Novel CALM3 Variant Causing Calmodulinopathy With Variable Expressivity in a 4-Generation Family.

BACKGROUND: CaM (calmodulin), encoded by 3 separate genes (CALM1, CALM2, and CALM3), is a multifunctional Ca2+-binding protein involved in many signal transduction events including ion channel regulation. CaM variants may present with early-onset long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia, or sudden cardiac death. Most reported variants occurred de novo. We identified a novel CALM3 variant, p.Asn138Lys (N138K), in a 4-generation family segregating with LQTS. The aim of this study was to elucidate its pathogenicity and to compare it with that of p.D130G-CaM-a variant associated with a severe LQTS phenotype. METHODS: We performed whole exome sequencing for a large, 4-generation family affected by LQTS. To assess the effect of the detected CALM3 variant, the intrinsic Ca2+-binding affinity was measured by stoichiometric Ca2+ titrations and equilibrium titrations. L-type Ca2+ and slow delayed rectifier potassium currents (ICaL and IKs) were recorded by whole-cell patch-clamp. Cav1.2 and Kv7.1 membrane expression were determined by optical fluorescence assays. RESULTS: We identified 14 p.N138K-CaM carriers in a family where 2 sudden deaths occurred in children. Several members were only mildly affected compared with CaM-LQTS patients to date described in literature. The intrinsic Ca2+-binding affinity of the CaM C-terminal domain was 10-fold lower for p.N138K-CaM compared with wild-type-CaM. ICaL inactivation was slowed in cells expressing p.N138K-CaM but less than in p.D130G-CaM cells. Unexpectedly, a larger IKs current density was observed in cells expressing p.N138K-CaM, but not for p.D130G-CaM, compared with wild-type-CaM. CONCLUSIONS: The p.N138K CALM3 variant impairs Ca2+-binding affinity of CaM and ICaL inactivation but potentiates IKs. The variably expressed phenotype of this variant compared with previously published de novo LQTS-CaM variants is likely explained by a milder impairment of ICaL inactivation combined with IKs augmentation.

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+] < 100 µM. Thus, they are expected to respond like wild-type CaM to Ca2+ spikes in excitable cells.

MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7.

Human voltage-gated sodium (NaV) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane ß-subunits. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel.