novel mutation of kcnq1 with severe long qt syndrome

Novel Mutation of KCNQ1 Potassium Channel at the Potassium Selectivity Filter in a Family with Severe Long QT Syndrome : 

Novel Mutation of KCNQ1 Potassium Channel at the Potassium Selectivity Filter in a Family with Severe Long QT Syndrome Authors: Taruna Ikrar 1, Haruo Hanawa 1, Hiroshi Watanabe 2, Shinsuke Okada 1, Yoshiyasu Aizawa 3 , Fumio Yamashita 1 , Satoru Komura 1 , Mahmoud M. Ramadan 1 , Masaomi Chinushi 1, Yoshifusa Aizawa 1 Affiliations: 1. Division of Cardiology, First Department of Internal Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2. Division of Clinical Pharmacology, Vanderbilt University School of Medicine, TN, USA. 3. Masonic Medical Research Laboratory, New York, USA

1. Introduction : 

1. Introduction LQTS is a cardiac disorder characterized by a prolonged QT interval, which predisposes to syncope, or sudden arrhythmic death. In this report, we investigated: -The function of K+ channel with a missense mutation of I313K at the Selectivity filter - Studied with details the structure of this channel.

2. Subjects and methods : 

2. Subjects and methods 2.1 Patient Three patients from the same Japanese family with a KCNQ1 mutation were selected for careful clinical and electrophysiological analysis. (after giving informed consent) Clinical evaluation: - Family history, - Clinical examination, -12-lead ECG, - Ambulatory ECG monitoring. The QT interval was evaluated by Bazett’s formula to identify prolonged QT or QTc interval.

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2.2 DNA isolation and mutation　analysis Genomic DNA was extracted from whole blood & isolated from leukocyte nuclei by conventional methods. For genetic screening, we used a direct DNA sequencing method for KCNQ1, KCNH2, SCN5A, KCNE1 & KCNE2 genes.

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2.3 Three-dimensional model analysis of KCNQ1 3-demensional computational models representing the position of selectivity filter were generated by homology models. As TIGYG sequence at the P-loop region (mutations of I313K ) was constructed into four repeat models of the pore region.

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2.4 Culture and transfection of COS7 cells COS-7 cell lines were cultured in DMEM supplemented with: - 10% FBS + 1% P/S antibiotic, - humidified 5% CO2 at 37oC, - cultured cells in dishes 60 mm. Transiently transfected with various plasmids by the Fugene-6 method. -1.0 or 0.5µg of WT-KCNQ1, -1.0 µg mutant-KCNQ1, - 0.5µg WT-KCNQ1+0.5µg mutant-KCNQ1.

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2.5 Electrophysiological experiments The whole-cell patch-clamp recording was performed using an Axoscope-9.2 patch-clamp amplifier 24-48 h after transfection. All experiments were conducted -At room temperature -The pipette resistance (4-6 MΩ ) -Internal solutions (in mM) -Normal Tyrodes solution (in mM) -With the Protocol (Slide no.13)

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2.6 Confocal imaging YFP-KCNQ1(WT) & CFP-KCNQ1(mutant) were transfected to COS-7 cells. (Using 0.25% trypsin for 60s to minimize the cell surface). Confocal imaging's of fluorescence were visualized using a FV500 confocal laser scanning microscope. (To excite the YFP (514 nm) & the CFP (458 nm))

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2.7 Statistical analysis Data analyses were performed with SPSS. (version 13) Comparison of means of grouping variable by Student’s t-test & one-way ANOVA. The significance level of 0.05 was used for all analyses.

3. Results 3.1 Clinical phenotypes : 

3. Results 3.1 Clinical phenotypes

Transthoracic 12-lead ECG : 

Transthoracic 12-lead ECG

3.2 The Site & Type of Mutation : 

3.2 The Site & Type of Mutation

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3.3 Representative IKs & Mutant Current

Current-Voltage Relationship : 

Current-Voltage Relationship

The peak and tail current density of IKs. In all groups, DNA was co-transfected with KCNE1. Data are presented as mean ±SEM. WT=wild type. * p<0.001 vs. WT 1 µg; † p<0.05 vs. WT 0.5 µg. : 

The peak and tail current density of IKs. In all groups, DNA was co-transfected with KCNE1. Data are presented as mean ±SEM. WT=wild type. * p<0.001 vs. WT 1 µg; † p<0.05 vs. WT 0.5 µg.

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3.4 Confocal imaging WT 1.0 μg/μl Mutant I313K μg/μl

4. Discussion : 

4. Discussion 4.1 De novo mutation: I313K The expression study of I313K channel showed a loss of KCNQ1 channel function; When co-expressed with the WT, a dominant negative suppression was confirmed. The mutation was not associated with a trafficking defect as reported earlier

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4.2 Site of mutation The site of mutation of I313K corresponded to the site of K+ selectivity located and at the center of K+ channel pore It is thought to play an essential role in selectivity and conductivity Mutations in the pore of the KCNQ1 channel have been thought to disrupt K+ transport

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4.3 Mechanism of loss of function of mutant-KCNQ1 We would like to have hypothesis. 1).The altered charge would affect the selectivity filter via impacting the transport of K+ through the channel pore. (When the amino acid residue is substituted from the neutral Ile to the positively-charged Lys) 2).I313K may cause an alteration in the pore size and hinder the passage of K+ ions.

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4.4 Conclusions Novel mutation of I313K-KCNQ1 was confirmed in a family of LQT1 with repetitive attacks of syncope. The mutant K+ channel showed a loss of function with a dominant-negative effect when co-expressed with the WT one.

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Thank You