Structural basis of ion channel function in Aquaporin-1

描述(由申请人提供)：通过水通道蛋白1(AQP1)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。这些通道对渗透性水的运动是必不可少的，对脑脉络丛和其他组织中cGMP依赖的信号转导具有潜在的重要作用。(AQP1)和环核苷酸门控(CNG)通道之间的序列相似性意味着羧基(C-)末端是结合域。我们的中心假设是cGMP与AQP1的C末端结合，并通过AQP1的中心孔控制阳离子电流，离子通道的活性进一步受蛋白-激酶相互作用的控制。我们使用膜片钳、电压钳、定点突变、蛋白质组学和蛋白质生物化学方法来研究AQPI在卵母细胞表达系统和在脉络丛中丰富表达AQP1的基本性质。已解决的AQP1的晶体结构数据允许知情地选择区域进行突变和进一步分析。 第一个目的是评估克隆的人AQP1 C-末端结构域保守残基在cGMP应答中的作用。与CNG通道专家J·卡彭博士合作，我们将使用光活化的共价配体测试野生型和突变型AQP1通道结合和通道活性的变化。第二个目标是定位离子孔，初步数据表明离子孔位于四重对称轴上四聚体的中心。第三个目的是评估受体酪氨酸激酶介导的磷酸化以及与EphB2受体的蛋白质-蛋白质相互作用在调节AQP1离子通道活性中的作用。第四个目的是通过发现大鼠脉络丛(原代培养)中是否存在AQP1离子电导来确定潜在的生理学相关性，脉络丛组织中大量表达天然AQP1。 我们的工作首次证明了AQP1是一个门控离子通道。CGMP依赖的离子通道活性的这种特性可能对大脑、外周神经系统、血管系统和心脏中的信号传递具有重要意义，并可能在一定程度上使癌症的病理生理生长，因为这些组织中表达AQP1。AQP1通道基本特性的发现可能为人类疾病的治疗干预打开机会，这些疾病涉及大脑和其他器官的水和盐失衡。

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.