severe long qt phenotypes associated with mutation of k+ channel

Severe Long QT Phenotypes Associated with Novel Mutation of I313K at the Centre of KCNQ1 Potassium Channel Pore : 

Severe Long QT Phenotypes Associated with Novel Mutation of I313K at the Centre of KCNQ1 Potassium Channel Pore Authors: Taruna Ikrar Division of Cardiology, First Department of Internal Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

1. Background : 

1. Background LQTS is a cardiac disorder characterized by a prolonged QT interval, which predisposes to syncope, and sudden arrhythmic death. In this report, we investigated: -The functional consequences effect of K+ channel with a missense mutation of I313K at the selectivity filter -The structure of this mutant channel in detail.

Schematic presentation of KCNQ1 Potassium channel : 

Schematic presentation of KCNQ1 Potassium channel

2. Subjects and methods : 

2. Subjects and methods Patient Three patients from the same Japanese family with a KCNQ1 mutation were selected for thorough clinical and electrophysiological analysis. 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.

Slide 5: 

DNA isolation, mutation　analysis, and Mutagenesis 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. For Mutagenesis, we used QuickChange site-directed mutagenesis kits to create the new mutant vectors; as 313G, 313V, 313E.

Slide 6: 

Three-dimensional model analysis of KCNQ1 3-demensional computational models representing the position of selectivity filter were generated by homology models. TIGYG sequence at the P-loop region the site of I313K mutation was constructed according to KcsA models of the pore region.

Three Dimensional Models : 

Three Dimensional Models The site of I313K mutation is positioned at the center of the channel pore

Slide 8: 

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

Slide 9: 

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 -With a pipette resistance (4-6 MΩ ) -With standard internal & bath solutions (in mM) -With a definite stimulant Protocol (Slide no.13)

3. Results : 

3. Results Clinical phenotypes ECG obtained from the affected proband (P1), a 64-year old Japanese woman from a family with a compound I313K mutation in KCNQ1

Slide 11: 

The pedigree of a family with I313K mutation. Assigned as males (squares) or females (circles); affected (black) or unaffected (white); reported LQT phenotype (gray) or unreported (slashes).

Coding region sequence around the KCNQ1 double point mutation from genomic DNA isolated from the three proband. : 

Coding region sequence around the KCNQ1 double point mutation from genomic DNA isolated from the three proband.

Slide 13: 

Electrophysiological studies Representative currents from whole-cell patch-clamp experiments performed on COS-7 cells.

Slide 14: 

Current-voltage relationship and current densities from whole-cell patch-clamp experiments performed on COS-7 cells

Peak Current Tail Current : 

Peak Current Tail Current Bar graphs representing the current densities obtained from the peak currents (+80 mV) or tail current (-40 mV) of cells transfected with the indicated amounts of pIRES2-EGFP-KCNQ1, pIRES2-EGFP-I313K, or KCNE1 expression plasmids.

Current Tracing without KCNE1 : 

Current Tracing without KCNE1

Current Tracing of single plasmid expression : 

Current Tracing of single plasmid expression Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT or mutant vectors, as present of KCNE1

Co-expression of WT + Mutant Vectors : 

Co-expression of WT + Mutant Vectors Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT + mutant vectors, as present of KCNE1

Slide 19: 

Confocal Imaging WT 1.0 μg Mutant I313K μg Subcellular localization of WT- and mutant-KCNQ1 in COS-7 cells. Confocal microscopic images of yellow fluorescent protein (YFP)-tagged pEGFP-N1-KCNQ1 and cyan fluorescent protein (CFP)-tagged pEGFP-N1-I313K expression plasmids are shown.

Slide 20: 

Double point mutation: I313K The expression of I313K mutant 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.

Slide 21: 

Site of mutation The site of mutation of I313K corresponded to the site of K+ selectivity filter located 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 potassium channel seem to disrupt K+ transport.

Slide 22: 

Mechanism of loss of function of mutant-KCNQ1 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 Isoleucine to the positively-charged Lysine I313K, or to the negative-charged Glutamic-acid I313E ) I313K may cause an alteration in the pore size and hinder the passage of K+ ions.

Possibility of Gain of Function : 

Possibility of Gain of Function If the amino acid residue is substituted from the neutral Isoleucine to the neutral-small Glycine (I313G), the pore size will get much bigger. This condition influences the selectivity and increase conductivity of K+ current, leading to gain of function of K+ channel.

Slide 24: 

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-KCNQ1. The importance of the charge and/or the pore size of the mutant K+ channel were suggested.

Slide 25: 

Thank You