Severe Long QT PhenotypesSevere Long QT Phenotypes Associated with Novel Associated with Novel Mutation of I313K at the Centre of KCNQ1 Potassium Mutation of I313K at the Centre of KCNQ1 Potassium

Channel PoreChannel Pore

Authors:Authors:Taruna IkrarTaruna Ikrar

Division of Multidisciplinary of Neuroscience, School of Division of Multidisciplinary of Neuroscience, School of Medicine, Universality of California, Irvine, CA 92617, USAMedicine, Universality of California, Irvine, CA 92617, USA

Division of Cardiology, First Department of Internal Medicine, Division of Cardiology, First Department of Internal Medicine, School of Medical Sciences, Niigata, JapanSchool of Medical Sciences, Niigata, Japan

1. Background1. Background

LQTS is a cardiac disorder characterized by a prolonged LQTS is a cardiac disorder characterized by a prolonged QT interval,QT interval, which predisposes to which predisposes to syncope, and sudden syncope, and sudden arrhythmic death.arrhythmic death.

In this report, we investigated: In this report, we investigated: -The functional consequences effect of K-The functional consequences effect of K++ channel with channel with a missense mutation of I313K at the selectivity filter a missense mutation of I313K at the selectivity filter -The structure of this mutant channel in detail.-The structure of this mutant channel in detail.

Schematic presentation of KCNQ1 Schematic presentation of KCNQ1 Potassium channelPotassium channel

2. Subjects and methods2. Subjects and methods PatientPatient

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

Clinical evaluation: - Family history,Clinical evaluation: - Family history, - Clinical examination, - Clinical examination, - 12-lead ECG, - 12-lead ECG, - Ambulatory ECG monitoring.- Ambulatory ECG monitoring.

The QT interval was evaluated by BazettThe QT interval was evaluated by Bazett ’’s formula to s formula to identify prolonged QT (or QTc) interval.identify prolonged QT (or QTc) interval.

DNA isolation, mutationDNA isolation, mutation 　　 analysis, and analysis, and

MutagenesisMutagenesis

Genomic DNA was extracted from whole blood & isolated Genomic DNA was extracted from whole blood & isolated from leukocyte nuclei by conventional methods. from leukocyte nuclei by conventional methods.

For genetic screening, we used a direct DNA sequencing For genetic screening, we used a direct DNA sequencing method for KCNQ1, KCNH2, SCN5A, KCNE1 & KCNE2 method for KCNQ1, KCNH2, SCN5A, KCNE1 & KCNE2 genes. genes.

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

Three-dimensional model analysis of Three-dimensional model analysis of KCNQ1KCNQ1

3-demensional computational models representing the 3-demensional computational models representing the position of selectivity position of selectivity filter filter were generated by were generated by homology models. homology models.

TIGYG sequence at the P-loop region the site of I313K TIGYG sequence at the P-loop region the site of I313K mutation was constructed according to KcsA models of mutation was constructed according to KcsA models of the pore region. the pore region.

Three Dimensional ModelsThree Dimensional Models

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

Culture and transfection of COS7 cells Culture and transfection of COS7 cells

COS-7 cells line were cultured in DMEM medium, COS-7 cells line were cultured in DMEM medium, supplemented with: supplemented with:

- 10% FBS + 1% P/S antibiotic, - 10% FBS + 1% P/S antibiotic,

- humidified 5% CO- humidified 5% CO22 at 37 at 37ooC. C.

Transiently transfected with various plasmids by the Transiently transfected with various plasmids by the Fugene-6 method. Fugene-6 method.

-1.0 or 0.5 -1.0 or 0.5 µµg of WT-KCNQ1, g of WT-KCNQ1, -1.0 -1.0 µµg mutant-KCNQ1, g mutant-KCNQ1, - 0.5 - 0.5 µµg WT-KCNQ1+0.5 g WT-KCNQ1+0.5 µµg mutant-KCNQ1g mutant-KCNQ1..

Electrophysiological experimentsElectrophysiological experiments

The whole-cell patch-clamp recording was performed The whole-cell patch-clamp recording was performed using an Axoscope-9.2 patch-clamp amplifier 24-48 h using an Axoscope-9.2 patch-clamp amplifier 24-48 h after transfection. after transfection.

All experiments were conductedAll experiments were conducted -At room temperature-At room temperature

-With a pipette resistance (4-6 MΩ )-With a pipette resistance (4-6 MΩ )-With standard internal & bath solutions (in mM) -With standard internal & bath solutions (in mM)

-With a definite stimulant Protocol (Slide no.13)-With a definite stimulant Protocol (Slide no.13)

3. Results3. Results Clinical phenotypesClinical phenotypes

ECG obtained from the affected proband (P1), a 64-year old Japanese ECG obtained from the affected proband (P1), a 64-year old Japanese

woman from a family with a compound I313K mutation in KCNQ1woman from a family with a compound I313K mutation in KCNQ1

The pedigree of a family with I313K mutation. Assigned as males The pedigree of a family with I313K mutation. Assigned as males (squares) or females (circles); affected (black) or unaffected (white); (squares) or females (circles); affected (black) or unaffected (white);

reported LQT phenotype (gray) or unreported (slashes). reported LQT phenotype (gray) or unreported (slashes).

Coding region sequence around the KCNQ1 double point mutation from Coding region sequence around the KCNQ1 double point mutation from

genomic DNA isolated from the three proband.genomic DNA isolated from the three proband.

Electrophysiological studiesElectrophysiological studies

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

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

Peak Current Tail CurrentPeak Current Tail Current

Bar graphs representing the current densities obtained from the peak Bar graphs representing the current densities obtained from the peak currents (+80 mV) or tail current (-40 mV) of cells transfected with currents (+80 mV) or tail current (-40 mV) of cells transfected with the indicated amounts of pIRES2-EGFP-KCNQ1, pIRES2-EGFP-the indicated amounts of pIRES2-EGFP-KCNQ1, pIRES2-EGFP-

I313K, or KCNE1 expression plasmidsI313K, or KCNE1 expression plasmids..

Current Tracing without KCNE1Current Tracing without KCNE1

Current Tracing of single plasmid expressionCurrent Tracing of single plasmid expression

Current-voltage relationship from whole-cell patch-clamp experiments , Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT or mutant vectors, as present of KCNE1transfected with WT or mutant vectors, as present of KCNE1

Co-expression of WT + Mutant VectorsCo-expression of WT + Mutant Vectors

Current-voltage relationship from whole-cell patch-clamp experiments , Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT + mutant vectors, as present of KCNE1transfected with WT + mutant vectors, as present of KCNE1

Confocal ImagingConfocal Imaging WT 1.0 μg Mutant I313K μgWT 1.0 μg Mutant I313K μg

Subcellular localization of WT- and mutant-KCNQ1 in COS-7 cells. Subcellular localization of WT- and mutant-KCNQ1 in COS-7 cells. Confocal microscopic images of yellow fluorescent protein (YFP)-Confocal microscopic images of yellow fluorescent protein (YFP)-tagged pEGFP-N1-KCNQ1 and cyan fluorescent protein (CFP)-tagged pEGFP-N1-KCNQ1 and cyan fluorescent protein (CFP)-

tagged pEGFP-N1-I313K expression plasmids are shown.tagged pEGFP-N1-I313K expression plasmids are shown.

Double point mutation: I313KDouble point mutation: I313K

The expression of I313K mutant channel showed a The expression of I313K mutant channel showed a loss ofloss of KCNQ1 KCNQ1 channel channel functionfunction; ;

When co-expressed with the When co-expressed with the WTWT, a dominant , a dominant negative suppression was confirmed. negative suppression was confirmed.

The mutation was not associated with a trafficking The mutation was not associated with a trafficking defect. defect.

Site of mutationSite of mutation

The site of mutation of I313K corresponded to the The site of mutation of I313K corresponded to the site of Ksite of K++ selectivity filter located at the center of K selectivity filter located at the center of K++ channel pchannel pore.ore.

It is thought to play an essential role It is thought to play an essential role inin selectivity and selectivity and conductivity. conductivity.

Mutations in the pore of the KCNQ1 potassium Mutations in the pore of the KCNQ1 potassium

channel seem to disrupt Kchannel seem to disrupt K++ transport. transport.

Mechanism of loss of function of mutant-Mechanism of loss of function of mutant-KCNQ1KCNQ1

The altered charge would affect the selectivity The altered charge would affect the selectivity filter via impacting the transport of K+ through filter via impacting the transport of K+ through the channel pore. the channel pore. (When the amino acid residue is substituted from the neutral (When the amino acid residue is substituted from the neutral

Isoleucine to the positively-charged Lysine Isoleucine to the positively-charged Lysine I313KI313K, or to the , or to the negative-charged Glutamic-acid negative-charged Glutamic-acid I313EI313E ) )

I313K may cause an alteration in the pore size I313K may cause an alteration in the pore size and hinder the passage of Kand hinder the passage of K++ ions. ions.

Possibility of Gain of FunctionPossibility of Gain of Function

If the amino acid residue is substituted from the If the amino acid residue is substituted from the neutral neutral IsoleucineIsoleucine to the neutral-small to the neutral-small GlycineGlycine (I313G),(I313G), the pore size will get much bigger. the pore size will get much bigger.

This condition influences the selectivity and increase This condition influences the selectivity and increase conductivity of Kconductivity of K++ current, leading to gain of function current, leading to gain of function

of Kof K+ + channel.channel.

ConclusionsConclusions

Novel mutation of I313K-KCNQ1Novel mutation of I313K-KCNQ1 was confirmed in was confirmed in a family of LQT1 with repetitive attacks of syncope. a family of LQT1 with repetitive attacks of syncope.

The mutant The mutant KK++ channel showed channel showed a loss of function a loss of function with awith a dominant-negative effect when co-expressed dominant-negative effect when co-expressed with the with the WT-KCNQ1WT-KCNQ1. .

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

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