Functions of calcium-activated ion channels in the heart

心脏中钙激活离子通道的功能

• 批准号: 6919301
• 负责人: Nipavan Chiamvimonvat
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919301
• 关键词: Adenoviridae; RNase protection assay; calcium channel; calcium flux; cardiac myocytes; clinical research; electrophysiology; gene expression; gene targeting; genetically modified animals; human tissue; immunofluorescence technique; in situ hybridization; laboratory mouse; polymerase chain reaction; potassium channel; protein isoforms; protein localization; protein structure function; single cell analysis; transfection /expression vector; voltage gated channel; western blottings

DESCRIPTION (provided by applicant): Recently, we have identified a novel Ca2+-activated K+ current (Ik,ca) in human and mouse atrial myocytes that contributes markedly towards the late phase of the atrial repolarization. The importance of the currents is underpinned by the fact that the late phase of the cardiac action potential is susceptible to aberrant excitation e.g. early after depolarization and arrhythmias. The channel is differentially expressed in atrial vs. ventricular myocytes. Finally, we have preliminary data, which suggest that the channel may co-localize with specific isoform of the voltage-gated Ca 2+ channels (VGCC). We hypothesize that the regional specific expression of the Ca 2+-activated K + channels (Kca) coordinates distinct functional roles in atria vs. ventricles. Specifically, we hypothesize that the channels are important in the repolarization process in atria, and alteration of the current can precipitate profound changes in the shape and duration of cardiac action potentials and possibly atrial arrhythmias. The following Specific Aims are proposed: 1. Functional identification of Ik, ca, in atrial and ventricular myocytes. We will determine the detail biophysical and pharmacologic properties of Ik,ca as well as quantifying the whole-cell current density in human and mouse myocytes to define the specific isoforms of Ik,ca, expressed in atria and ventricles. To conclusively establish that the activity of Ik,ca contributes towards the repolarization process in the heart, we will employ in-vitro adenoviral-mediated gene transfer to single isolated human and mouse cardiac myocytes using dominant-negative (DN) functional knockout strategies. 2. Biochemical identification of the specific isoform encoding the cardiac Ik,ca, The functional study will be corroborated using Western blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence techniques and in-situ hybridization. 3. Co-localization of cardiac Kca, channel with specific isoform of VGCC. To test the hypothesis that the channel colocalizes with specific isoform of VGCC, immunofluorescence techniques using double labeling will be used. In addition, we will use a gene targeted eqD Ca2+ channel null mutant mice as a tool to specifically examine the co-localization of the Kca channel with the different isoforms of VGCC. 4. Determination of the functional roles of Ik,ca in whole animals. In-vivo adenoviral-mediated gene transfer to overexpress the functional Kca channel or engineered DN constructs to mice and in vivo electrophysiologic studies will be used to assess the functional roles of the channel in whole animals. Complimentary experiments using transgenic mice of specific SK channel will be performed. Transgenic mice with conditional expression for each of the three SK channel genes have already been constructed. These mouse models will be used to study the functional roles of the Kca channel in the heart. ECG, telemetric ECG recordings and electrophysiologic studies will be performed. Single isolated myocytes will be studied using patch-clamp techniques as in Aim 1.

描述（由申请人提供）：最近，我们在人和小鼠心房心肌细胞中确定了一种新型的Ca2+活化的K+电流（IK，CA），该肌细胞对心房复置的后期显着贡献。电流的重要性是基于以下事实：心脏作用电位的后期容易受到异常激发的影响，例如去极化和心律不齐后的早期。该通道在心房与心室肌细胞中差异表达。最后，我们有初步数据，这表明该通道可以与电压门控Ca 2+通道（VGCC）的特定同工型共定位。我们假设Ca 2 +活化的K +通道（KCA）的区域特异性表达在心房与心室中的不同功能作用。具体而言，我们假设通道在心房的复极化过程中很重要，而电流的改变会导致心脏作用电位的形状和持续时间的深刻变化以及可能的心律失常。提出了以下具体目标： 1。在心房和心室心肌细胞中IK，CA的功能鉴定。我们将确定IK，CA的细节生物物理和药理学特性，并量化人和小鼠肌细胞中的全细胞电流密度，以定义在心房和心室中表达的IK，CA的特定同工型。为了最终确定IK的活性，CA有助于心脏中的复极化过程，我们将使用优势 - 阴性（DN）功能敲除策略来利用腺病毒介导的基因转移到单个孤立的人和小鼠心肌细胞。 2。编码心脏IK，CA的特定同工型的生化鉴定，功能研究将使用蛋白质印迹分析，核糖核酸酶保护测定法（RPA），逆转录酶 - 聚合酶链链（RT-PCR），免疫荧光技术和免疫杂化技术和免疫杂化技术和含situ杂交杂交。 3。心脏KCA的共定位，通道，具有特定的VGCC同工型。为了测试通道与VGCC的特定同工型共定位的假设，将使用使用双重标记的免疫荧光技术。此外，我们将使用具有基因的EQD CA2+通道null突变小鼠作为一种工具，以专门检查具有VGCC的不同同工型KCA通道的共定位。 4。确定IK，CA在整个动物中的功能作用。体内腺病毒介导的基因转移以过表达功能性KCA通道或工程DN构建体向小鼠和体内电生理学研究用于评估该通道在整个动物中的功能作用。将进行使用特定SK通道的转基因小鼠的免费实验。已经构建了三个SK通道基因中每个SK通道基因的条件表达的转基因小鼠。这些小鼠模型将用于研究KCA通道在心脏中的功能作用。将进行心电图，遥测心电图记录和电生理研究。单个隔离的肌细胞将使用AIM 1中的贴片钳技术研究。