Control Mechanisms of Human Voltage Gated Proton Channels, hHv1

项目总结/摘要 电压门控质子通道（HV 1）存在于人体的多种组织中，并发挥着重要的作用 对人类健康的影响。例如，它有助于通过白色血细胞、精子 成熟和活动性、嗜碱性粒细胞释放组胺、B淋巴细胞信号传导和气道液 调控异常的HV 1功能与乳腺癌转移、脑损伤、肿瘤坏死因子相关。 缺血性中风和慢性淋巴细胞白血病恶化。由于其基因尚未报道 直到2006年，HV 1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重要组成部分。这个新人的身份， 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HV 1 功能和功能障碍非常显著。 直接转化研究将评估报告的HV 1参与乳腺癌生长的情况 和转移。将使用具有不同HV 1表达的细胞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）。我们 目前的工作假设是HV 1作为转换膜电位变化的开关 细胞病理学。我们还将建立在我们发现的HV 1参与人类B细胞的基础上 和慢性淋巴细胞白血病。一种新的方法将是确定突变的影响 在患有B细胞恶性肿瘤的人类受试者中鉴定。 DeCoursey实验室一直深入参与HV 1的研究，从发现其 在哺乳动物和人类细胞中的存在，以确定其在许多人类细胞中的作用， 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，我们打算继续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，我们发现产生质子的机制 选择性传导需要孔中心的天冬氨酸。我们将测试A 疏水区在使用诱变、膜片钳和分子生物学技术中起着额外的关键作用， 动力学仿真我们将攻击的电压门控机制和独特的pH值 依赖性门控，这是必不可少的所有功能的这种分子使用类似的方法，但 包括详细的机理模型以及新改进的分子动力学方法 决定质子化的经验，而不是假设它。我们将不断完善我们的 使用组氨酸扫描，了解封闭和开放HV 1通道的结构 诱变和NMR。结构-功能知识对于理解机制和 和药物设计。

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.