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

• 批准号: 9916761
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916761
• 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

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）存在于许多人体组织中，并且起着许多重要作用 对人类健康。例如，它导致细菌被白细胞杀死，精子 成熟和迁移率，嗜碱性粒细胞释放组胺，B淋巴细胞信号和气道流体 规定。 HV1功能异常已在乳腺癌转移中浸渍，脑损伤 缺血性中风，加剧了慢性淋巴细胞性白血病。因为没有报道其基因 直到2006年，HV1是电压封闭离子通道家族的新来者。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。这个新来的状态，它 独特的结构及其在人类健康和疾病中的重要作用使人们了解HV1 功能和功能障碍高度显着。 直接翻译的研究将评估报告的HV1参与乳腺癌生长 和转移。将使用具有不同HV1表达的细胞检查小鼠的肿瘤生长 从完全敲除（CRISPR/CAS9）到降低（shRNA）到正常（WT）的水平。我们的 当前的工作假设是HV1充当传递膜电位变化的开关 进入细胞病理学。我们还将建立在HV1参与人B细胞的基础上 在慢性淋巴细胞性白血病中。一种新颖的方法是确定突变的影响 在患有B细胞恶性肿瘤的人类受试者中被批准。 Decoursey Lab已深入研究HV1的研究，从发现其 在哺乳动物和人类细胞中存在，以确定其在许多人类细胞中的作用，以及 组织，最终剖析分子本身，以识别哪些部分执行主要功能。 在接下来的五年中，我们打算追求我们对这一重要分子的了解 以我们最近的进步为基础的多个级别。我们发现产生质子的机制 选择性传导需要在孔的中心天冬氨酸。我们将测试是否 疏水区使用诱变，斑块夹和分子起着额外的关键作用 动力学模拟。我们将攻击电压门的机理和独特的∆PH 使用类似方法对该分子的所有功能至关重要的依赖门控，但是 包括详细的机械模型以及新改进的分子动力学方法 从经验上决定质子化而不是假设质子化。我们将不断完善我们的 使用组氨酸扫描，了解关闭和开放HV1通道的结构的知识 诱变和NMR。结构功能知识对于理解机制至关重要 和药物设计。