logging in or signing up Insight into Gene Therapy For Severe Long QT Phenotype TarunaIkrar Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: Embed: Flash iPad Dynamic Copy Does not support media & animations Automatically changes to Flash or non-Flash embed WordPress Embed Customize Embed URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 152 Category: Science & Tech.. License: All Rights Reserved Like it (1) Dislike it (0) Added: July 20, 2010 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript Functional Impact of Altered the Amino Acid Residue atthe Selectivity Filter of KCNQ1 Potassium Channel: Insight into Gene Therapy For Severe Long QT PhenotypeBy:Ikrar T, Aizawa Y, Hanawa H, Furushima H : Functional Impact of Altered the Amino Acid Residue atthe Selectivity Filter of KCNQ1 Potassium Channel: Insight into Gene Therapy For Severe Long QT PhenotypeBy:Ikrar T, Aizawa Y, Hanawa H, Furushima H 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 mutation at the site of I313 residue of KCNQ1 selectivity filter -The structure of these mutants channel in detail. Cardiac action potential. upstroke of rapid depolarization (phase 0), which is mostly the result of rapid inflow of sodium; rapid repolarization after the peak (phase 1) is primarily the result of an outward repolarizing chloride current; the plateau (phase 2); rapid repolarization after the plateau (phase 3), predominantly caused by outward potassium current (IKs); and the period between the maximum negativity (maximum diastolic potential) and the upstroke of the next action potential (phase 4) the balance between slow inward sodium and outward potassium current. : Cardiac action potential. upstroke of rapid depolarization (phase 0), which is mostly the result of rapid inflow of sodium; rapid repolarization after the peak (phase 1) is primarily the result of an outward repolarizing chloride current; the plateau (phase 2); rapid repolarization after the plateau (phase 3), predominantly caused by outward potassium current (IKs); and the period between the maximum negativity (maximum diastolic potential) and the upstroke of the next action potential (phase 4) the balance between slow inward sodium and outward potassium current. 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 6: Molecular Biology and Electrophysiology Genomic DNA was isolated from leukocyte nuclei by conventional methods. For Mutagenesis, we used QuickChange site-directed mutagenesis kits to create the new mutant vectors; as 313K, 313G, 313V, 313E. Transiently transfected with various plasmids by the Fugene-6 method. The whole-cell patch-clamp recording was performed using an Axoscope-9.2 patch-clamp amplifier 24-48 h after transfection. Slide 7: Confocal imaging and 3-dimensional models Confocal imaging's of fluorescence were visualized using a FV500 confocal laser scanning microscope. [To excite the YFP (514 nm) & the CFP (458 nm)] 3-demensional computational models representing the position of selectivity filter were generated by homology models. 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 9: 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 11: Electrophysiological studies Representative currents from whole-cell patch-clamp experiments performed on COS-7 cells. Slide 12: 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 of the virtual mutants channel vectors : Current of the virtual mutants channel vectors Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT or mutant vectors, as present of KCNE1 Co-expression of WT + Virtual Mutant Vectors : Co-expression of WT + Virtual Mutant Vectors Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT + mutant vectors, as present of KCNE1 Slide 18: 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. Three dimensional models : Three dimensional models The site of I313K mutation is positioned at the center of the channel pore Slide 20: Site of mutation The site of mutation at I313 residue 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 21: Mechanism of loss or Gain of function the 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 Ile to the positively (I313K) or negatively-charged (I313E)]. The changed the size of amino acid residue may cause an alteration in the pore size, will hinder the passage or make augmented of K+ ions. [When the amino acid residue is substituted from the normal Ile to homology size (I313V) or the smallest (I313G)]. Gene Therapy Perspective : Gene Therapy Perspective These new finding can enrich the studies on channel properties and functions. As new perspective related to the strategies for diagnostic and gene specific risk stratification Then, for gene therapy directed to in hereditary diseases of LQTS Slide 24: Conclusions Double-point mutation of I313K was confirmed in a family of LQT1 with repetitive attacks of syncope. Charged residues (I313K, I313E) at the pore center of KCNQ1 resulted in a loss of K+ channel function and a dominant-negative pattern when co-transfected with the wild type channel. The neutral residues (I313G, I313V) showed zero or a small reduciton of the K+ current and some mutants might show a gain-of-function (may become SQTS) when co-expressed with the wild type channel. Slide 25: Thank You You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
Insight into Gene Therapy For Severe Long QT Phenotype TarunaIkrar Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: Embed: Flash iPad Dynamic Copy Does not support media & animations Automatically changes to Flash or non-Flash embed WordPress Embed Customize Embed URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 152 Category: Science & Tech.. License: All Rights Reserved Like it (1) Dislike it (0) Added: July 20, 2010 This Presentation is Public Favorites: 0 Presentation Description No description available. Comments Posting comment... Premium member Presentation Transcript Functional Impact of Altered the Amino Acid Residue atthe Selectivity Filter of KCNQ1 Potassium Channel: Insight into Gene Therapy For Severe Long QT PhenotypeBy:Ikrar T, Aizawa Y, Hanawa H, Furushima H : Functional Impact of Altered the Amino Acid Residue atthe Selectivity Filter of KCNQ1 Potassium Channel: Insight into Gene Therapy For Severe Long QT PhenotypeBy:Ikrar T, Aizawa Y, Hanawa H, Furushima H 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 mutation at the site of I313 residue of KCNQ1 selectivity filter -The structure of these mutants channel in detail. Cardiac action potential. upstroke of rapid depolarization (phase 0), which is mostly the result of rapid inflow of sodium; rapid repolarization after the peak (phase 1) is primarily the result of an outward repolarizing chloride current; the plateau (phase 2); rapid repolarization after the plateau (phase 3), predominantly caused by outward potassium current (IKs); and the period between the maximum negativity (maximum diastolic potential) and the upstroke of the next action potential (phase 4) the balance between slow inward sodium and outward potassium current. : Cardiac action potential. upstroke of rapid depolarization (phase 0), which is mostly the result of rapid inflow of sodium; rapid repolarization after the peak (phase 1) is primarily the result of an outward repolarizing chloride current; the plateau (phase 2); rapid repolarization after the plateau (phase 3), predominantly caused by outward potassium current (IKs); and the period between the maximum negativity (maximum diastolic potential) and the upstroke of the next action potential (phase 4) the balance between slow inward sodium and outward potassium current. 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 6: Molecular Biology and Electrophysiology Genomic DNA was isolated from leukocyte nuclei by conventional methods. For Mutagenesis, we used QuickChange site-directed mutagenesis kits to create the new mutant vectors; as 313K, 313G, 313V, 313E. Transiently transfected with various plasmids by the Fugene-6 method. The whole-cell patch-clamp recording was performed using an Axoscope-9.2 patch-clamp amplifier 24-48 h after transfection. Slide 7: Confocal imaging and 3-dimensional models Confocal imaging's of fluorescence were visualized using a FV500 confocal laser scanning microscope. [To excite the YFP (514 nm) & the CFP (458 nm)] 3-demensional computational models representing the position of selectivity filter were generated by homology models. 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 9: 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 11: Electrophysiological studies Representative currents from whole-cell patch-clamp experiments performed on COS-7 cells. Slide 12: 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 of the virtual mutants channel vectors : Current of the virtual mutants channel vectors Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT or mutant vectors, as present of KCNE1 Co-expression of WT + Virtual Mutant Vectors : Co-expression of WT + Virtual Mutant Vectors Current-voltage relationship from whole-cell patch-clamp experiments , transfected with WT + mutant vectors, as present of KCNE1 Slide 18: 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. Three dimensional models : Three dimensional models The site of I313K mutation is positioned at the center of the channel pore Slide 20: Site of mutation The site of mutation at I313 residue 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 21: Mechanism of loss or Gain of function the 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 Ile to the positively (I313K) or negatively-charged (I313E)]. The changed the size of amino acid residue may cause an alteration in the pore size, will hinder the passage or make augmented of K+ ions. [When the amino acid residue is substituted from the normal Ile to homology size (I313V) or the smallest (I313G)]. Gene Therapy Perspective : Gene Therapy Perspective These new finding can enrich the studies on channel properties and functions. As new perspective related to the strategies for diagnostic and gene specific risk stratification Then, for gene therapy directed to in hereditary diseases of LQTS Slide 24: Conclusions Double-point mutation of I313K was confirmed in a family of LQT1 with repetitive attacks of syncope. Charged residues (I313K, I313E) at the pore center of KCNQ1 resulted in a loss of K+ channel function and a dominant-negative pattern when co-transfected with the wild type channel. The neutral residues (I313G, I313V) showed zero or a small reduciton of the K+ current and some mutants might show a gain-of-function (may become SQTS) when co-expressed with the wild type channel. Slide 25: Thank You