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Control Mechanisms of Human Voltage Gated Proton Channels, hHv1

人类电压门控质子通道的控制机制，hHv1

基本信息

批准号：
10394280
负责人：
THOMAS E DECOURSEY
金额：
$ 36.9万
依托单位：
RUSH UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10394280
关键词：
Aspartate Autoimmune Diseases B lymphoid malignancy B-Lymphocytes Bacteria Basophils Brain Injuries Breast cancer metastasis CRISPR/Cas technology Cells Chronic Lymphocytic Leukemia Disease Drug Design Family Functional disorder Genes Growth Health Histamine Release Histidine Human Hydrophobicity Ion Channel Gating Ischemic Stroke Knock-out Knowledge Leukocytes Liquid substance Membrane Potentials Modeling Mus Mutagenesis Mutation Neoplasm Metastasis Play Proteins Protons Regulation Reporting Role Scanning Signal Transduction Sperm Maturation Sperm Motility Stroke Structure Testing Tissues Work cell killing cell type cellular pathology human subject human tissue improved leukemia malignant breast neoplasm molecular dynamics novel strategies patch clamp protonation small hairpin RNA sperm cell translational study tumor growth voltage zygote

项目摘要

Project Summary/Abstract The voltage gated proton channel (HV1) exists in many human tissues and plays numerous roles vital to human health. For example, it contributes to bacterial killing by white blood cells, sperm maturation and mobility, histamine release by basophils, B lymphocyte signaling, and airway fluid regulation. Abnormal HV1 function has been implicated in breast cancer metastasis, brain damage in ischemic stroke, and exacerbation of chronic lymphocytic leukemia. As its gene was not reported until 2006, HV1 is a newcomer to the voltage gated ion channel family. Finally, its structure is unique in resembling a crucial component of all voltage-gated ion channels. This newcomer status, its unique structure, and its essential roles in human health and disease make understanding HV1 function and dysfunction highly significant. Directly translational studies will evaluate reported involvement of HV1 in breast cancer growth and metastasis. Tumor growth in mice will be examined using cells with different HV1 expression levels, ranging from complete knock-out (CRISPR/Cas9) to reduced (shRNA) to normal (WT). Our current working hypothesis is that HV1 acts as a switch that transduces membrane potential changes into cellular pathology. We will also build on our discovery of the involvement of HV1 in human B cells and in chronic lymphocytic leukemia. A novel approach will be to determine the effects of mutations indentified in human subjects with B cell malignancy. The DeCoursey lab has been deeply involved in the study of HV1, from discovering its existence in mammalian and human cells, to identifying its role in a number of human cells and tissues, to finally dissecting the molecule itself to identify which parts perform the major functions. Over the next five years we intend to pursue expanding our knowledge of this important molecule at multiple levels, building on our recent progress. We found that the mechanism producing proton selective conduction requires an aspartate in the center of the pore. We will test whether a hydrophobic region plays an additional critical role using mutagenesis, patch-clamp, and molecular dynamics simulations. We will attack the mechanisms of voltage-gating and the unique ∆pH dependent gating that is essential to all functions of this molecule using similar approaches, but including a detailed mechanistic model as well as a newly improved molecular dynamics approach that determines protonation empirically rather than assuming it. We will continually refine our knowledge of the structures of both closed and open HV1 channels, using histidine scanning mutagenesis and NMR. Structure-function knowledge is crucial both for understanding mechanisms and for drug design.

项目摘要/摘要电压门控质子通道(Hv1)存在于人体多种组织中，发挥着多种重要作用。为人类健康干杯。例如，它有助于白细胞、精子等细菌的杀灭成熟度和流动性、嗜碱性粒细胞释放组胺、B淋巴细胞信号转导和气道液监管。人类免疫缺陷病毒1功能异常与乳腺癌转移、脑损伤有关缺血性中风和慢性淋巴细胞白血病的恶化。由于其基因未见报道直到2006年，HV1还是电压门控离子通道家族的新成员。最后，它的结构是独特的类似于所有电压门控离子通道的关键组件。这个新人的身份，它独特的结构及其在人类健康和疾病中的重要作用使我们能够理解人类免疫缺陷病毒1 功能和功能障碍非常显著。直接翻译研究将评估有关人类免疫缺陷病毒1参与乳腺癌生长的报道和转移。将使用具有不同HV1表达的细胞来检测小鼠的肿瘤生长水平，从完全敲除(CRISPR/Cas9)到减少(ShRNA)到正常(WT)不等。我们的目前的工作假说是，HV1充当转导膜电位变化的开关转化为细胞病理学。我们还将在人类B细胞中发现人类免疫缺陷病毒1的基础上再接再厉在慢性淋巴细胞性白血病中。一种新的方法将是确定突变的影响在患有B细胞恶性肿瘤的受试者中被鉴定。德库西实验室一直在深入参与人类免疫缺陷病毒1的研究，从发现它的在哺乳动物和人类细胞中的存在，以确定其在一些人类细胞和最后解剖分子本身，以确定哪些部分执行主要功能。在接下来的五年里，我们打算通过以下途径扩大我们对这种重要分子的了解多层次，以我们最近的进展为基础。我们发现产生质子的机制选择性传导需要在气孔中心有一个天冬氨酸。我们将测试一个疏水性区域通过突变、膜片钳和分子起到额外的关键作用动力学模拟。我们将攻击电压门控的机制和独特的∆pH 依赖的门控对于这个分子的所有功能都是必不可少的，但使用类似的方法，包括一个详细的机理模型以及一个新改进的分子动力学方法这是根据经验而不是假设来决定质子化的。我们将不断完善我们的使用组氨酸扫描了解闭合和开放的HV1通道的结构诱变和核磁共振。结构-功能知识对于理解机制是至关重要的以及药物设计。