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）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的运动是必不可少的，并且对于脑脉络膜丛和其他组织中cgmp依赖的信号传导可能很重要。（AQP1）和环核苷酸门控（CNG）通道之间的序列相似性暗示羧基（C-）末端是结合域。我们的中心假设是cGMP与c端结合，并通过AQP1的中心孔形成阳离子电流，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丰富表达AQP1的脉络膜丛中的基本特性。已解决的AQP1晶体结构数据允许知情的突变区域选择和进一步的分析。

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.