Potassium channels, membrane potential, and CHD

项目摘要 先天性心脏病(CHD)在美国和世界范围内对儿童造成严重的发病率和死亡率。 尽管这对儿童健康有影响，但我们根本不了解CHD的遗传原因。最近，三人组 基于全外显子组测序发现了一类电压门控性钾通道(Multiple KCNH 家庭成员)作为CHD的候选对象，特别是异位畸形，是一种左右(LR)模式的紊乱， 对心脏功能有严重影响。然而，连接钾通道和结构的分子作用 心脏病和异位症是史无前例的。 我们提出，我们的初步数据支持，KCNH6定义了细胞信号转导的新范式 早期胚胎细胞。我们的数据支持一个电生理模型，在这个模型中，特定的生殖层 (近轴中胚层和外胚层)依赖于离子通道网络。我们最重要的假设是 K+通道定义了电膜电位，并调节电压门控钙通道 从多能性向特定的细胞命运、原肠形成和LR模式的退出提供了一种看似合理的 我们的HTX和CHD患者的机制。我们的电生理通路然后整合到 决定胚胎中特定细胞命运的生化信号通路。 在这项提案修订中，我们将重点关注KCNH6，以了解基因缺失是否导致LR图案缺陷 非洲爪哇。此外，我们将测试KCNH6在LR图案级联中的作用。然后，使用 一系列精心选择的化学和离子扰动，我们将测试膜电位是否真的 对多能性、细胞命运和钙调节至关重要。由于这个项目的新颖性，我们还将 进行无偏基因组学(RNAseq)以发现Vm的转录靶点。最后，我们会 用全细胞电压钳和细胞内电压钳测量电生理特性 记录并确定定义早期生殖细胞膜电位的各种电流。 我们建议的一个主要优势是我们的专业知识；我们与Xenopus之间的合作 发育生物学家和电生理学家将使我们能够严格研究细胞膜 作为胚胎构图机制的潜力。

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.