Potassium channels, membrane potential, and CHD

项目摘要 先天性心脏病（CHD）导致严重的发病率和死亡率的儿童在美国和世界各地。 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，Trio 基于全外显子组测序已经确定了一类电压门控钾通道（多KCNH 家族成员）作为CHD的候选者，特别是异位症，一种左右（LR）模式紊乱， 会严重影响心脏功能然而，连接钾通道与结构性钾通道的分子作用， 心脏病和内脏异位是前所未有的。 我们提出，我们的初步数据支持，KCNH 6定义了一个新的范例，细胞信号转导， 早期胚胎细胞我们的数据支持一个电生理模型，其中特定的胚层命运 （近轴中胚层和外胚层）依赖于离子通道网络。我们的首要假设是 K+通道定义膜电位，并调节电压门控Ca 2+通道， 从多能性向特定细胞命运、原肠胚形成和LR模式的退出提供了一个合理的解释。 Htx和CHD患者的发病机制。我们的电生理通路，然后整合 决定胚胎中特定细胞命运的生化信号通路。 在这个建议修订中，我们将重点关注KCNH 6，看看基因缺失是否会导致LR模式缺陷， 爪蟾此外，我们将测试KCNH 6在LR图案化级联中的何处发挥作用。然后，使用 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是 对多能性、细胞命运和钙调节至关重要。由于该项目的新奇，我们还将 进行无偏基因组学（RNAseq），用于发现HPVm的转录靶点。最后我们将 使用全细胞电压钳和细胞内电压钳测量电生理学的电特性 记录并确定定义早期生殖细胞膜电位的各种电流。 我们的建议的一个主要优势是我们的专业知识;我们已经建立了一个合作， 发展生物学家和电生理学家，这将使我们能够严格研究膜 作为胚胎模式化机制的潜力。

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.