Structural basis of ion channel function in Aquaporin-1

Aquaporin-1 离子通道功能的结构基础

• 批准号: 6767585
• 负责人: ANDREA J YOOL
• 金额: $ 28.79万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6767585
• 关键词: Xenopus oocyte; biological signal transduction; cations; choroid plexus; cyclic AMP; cyclic GMP; electrical conductance; electrophysiology; gene mutation; genetic manipulation; intermolecular interaction; membrane channels; membrane permeability; mutant; nucleotides; phosphorylation; protein binding; protein kinase A; protein protein interaction; protein structure function; proteomics; site directed mutagenesis; tetraethylammonium compound; tissue /cell culture; voltage /patch clamp; water channel; water flow

DESCRIPTION (provided by applicant): Permeability to both water and cations through Aquaporin-1 (AQP1) channels suggests an impressive multifunctional capacity. These channels are essential for osmotic water movement, and potentially important for cGMP-dependent signaling in brain choroid plexus and other tissues. Sequence similarity between (AQP1) and cyclic-nucleotide-gated (CNG) channels implicates the carboxy (C-) terminus as the binding domain. Our central hypothesis is that cGMP binds to the C-terminus and gates cationic current through a central pore in AQP1, and that ion channel activity is further governed by protein-kinase interactions. We use patch clamp, voltage clamp, site-directed mutagenesis, proteomic and protein biochemistry methods to address fundamental properties of AQPI in an oocyte expression system, and in choroid plexus, which abundantly expresses AQP1. Solved crystal structure data for AQP1 allow the informed selection of regions for mutagenesis and further analysis. The first aim is to evaluate the role of conserved residues in the C-terminal domain of cloned human AQP1 in the response to cGMP. In collaboration with Dr. J. Karpen, an expert in CNG channels, we will test for alterations in binding and channel activity in wild type and mutant AQP1 channels using a photoactivated covalent ligand. The second aim is to locate the ion pore, suggested by preliminary data to be in the center of the tetramer at the four-fold axis of symmetry. The third aim is to assess the roles of receptor tyrosine kinase-mediated phosphorylation and protein-protein interaction with the ephrin receptor EphB2 in governing activity of AQP1 ion channels. The fourth aim is to determine potential physiological relevance by discovering whether an AQP1 ionic conductance is present in rat choroid plexus (primary cultures), a tissue in which native AQP1 is abundantly expressed. Our work was the first to show that AQP1 is a gated ion channel. This property of cGMP-dependent ion channel activity may be significant to signaling in the brain, peripheral nervous system, vascular system and heart, and may in part enable pathophysiological growth in cancer, since these are tissues in which AQP1 is expressed. Discovery of the fundamental properties of AQP1 channels may open opportunities for therapeutic intervention in human diseases involving fluid and salt imbalance in the brain and other organs.

描述（由申请人提供）：水通道蛋白-1 (AQP1) 通道对水和阳离子的渗透性表明其具有令人印象深刻的多功能能力。这些通道对于渗透水运动至关重要，并且对于脑脉络丛和其他组织中的 cGMP 依赖性信号传导也可能很重要。 (AQP1) 和环核苷酸门控 (CNG) 通道之间的序列相似性表明羧基 (C-) 末端是结合域。我们的中心假设是 cGMP 与 C 末端结合并通过 AQP1 的中心孔控制阳离子电流，并且离子通道活性进一步受到蛋白激酶相互作用的控制。我们使用膜片钳、电压钳、定点诱变、蛋白质组和蛋白质生物化学方法来解决卵母细胞表达系统和大量表达 AQP1 的脉络丛中 AQPI 的基本特性。解决的 AQP1 晶体结构数据允许明智地选择用于诱变和进一步分析的区域。 第一个目的是评估克隆人 AQP1 C 端结构域中保守残基在 cGMP 响应中的作用。我们将与 CNG 通道专家 J. Karpen 博士合作，使用光激活共价配体测试野生型和突变型 AQP1 通道中结合和通道活性的变化。第二个目标是定位离子孔，初步数据表明该离子孔位于四重对称轴处的四聚体中心。第三个目的是评估受体酪氨酸激酶介导的磷酸化以及与肝配蛋白受体 EphB2 的蛋白质-蛋白质相互作用在控制 AQP1 离子通道活性中的作用。第四个目标是通过发现大鼠脉络丛（原代培养物）中是否存在 AQP1 离子电导来确定潜在的生理相关性，大鼠脉络丛是天然 AQP1 大量表达的组织。 我们的工作首次证明 AQP1 是门控离子通道。 cGMP 依赖性离子通道活性的这种特性可能对大脑、周围神经系统、血管系统和心脏中的信号传导具有重要意义，并且可能在一定程度上促进癌症的病理生理生长，因为这些是表达 AQP1 的组织。 AQP1 通道基本特性的发现可能为涉及大脑和其他器官中液体和盐失衡的人类疾病的治疗干预提供机会。