Inhibition of Nonsense-Mediated mRNA Decay by Antisense Morpholino Oligonucleotides Restores Functional Expression of hERG Nonsense and Frameshift Mutations in Long-QT Syndrome

Mutations in the human ether-a-go-go-related gene (hERG) cause long-QT syndrome type 2 (LQT2). We previously described a homozygous LQT2 nonsense mutation Q1070X in which the mutant mRNA is degraded by nonsense-mediated mRNA decay (NMD) leading to a severe clinical phenotype. The degradation of the Q1070X transcript precludes the expression of truncated but functional mutant channels. In the present study, we tested the hypothesis that inhibition of NMD can restore functional expression of LQT2 mutations that are targeted by NMD. We showed that inhibition of NMD by RNA interference-mediated knockdown of UPF1 increased Q1070X mutant channel protein expression and hERG current amplitude. More importantly, we found that specific inhibition of downstream intron splicing by antisense morpholino oligonucleotides prevented NMD of the Q1070X mutant mRNA and restored the expression of functional Q1070X mutant channels. The restoration of functional expression by antisense morpholino oligonucleotides was also observed in LQT2 frameshift mutations. Our findings suggest that inhibition of NMD by antisense morpholino oligonucleotides may be a potential therapeutic approach for some LQT2 patients carrying nonsense and frameshift mutations

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K+ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K+ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

向精神薬服用患者の突然死症例におけるカリウムイオンチャネルに関する分子生物学的解析：QT延長症候群関連遺伝子の多型が危険因子となり得るか？

Psychotropic drugs can pose the risk of acquired long QT syndrome (LQTS). Unexpected autopsy-negative sudden death in patients taking psychotropic drugs may be associated with prolonged QT intervals and life-threatening arrhythmias. We analyzed genes that encode for cardiac ion channels and potentially associated with LQTS, examining specifically the potassium channel genes KCNQ1 and KCNH2 in 10 cases of sudden death involving patients administered psychotropic medication in which autopsy findings identified no clear cause of death. We amplified and sequenced all exons of KCNQ1 and KCNH2, identifying G643S, missense polymorphism in KCNQ1, in 6 of the 10 cases. A study analysis indicated that only 11% of 381 healthy Japanese individuals carry this polymorphism. Reports of previous functional analyses indicate that the G643S polymorphism in the KCNQ1 potassium channel protein causes mild IKs channel dysfunction. Our present study suggests that administering psychotropic drug therapy to individuals carrying the G643S polymorphism may heighten the risk of prolonged QT intervals and life-threatening arrhythmias. Thus, screening for the G643S polymorphism before prescribing psychotropic drugs may help reduce the risk of unexpected sudden death2013博士（歯学）松本歯科大

Sudden death and cardiac arrest without phenotype: the utility of genetic testing.

Approximately 4% of sudden cardiac deaths are unexplained [the sudden arrhythmic death syndrome (SADS)], and up to 6-10% of survivors of cardiac arrest do not have an identifiable cardiac abnormality after comprehensive clinical evaluation [idiopathic ventricular fibrillation (IVF)]. Genetic testing may be able to play a role in diagnostics and can be targeted to an underlying phenotype present in family members following clinical evaluation. Alternatively, post-mortem genetic testing (the "molecular autopsy") may diagnose the underlying cause if a clearly pathogenic rare variant is found. Limitations include a modest yield, and the high probability of finding a variant of unknown significance (VUS) leading to a low signal-to-noise ratio. Next generation sequencing enables cost-efficient high throughput screening of a larger number of genes but at the expense of increased genetic noise. The yield from genetic testing is even lower in IVF in the absence of any suggestion of another phenotype in the index case or his/her family, and should be actively discouraged at this time. Future improvements in diagnostic utility include optimization of the use of variant-calling pipelines and shared databases as well as patient-specific models of disease to more accurately assign pathogenicity of variants. Studying "trios" of parents and the index case may better assess the yield of sporadic and recessive disease

Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

Ca2+/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca2+ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within CaV1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a “hinged lid–shield” mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a “shield” in CaV1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca2+ channelopathies involving S6 mutations

Facilitating arrhythmia simulation: the method of quantitative cellular automata modeling and parallel running

BACKGROUND: Many arrhythmias are triggered by abnormal electrical activity at the ionic channel and cell level, and then evolve spatio-temporally within the heart. To understand arrhythmias better and to diagnose them more precisely by their ECG waveforms, a whole-heart model is required to explore the association between the massively parallel activities at the channel/cell level and the integrative electrophysiological phenomena at organ level. METHODS: We have developed a method to build large-scale electrophysiological models by using extended cellular automata, and to run such models on a cluster of shared memory machines. We describe here the method, including the extension of a language-based cellular automaton to implement quantitative computing, the building of a whole-heart model with Visible Human Project data, the parallelization of the model on a cluster of shared memory computers with OpenMP and MPI hybrid programming, and a simulation algorithm that links cellular activity with the ECG. RESULTS: We demonstrate that electrical activities at channel, cell, and organ levels can be traced and captured conveniently in our extended cellular automaton system. Examples of some ECG waveforms simulated with a 2-D slice are given to support the ECG simulation algorithm. A performance evaluation of the 3-D model on a four-node cluster is also given. CONCLUSIONS: Quantitative multicellular modeling with extended cellular automata is a highly efficient and widely applicable method to weave experimental data at different levels into computational models. This process can be used to investigate complex and collective biological activities that can be described neither by their governing differentiation equations nor by discrete parallel computation. Transparent cluster computing is a convenient and effective method to make time-consuming simulation feasible. Arrhythmias, as a typical case, can be effectively simulated with the methods described

Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

Inherited human long-QT2 syndrome (LQTS) results from mutations in the gene encoding the HERG channel. Several LQT2-associated mutations have been mapped to the amino terminal cytoplasmic Per-Arnt-Sim (PAS) domain of the HERG1a channel subunit. Here we have characterized the trafficking properties of some LQT2-associated PAS domain mutants and analyzed rescue of the trafficking mutants by low temperature (27°C) or by the pore blocker drug E4031. We show that the LQT2-associated mutations in the PAS domain of the HERG channel display molecular properties that are distinct from the properties of LQT2-associated mutations in the trans-membrane region. Unlike the latter, many of the tested PAS domain LQT2-associated mutations do not result in trafficking deficiency of the channel. Moreover, the majority of the PAS domain mutations that cause trafficking deficiencies are not rescued by a pore blocking drug. We have also explored the in vitro folding stability properties of isolated mutant PAS domain proteins using a thermal unfolding fluorescence assay and a chemical unfolding assay

N-Terminal Arginines Modulate Plasma-Membrane Localization of Kv7.1/KCNE1 Channel Complexes

BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks)) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks) and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I(Ks) modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks) resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA') were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks). Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex