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

Prakash, Ohm; Gupta, Nitika; Milburn, Amy; McCormick, Liam F.; Deugi, Vishvangi; Fisch, Pauline; Wyles, Jacob; Thomas, N. Lowri; Antonyuk, S.V.; Dart, Caroline; Helassa, Nordine

doi:10.1016/j.jbc.2022.102777

Cited by 3 publications

(6 citation statements)

References 114 publications

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“…These findings agree with other studies which have demonstrated a site-dependent, ranging degree of chemical shift perturbation across LQTS-CaM variants, which present more apparently when variants are compared with WT in their calcified states (Holt et al, 2020;Pipilas et al, 2016;Wang et al, 2020;Wren et al, 2019). Additionally, we demonstrated that LQTS-associated CaM variants displayed altered susceptibility to protease hydrolysis, consistent with other works (Crotti et al, 2013;Dal Cortivo et al, 2022;Prakash et al, 2023). This was more apparent when mutants were compared in Ca 2+ -saturating conditions, reflecting the more distinct Ca 2+ -bound conformations of mutants, rather than their more subtly dissimilar apo-conformations.…”

Section: Discussionsupporting

confidence: 93%

“…Such modulation would reduce the repolarisation capacity of IKs, extending the APD and contributing to the LQTS phenotype which presented in patients harbouring said CaM mutations. LQTS-associated CaM variants have been shown to perturb a range of other cardiac ion channels, including reducing the Ca 2+ -dependent inactivation of Cav1.2 (Gomez-Hurtado et al, 2016;Limpitikul et al, 2014;Prakash et al, 2023;Yin et al, 2014), altered inhibition of RyR2 (Nomikos et al, 2014;Vassilakopoulou et al, 2015) and activation of CaMKIIδ (Berchtold et al, 2016;Prakash et al, 2023). The large repertoire of targets which CaM interacts with, combined with the limited clinical data from CaM-driven LQTS patients, make it difficult to establish a direct correlation between CaM mutation and disease severity.…”

Section: Discussionmentioning

confidence: 99%

“…For biophysical and structural biology experiments. The sequence of human wild-type (WT) CaM was subcloned into the pE-SUMOPro-Kan vector (LifeSensors, USA) as previously described (Prakash et al, 2021(Prakash et al, , 2023. A series of site-directed mutagenesis (SDM) reactions were performed using the QuikChangeII kit (Agilent Technologies, USA), according to the manufacturer's recommendation, in order to generate LQTS-associated CaM mutants D95V, N97I and D131H.…”

Section: Molecular Biologymentioning

confidence: 99%

“…CaM proteins were recombinantly expressed in Escherichia coli BL21 (DE3) STAR, purified using affinity and size-exclusion chromatography, as previously described (Prakash et al, 2021(Prakash et al, , 2023. For nucleic magnetic resonance (NMR) experiments, uniformly isotopic-labelled CaM proteins were grown in minimal medium containing 88 mM Na 2 HPO4, 55 mM KH 2 PO 4 , 30 μM thiamine-HCl, 136 μM CaCl 2 , 1 mM MgSO 4 , 19 mM 15 NH 4 Cl (Cambridge Isotope Laboratories) and 22 mM glucose.…”

Section: Recombinant Protein Expression and Purification Of Cam Variantsmentioning

confidence: 99%

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Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

McCormick

Wadmore

Milburn

et al. 2023

The Journal of Physiology

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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. imageKey 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.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Molecular Biologymentioning

confidence: 99%

Section: Recombinant Protein Expression and Purification Of Cam Variantsmentioning

confidence: 99%

See 2 more Smart Citations

Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

McCormick

Wadmore

Milburn

et al. 2023

The Journal of Physiology

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“…unc-43 encodes a homolog of the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) which is considered a general regulator of synaptic plasticity ( 35 , 45 ). There are few studies available on the effects of arrhythmogenic calmodulin mutations on CaMKII, and the results are inconsistent ( 16 , 46 , 47 ). Therefore, further studies are required to elucidate whether hcmd-1(D96V) mutants experience whole body convulsions due to defective signaling via unc-43 or GABAergic signaling.…”

Section: Discussionmentioning

confidence: 99%

Human calmodulin mutations cause arrhythmia and affect neuronal function in C. elegans

Jensen

Frantzen

Wesseltoft

et al. 2023

Human Molecular Genetics

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In humans, mutations in calmodulin cause cardiac arrhythmia. These mutations disrupt the ability of calmodulin to sense calcium concentrations and correctly regulate two central calcium channels, together obstructing heart rhythm. This correlation is well established, but also surprising since calmodulin is expressed in all tissues and interacts with hundreds of proteins. Until now, most studies have focused on cardiac cell function and regulation of specific cardiac targets, and thus potential other effects of these mutations have largely been unexplored. Here, we introduce the nematode Caenorhabditis elegans as an in vivo model to study effects of three human calmodulin mutations with different impairment on calcium binding. We find that arrhythmic effects of the calmodulin mutations N54I and D96V can be recapitulated in disruption of two rhythmic behaviors, pharynx pumping and defecation motor program. Interestingly, we also find that these mutations affect neuronal function, but in different ways. Whereas D96V sensitizes signaling at the neuromuscular junction, N54I has a protective effect. The mutation N98S did not affect rhythmic behavior, but impaired chemosensing. Therefore, pathogenic calmodulin mutations act through different mechanisms in rhythmic behavior and neuronal function in C. elegans, emphasizing the strength of using live multicellular models. Finally, our results support the hypothesis that human calmodulin mutations could also contribute to neurological diseases.

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Cardiac Ion Channels and Heart Rate and Rhythm

Tripathi

2023

Heart Rate and Rhythm

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

Cited by 3 publications

References 114 publications

Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

Human calmodulin mutations cause arrhythmia and affect neuronal function in C. elegans

Cardiac Ion Channels and Heart Rate and Rhythm

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