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.

项目概要 先天性心脏病 (CHD) 导致美国和全世界儿童严重发病和死亡。 尽管这对儿童健康有影响，但我们根本不了解冠心病的遗传原因。 基于全外显子组测序已经鉴定出一类电压门控钾通道（多个 KCNH 家庭成员）作为先心病（CHD）的候选者，特别是异位症，一种左右（LR）模式障碍， 然而，将钾通道与结构连接起来的分子作用。 心脏病和异质性是前所未有的。 我们建议，并且我们的初步数据支持，KCNH6 定义了细胞信号传导的新范式 我们的数据支持特定胚层命运的电生理模型。 （轴旁中胚层和外胚层）依赖于离子通道网络。我们的总体假设是。 K+ 通道定义电膜电位并调节电压门控 Ca2+ 通道，从而建立 从多能性向特定细胞命运、原肠胚形成和 LR 模式的退出提供了合理的 然后，我们的电生理通路与 Htx 和 CHD 患者的机制相结合。 定义胚胎中特定细胞命运的生化信号通路。 在本次提案修订中，我们将重点关注 KCNH6，看看基因缺失是否会导致 LR 模式缺陷 此外，我们将测试 KCNH6 在 LR 模式级联中发挥的作用。 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 对于多能性、细胞命运和钙调节至关重要。由于该项目的新颖性，我们还将 执行无偏基因组学 (RNAseq) 来发现  Vm 的转录靶标。 使用全细胞电压钳和细胞内电生理学测量电特性 记录并确定定义早期生殖细胞膜电位的各种电流。 我们提案的一个主要优势是我们的专业知识；我们与 Xenopus 之间建立了合作关系； 发育生物学家和电生理学家将使我们能够严格研究膜 作为胚胎模式机制的潜力。