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Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation.

ABSTRACT: Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca²⁺-sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Ca_v1.2 Ca²⁺/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca²⁺-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Ca_v1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Ca_v1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.

SUBMITTER: Prakash O

PROVIDER: S-EPMC9830374 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation.

Prakash Ohm O Gupta Nitika N Milburn Amy A McCormick Liam L Deugi Vishvangi V Fisch Pauline P Wyles Jacob J Thomas N Lowri NL Antonyuk Svetlana S Dart Caroline C Helassa Nordine N

The Journal of biological chemistry 20221208 1

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca<sup>2+</sup>-sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determi ...[more]

PMID: 36496072

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Project description:Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca2+ concentrations (1 μm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype.

Project description:Short QT syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death. Thus far, mutations in five different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for an L-type calcium channel subunit.The electrocardiogram of the affected member of a single family revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and symmetrical T-waves. Invasive electrophysiological testing showed short ventricular refractory periods and increased vulnerability to induce ventricular fibrillation. DNA screening of the patient identified no mutation in previously known SQTS genes; however, a new variant at a heterozygous state was identified in the CACNA2D1 gene (nucleotide c.2264G > C; amino acid p.Ser755Thr), coding for the Ca(v)?(2)?-1 subunit of the L-type calcium channel. The pathogenic role of the p.Ser755Thr variant of the CACNA2D1 gene was analysed by using co-expression of the two other L-type calcium channel subunits, Ca(v)1.2?1 and Ca(v)?(2b), in HEK-293 cells. Barium currents (I(Ba)) were recorded in these cells under voltage-clamp conditions using the whole-cell configuration. Co-expression of the p.Ser755Thr Ca(v)?(2)?-1 subunit strongly reduced the I(Ba) by more than 70% when compared with the co-expression of the wild-type (WT) variant. Protein expression of the three subunits was verified by performing western blots of total lysates and cell membrane fractions of HEK-293 cells. The p.Ser755Thr variant of the Ca(v)?(2)?-1 subunit was expressed at a similar level compared with the WT subunit in both fractions. Since the mutant Ca(v)?(2)?-1 subunit did not modify the expression of the pore-forming subunit of the L-type calcium channel, Ca(v)1.2?1, it suggests that single channel biophysical properties of the L-type channel are altered by this variant.In the present study, we report the first pathogenic mutation in the CACNA2D1 gene in humans, which causes a new variant of SQTS. It remains to be determined whether mutations in this gene lead to other manifestations of the J-wave syndrome.

Dataset Information

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation.

Publications

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation.

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