Conflict of interest:MEA is a named inventor on patents claiming to treat heart disease by CaMKII inhibition. No income derives from these activities.

Background: CaM (calmodulin), encoded by 3 separate genes ( CALM1 , CALM2 , and CALM3 ), is a multifunctional Ca 2+ -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 Ca 2+ -binding affinity was measured by stoichiometric Ca 2+ titrations and equilibrium titrations. L-type Ca 2+ and slow delayed rectifier potassium currents (I CaL and I Ks ) 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 Ca 2+ -binding affinity of the CaM C-terminal domain was 10-fold lower for p.N138K-CaM compared with WT-CaM. I CaL inactivation was slowed in cells expressing p.N138K-CaM but less than in p.D130G-CaM cells. Unexpectedly, a larger I Ks current density was observed in cells expressing p.N138K-CaM, but not for p.D130G-CaM, compared with WT-CaM. Conclusions: The p.N138K CALM3 variant impairs Ca 2+ -binding affinity of CaM and I CaL inactivation but potentiates I Ks . The variably expressed phenotype of this variant compared with previously published de novo LQTS-CaM variants is likely explained by a milder impairment of I CaL inactivation combined with I Ks augmentation.

Human voltage-gated sodium (Na) 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 β-subunits. Na 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 Na 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 Na1.2, Na1.5, and Na1.6 showed that CaM binding was mediated by the C-domain of CaM (CaM), while the N-domain (CaM) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other Na isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of Na1.1, Na1.4, and Na1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaM; resonances of CaM were identical to free CaM. Thus, CaM residues contribute to the interface with the IQ motif, while CaM is available to interact elsewhere on the channel.

Na V 1.2 EFL domain allosterically enhances Ca 2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD Graphical abstract Highlights d In solution, only the IQ motif of Na V 1.2 CTD(1,777-1,937) contacts CaM ± Ca 2+ d Ca 2+ changes interfaces of CaM + Na V 1.2-IQ p and CaM + Na V 1.2-CTD complexes identically d Ca 2+ binds to N-domain sites of CaM + Na V 1.2-CTD preferentially relative to free CaM d Apo pathogenic CaM mutants bind the Na V 1.2 CTD as well or better than WT CaM

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