutations associated with congenital disorders of heart rhythm, known as long QT-syndrome (LQTS). LQTS patients suffer from prolonged ventricular recovery times (QT-interval) which increases their risk of significant cardiac events. Loss of function KV7.1 mutations are the largest cause of LQTS, accounting for >50% of cases. CaM facilitates Ca2+-sensitivity to KV7.1 in producing IKs, Kv7.1 mutations which reduce CaM binding promote LQTS pathology. However, the effects of LQTS-associated CaM mutations on Kv7.1 function remain unknown. Purpose To determine the biophysical consequences of congenital LQTS-associated CaM mutations and how they alter modulation of Kv7.1 in producing the ventricular repolarising IKs current. Methods WT and mutant CaM proteins were recombinantly expressed and purified for biophysical characterisation. Using circular dichroism, secondary structures and thermostability of proteins were quantified. Isothermal titration calorimetry was used to quantitatively measure interactions between CaM proteins and binding sites of KV7.1 (Helix B). NMR was employed to study the conformations of target-bound WT and mutant proteins. Whole cell currents were determined using voltage clamp electrophysiology in HEK cells. Results Mutations significantly changed the thermostability and secondary structure distributions of CaM, and also caused site-dependent increases in susceptibility to protease digestion. CaM interacted with Helix B (KV7.1) via both Ca2+-dependent and independent mechanisms. Ca2+ dependent binding to Helix B was much higher affinity than Ca2+-independent binding, with >2000-fold reduction in dissociation constant measured. LQTS-CaM variants reduced Helix B affinity with the largest reductions found in EF-hand IV mutants. These mutants also adopted most distinct conformations when Helix B-bound. Calmodulation of the KV7.1 channel produced larger (IKs) currents without altering channel activation kinetics. IKs exhibited Ca2+-sensitivity, in response to increased cytosolic Ca2+, larger currents were generated. Modulation by CaM mutants reduced current density at systolic Ca2+-concentrations (1000 nM), within physiological time periods (0.35 s), revealing a direct QT-prolonging modulatory effect. Conclusions Provided here are mechanistic insights as to how LQTS-associated CaM variants contribute to electrical disease of the heart. Mutations in the highly conserved structure of CaM disrupt protein conformation and perturb complex formation with KV7.1. This results in aberrant Ca2+-sensitivity of Kv7.1, reducing IKs generation. This ultimately decreases the repolarisation capacity of cells and would extend the QT interval of myocytes. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship

Calcium Ion Binding to the Mutants of Calmodulin: A Structure-Based Computational Predictive Model of Binding Affinity Using a Charge Scaling Approach in Molecular Dynamics Simulation.

The binding of calcium ions (Ca2+) to the calcium-binding proteins (CBPs) controls a plethora of regulatory processes. Among the roles played by CBPs in several diseases, the onset and progress of some cardiovascular diseases are caused by mutations in calmodulin (CaM), an important member of CBPs. Rationalization and prediction of the binding affinity of Ca2+ ions to the CaM can play important roles in understanding the origin of cardiovascular diseases. However, there is no robust structure-based computational method for predicting the binding affinity of Ca2+ ions to the different forms of CBPs in general and CaM in particular. In the current work, we have devised a fast yet accurate computational technique to accurately calculate the binding affinity of Ca2+ to the different forms of CaM. This method combines the well-known molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method and a charge scaling approach developed by previous authors that takes care of the polarization of CaM and Ca2+ ions. Our detailed analysis of the different components of binding free energy shows that subtle changes in electrostatics and van der Waals contribute to the difference in the binding affinity of mutants from that of the wild type (WT), and the charge scaling approach is superior in calculating these subtle changes in electrostatics as compared to the nonpolarizable force field used in this work. A statistically significant regression model made from our binding free energy calculations gives a correlation coefficient close to 0.8 to the experimental results. This structure-based predictive model can open up a new strategy to understand and predict the binding of Ca2+ to the mutants of CBPs, in general.

PO-617-06 BIDIRECTIONAL NEAREST NEIGHBOR COLOCALIZATION IDENTIFIES CARDIOMYOCYTE MICROTRANSLATOMES PRODUCING CALMODULIN AND ITS EFFECTOR TARGETS

Calmodulin (CaM), which regulates myriad proteins in cardiac myocytes, features translational redundancy with three encoding genes (CALM1-3). Mutations in CaM result in calmodulinopathies, characterized by arrhythmia and myopathy. However, the specific roles of the 3 CaM genes and the structural underpinnings of their function remain unclear.

PO-614-08 SODIUM CHANNEL NAV1.6 AND NA-CA EXCHANGER REMODELING CONTRIBUTES TO ARRHYTHMOGENIC LATE SODIUM CURRENT AND CA2+ SPARKS IN THE PRESENCE OF D96V MUTANT CALMODULIN

Calmodulin (CaM) facilitates sodium channel (NaV) inactivation, thereby preventing proarrhythmic late sodium current (INa). To date, a link between arrhythmogenic mutations in CaM and NaV dysfunction is not well established. Outside of NaV1.5, dysfunctional inactivation of Nav1.6 promotes late INa-mediated arrhythmias.

DEER experiments reveal fundamental differences between calmodulin complexes with IQ and MARCKS peptides in solution

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure.

Disparate effects of CaM N- and C-lobe CPVT-linked mutations on SK channel-CaM assembly

Small conductance Ca2+-activated K+ (SK) channels are voltage independent and their gating is governed by Ca2+ via calmodulin (CaM) constitutively bound to the C-terminal CaM-binding domain (CaMBD). Recently, several CaM mutations have been linked to a hereditary arrhythmia syndrome, Catecholaminergic Polymorphic Ventricular Tachycardia. Although in vitro studies demonstrated that mutations in both terminuses of CaM cause loss of SK-channel complex function, the exact mechanisms remain elusive. We hypothesize that N-terminus and C-terminus CaM mutations affect SKs differently given that CaM associates with SK via its C-terminus lobe, while CaM’s N-terminus lobe serves as a Ca2+ sensor for the complex.

Orai1 Boosts SK3 Channel Activation.

Simple SummaryBreast, colon, and prostate cancer account for about a third of cancer cases and a fifth of cancer deaths. At the molecular level, one reason for the development of cancer is the dysfunction or altered co-regulation of cellular proteins. In this study, we focused on the co-regulation of ion channels, specifically the prominent Ca2+ ion channel Orai1 and the Ca2+ activated K+ ion channel SK3. It has recently been reported that their interplay promotes the growth of breast and colon cancer cells, but the molecular determinants for their co-regulation have remained elusive. In this study, we set out to characterize their interplay and the crucial regions therefore required. Moreover, we found that the function of prostate cancer cells is also controlled by the interplay of Ca2+ and the Ca2+ sensitive K+ channels. Our findings provide a better understanding of the co-regulation of these ion channels, which could be used in the future for the development of novel therapeutics.The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3–Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3–Orai1 interplay by significantly decreasing their co-localization. Forced STIM1–Orai1 activity and associated Ca2+ influx promote SK3 K+ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model.

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

Abstract 12302: Na V 1.6 Dysregulation by Mutant Calmodulin Enhances Late Sodium Current in Human iPSC Cardiomyocytes and Promotes Arrhythmias in D96V Calmodulin-Expressing Transgenic Mice

Calmodulin (CaM) mutants are associated with severe arrhythmias (calmodulinopathy). While dysregulation of Ca 2+ handling proteins (RyR2, Ca V 1.2) are known mediators of calmodulinopathy, the role of Na + channels (Na V s) remains unclear. Interestingly, effects on Ca V 1.2 do not fully explain action potential (AP) prolongation by the long QT syndrome CaM mutant D96V. We investigated Na V dysregulation by D96V-CaM and its arrhythmogenic impact. In wild-type (WT) murine cardiomyocytes (CM) dialysis of D96V-CaM (6.5 μM) via a patch pipette prolonged AP, induced early and delayed afterdepolarizations (EADs and DADs) and increased Ca 2+ waves compared to WT-CaM. The effects of D96V-CaM were abrogated by a Na V 1.6 blocker, 4,9-anhydrotetrodotoxin (4,9ahTTX, 300 nM; Fig. 1A ). Dialysis of D96V-CaM dysregulated inactivation of 4,9ahTTX-sensitive Na + current (I Na ) promoting late I Na in WT murine CM ( Fig. 1B ) and in Chinese Hamster Ovary (CHO) cells expressing Na V 1.6 but not Na V 1.5. D96V-CaM, but not WT-CaM, also promoted late I Na in human induced pluripotent stem cell (iPSC) - derived ventricular myocytes, underscoring translational relevance ( Fig. 1C ). To examine the arrhythmia potential of Na V 1.6 dysregulation by D96V-CaM, we generated mice with cardiac-specific expression of D96V-CaM (cD96V; tgD96V-CaM x β-MHC-Cre). cD96V mice exhibited increased late I Na and Ca 2+ waves resembling those in D96V-CaM-dialyzed WT CMs. In vivo cD96V mice evidenced prolonged QTc intervals and ventricular tachycardia (VT) during bradycardia (carbachol 0.5 mg/kg intraperitoneal). Cardiac-specific Na V 1.6 knockout protected cD96V mice from impaired I Na inactivation and arrhythmogenic Ca 2+ waves on cellular level as well as long QT and VT in vivo ( Fig. 1D, E ). In summary, D96V-CaM promotes arrhythmias by dysregulating Na V 1.6 and thereby, facilitating aberrant Ca 2+ release. Importantly, Na V 1.6 may be a therapeutic target for management of CaM-related arrhythmias.

Mutant D96V calmodulin induces unexpected remodeling of cardiac nanostructure and physiology

Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized <100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided "smart" patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized <100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.

Calmodulin acetylation: A modification to remember

The formation of new memories appears to require alterations in the shape and strength of synapses within the hippocampus, yet our knowledge of the molecular mechanisms underlying these changes remains incomplete. Zhang and colleagues provide new understanding of memory formation by uncovering the lysine acetyltransferase SRC3 as the key driver of the novel posttranslational modification of calmodulin (CaM) acetylation, which regulates CaM's activity and subsequent activation of CaMKII. This new pathway is demonstrated to be both necessary and sufficient for CA3→CA1 synapse long-term potentiation (LTP) and fear memory formation, and this approach may act as a blueprint for future investigation of the role of acetylation of other proteins in neuronal functions.

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+–calmodulin binding

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+–CaM required the cytoplasmic amino acid stretch E33–Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+–CaM.

Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell-Cell Communication?

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

The heart arrhythmia-linked D130G calmodulin mutation causes premature inhibitory autophosphorylation of CaMKII

The Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) is well known for transmitting Ca2+-signals, which leads to a multitude of physiological responses. Its functionality is believed to involve CaMKII holoenzyme dynamics where trans-autophosphorylation of the crucial phosphorylation site, T286 occurs. Phosphorylation of this site does not occur when stimulated exclusively with the arrhythmia associated D130G mutant form of CaM in vitro. Here, we present evidence that the loss-of-CaMKII function correlates with premature phosphorylation of its inhibitory phosphosite T306 in CaMKIIα and T307 in CaMKIIδ as this site was up to 20-fold more phosphorylated in the presence of D130G CaM compared to wildtype CaM. Indeed, changing this phosphosite to a non-phosphorylatable alanine reversed the inhibitory effect of D130G both in vitro and in live cell experiments. In addition, several phosphosites with so far undescribed functions directing the Ca2+-sensitivity of the CaMKII sensor were also affected by the presence of the D130G mutation implicating a role of several additional autophosphosites (besides T286 and T306/T307) so far not known to regulate CaMKII Ca2+ sensitivity. Furthermore, we show that introducing a D130G mutation in the CALM2 gene of the P19CL6 pluripotent mouse embryonic carcinoma cell line using CRISPR/Cas9 decreased the spontaneous beat frequency compared to wildtype cells when differentiated into cardiomyocytes supporting an alteration of cardiomyocyte physiology caused by this point mutation. In conclusion, our observations shed for the first time light on how the D130G CaM mutation interferes with the function of CaMKII and how it affects the beating frequency of cardiomyocyte-like cells.

NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD

Neuronal voltage-gated sodium channel NaV1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaMN) is structurally independent of the C-domain (CaMC) whether CaM is bound to the NaV1.2IQp (1,901-1,927) or NaV1.2CTD (1,777-1,937) with or without calcium. However, in the CaM+ NaV1.2CTD complex, the Ca2+ affinity of CaMN was more favorable than in free CaM, while Ca2+ affinity for CaMC was weaker than in the CaM+ NaV1.2IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the NaV1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaMN sites. However, only N53I and A102V bound calcium at CaMC sites at [Ca2+]< 100μM. Thus, they are expected to respond like wild-type CaM to Ca2+ spikes in excitable cells.

The Crystal Structure of Calmodulin Bound to the Cardiac Ryanodine Receptor (RyR2) at Residues Phe4246-Val4271 Reveals a Fifth Calcium Binding Site.

Calmodulin (CaM) regulates the activity of a Ca2+ channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca2+-bound CaM bound to RyR2 CaMBD3. The structure reveals Ca2+ bound to the four EF-hands of CaM as well as a fifth Ca2+ bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca2+ and weakens the binding of CaMBD3 to Ca2+-bound CaM. Thus, the binding of the fifth Ca2+ is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca2+ to CaM may contribute to the regulation of RyR2 channel function.

Assessing the Impact of Calmodulinopathic Mutations on Neuronal Ion Channel Regulation

Calmodulin (CaM) represents an essential component of the cellular response to calcium influx within multiple excitable cell types, including neurons and cardiac myocytes. Calcium binding to CaM is known to mediate the opposing phenomena of calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), in different voltage-gated calcium channels (VGCCs). In humans, mutations within CaM can lead to clinical disorders known as calmodulinopathies, in which patients suffer from severe cardiac symptoms such as long-QT syndrome (LQTS). LQTS in calmodulinopathy patients can be explained by a loss of calcium binding to mutant CaM, which in turn causes a deficit in CDI of the cardiac CaV1.2 L-type calcium channel. However, CaM-mediated regulation is a hallmark of numerous VGCCs, and disruption of these processes in channels outside the heart may explain why calmodulinopathy patients exhibit a high incidence of neurological impairments including developmental delay, seizures, and autism. We therefore probed the effect of pathogenic CaM mutants on VGCCs which are critical to normal neurological function. To begin, we examined the effect of CaM mutants on the robust CDI of CaV1.3 L-type calcium channels, identifying CDI deficits similar to what has been seen in CaV1.2. We next examined the effect of the CaM mutants on the regulation of CaV2.1 channels, which display both CDI and CDF. The CaM mutants caused a significant disruption of CDF in CaV2.1, however the pattern of severity differed from that of the two L-type channels. Thus, the variability of neurologic phenotypes across calmodulinopathy patients may be due, in part, to a differential effect of CaM mutations on the myriad of VGCCs in the brain. Overall, these results suggest a multifaceted role for calmodulinopathic mutations in neuronal dysfunction through disrupted calcium dependent feedback of VGCCs.

Calmodulinopathy Correction using CRISPR Interference in a Cardiac Microtissue Model

Calmodulinopathies, caused by mutations in calmodulin (CaM), a ubiquitous Ca2+ sensor, can lead to life-threatening cardiac arrhythmias, such as long QT syndrome (LQTS). In this study, we created a calmodulinopathy model in 3D cardiac microtissues (CMTs), using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) with the D130G-CALM2 mutation, and used CRISPR interference (CRISPRi) to rescue the disease phenotype. Lentiviruses that encode a guide RNA (gRNA) targeting CALM2 (both wildtype and mutant) and a Krüppel associated box (KRAB) transcriptional repressor were transduced to CMTs and their electrophysiological and mechanical properties were analyzed. Results show that D130G-CALM2 CMTs reproduced key features of calmodulinopathy associated LQTS, including prolongation of action potential duration (APD), electrical alternans and longer pacing cycle lengths at which loss of 1:1 capture occurred compared to uncorrected mutant CMTs. After transduction with CALM2 gRNA and KRAB lentivirus, we observed significant knockdown of CALM2 mRNA, shortened APD, increased loading rate during contraction and relaxation, and decreased time to peak and relaxation time over a range of pacing frequencies in CMTs. In conclusion, by demonstrating correction of disease electrophysiological and mechanical phenotype by CRISPRi in 3D tissue models, this study provides a proof of concept for the utility of using viral transduction for CRISPRi in cardiac muscle to correct the tissue phenotype.