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：
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
项目类别：
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

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.

项目概要/摘要电压门控质子通道 (HV1) 存在于许多人体组织中，发挥着多种重要作用例如，它有助于白细胞、精子杀死细菌。成熟和流动性、嗜碱性粒细胞释放组胺、B 淋巴细胞信号传导和气道液体 HV1 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，因为其基因尚未报道。直到2006年，HV1还是电压门控离子通道家族的新成员。最后，它的结构是独一无二的。在重新组装所有电压门控离子通道的关键组件时，它是新来者。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了解 HV1 功能和功能障碍非常重要。直接转化研究将评估 HV1 参与乳腺癌生长的报道将使用具有不同 HV1 表达的细胞来检查小鼠中的肿瘤生长。水平，范围从完全敲除 (CRISPR/Cas9) 到减少 (shRNA) 到正常 (WT)。目前的工作假设是 HV1 作为转换膜电位变化的开关我们还将进一步研究 HV1 参与人类 B 细胞的发现。在慢性淋巴细胞白血病中，一种新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鉴定出。 DeCoursey实验室从发现HV1以来一直深入参与HV1的研究存在于哺乳动物和人类细胞中，以确定其在许多人类细胞中的作用和组织，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，我们打算扩大我们对这一重要分子的了解在我们最近取得的进展的基础上，我们发现了产生质子的机制。选择性传导需要孔中心有天冬氨酸，我们将测试是否有。疏水区利用诱变、膜片钳和分子生物学发挥着额外的关键作用我们将研究电压门控机制和独特的 ΔpH。依赖门控对于使用类似方法的该分子的所有功能至关重要，但是包括详细的力学模型以及新改进的分子动力学方法凭经验决定质子化，而不是假设它，我们将不断完善我们的理论。使用组氨酸扫描了解封闭和开放 HV1 通道的结构诱变和核磁共振知识对于理解机制至关重要。以及药物设计。