Potassium channels, membrane potential, and CHD

钾通道、膜电位和 CHD

• 批准号: 10439505
• 负责人: Mustafa K Khokha
• 金额: $ 55.37万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10439505
• 关键词: Affect; Biochemical; Calcium; Calcium Channel; Calcium Signaling; Candidate Disease Gene; Cardiac; Cardiac development; Cell membrane; Cell model; Cells; Chemicals; Child; Child Health; Closure by clamp; Collaborations; Congenital Abnormality; Data; Defect; Dependence; Development; Disease; Ectoderm; Ectoderm Cell; Electrophysiology (science); Embryo; Europe; Exhibits; Family member; Fetus; Fluorescence; Gap Junctions; Gastrula; Genes; Genetic; Genetic Transcription; Genomic approach; Genomics; Germ Cells; Germ Layers; Heart; Image; Individual; Infant Mortality; Ion Channel; Kinetics; Lead; Left; Ligands; Measurement; Measures; Membrane; Membrane Potentials; Mesoderm; Mesoderm Cell; Methods; Modeling; Molecular; Monitor; Morbidity - disease rate; Neurons; Optics; Paraxial Mesoderm; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Play; Potassium; Potassium Channel; Property; Reading; Regulation; Role; Series; Signal Pathway; Signal Transduction; Situs Inversus; Structure; Testing; Transducers; Voltage-Gated Potassium Channel; Work; Xenopus; base; blastocyst; blastomere structure; cardiogenesis; congenital heart disorder; electrical property; embryo cell; exome sequencing; experimental study; gastrulation; genetic analysis; genetic manipulation; heart function; infant death; inhibitor; intercellular communication; mortality; nodal myocyte; pluripotency; receptor; structural heart disease; transcription factor; transcriptome sequencing; voltage; voltage clamp

Project Summary Congenital heart disease (CHD) leads to severe morbidity and mortality to children in the US and worldwide. Despite this impact on child health, we simply do not understand the genetic causes of CHD. Recently, trio based whole exome sequencing has identified a class of voltage-gated potassium channels (multiple KCNH family members) as candidates for CHD and, specifically heterotaxy, a disorder of left-right (LR) patterning that has a severe effect on cardiac function. However, a molecular role connecting potassium channels to structural heart disease and heterotaxy is unprecedented. We propose, and our preliminary data support, that KCNH6 defines a new paradigm for cell signaling in early embryonic cells. Our data support an electrophysiological model where specific germ layers fates (paraxial mesoderm and ectoderm) are dependent on an ion channel network. Our overarching hypothesis is that K+ channels define electrical membrane potential and regulate voltage gated Ca2+ channels that establish an exit from pluripotency towards specific cell fates, gastrulation, and LR patterning providing a plausible mechanism for our patients with Htx and CHD. Our electrophysiological pathway then integrates with biochemical signaling pathways that define specific cell fates in the embryo. In this proposal revision, we will focus on KCNH6 to see if gene depletion leads to LR patterning defects in Xenopus. In addition, we will test where in the LR patterning cascade, KCNH6 plays a role. Then, using a series of judiciously chosen chemical and ionic perturbations, we will test if membrane potential is indeed essential for pluripotency, cell fate, and calcium regulation. Due to the novelty of this project, we will also perform unbiased genomics (RNAseq) for discovery of transcriptional targets of  Vm. Finally, we will measure electrical properties electrophysiologically using both whole-cell voltage clamp and intracellular recordings and determine the various currents that define membrane potential in early germ cells. A major strength of our proposal is our expertise; we have forged a collaboration between Xenopus developmental biologists and electrophysiologists that will allow us to rigorously investigate membrane potential as an embryonic patterning mechanism.

项目摘要 先天性心脏病（CHD）导致美国和全球儿童的严重发病率和死亡率。 尽管对儿童健康有影响，但我们根本不了解CHD的遗传原因。最近，三重奏 基于整个外显子组测序已经确定了一类电压门控钾通道（多个KCNH 家庭成员）作为冠心病的候选人，尤其是异性疾病，是一种左右（LR）的疾病 对心脏功能有严重影响。但是，将钾通道连接到结构的分子角色 心脏病和杂质是前所未有的。 我们提出的以及我们的初步数据支持KCNH6定义了用于细胞信号的新范式 早期胚胎细胞。我们的数据支持特定细菌层命运的电生理模型 （近期中胚层和外胚层）取决于离子通道网络。我们的总体假设是 K+通道定义了电膜电位，并调节建立的电压门控Ca2+通道 从多能性的出口向特定的细胞命运，过口和LR模式的出口提供了合理的 我们患有HTX和CHD患者的机制。然后，我们的电生理途径与 定义胚胎中特定细胞命运的生化信号通路。 在此提案修订中，我们将专注于KCNH6，以查看基因耗竭是否导致LR模式缺陷 Xenopus。此外，我们将测试在LR模式级联反应中，KCNH6起作用。然后，使用一个 一系列明智选择的化学和离子扰动，我们将测试膜电位是否确实是 多能性，细胞命运和钙调节所必需的。由于这个项目的新颖性，我们也将 执行无偏基因组学（RNASEQ）来发现VM的转录靶标。最后，我们会的 使用全细胞电压夹和细胞内测量电生理的电特性 记录并确定在早期生殖细胞中定义膜电位的各种电流。 我们建议的主要优势是我们的专业知识。我们忘记了Xenopus之间的合作 正在开发生物学家和电生理学家，这将使我们能够严格研究膜 作为胚胎图案机制的潜力。