Structure Analysis of Aquaporin Membrane Channels

描述(由申请人提供)：MIP家族的水选择跨膜孔蛋白，俗称“水通道蛋白”，负责水在细胞膜上的快速移动，因此对溶质运输和细胞、组织和生物渗透调节至关重要。这些蛋白质中的分子缺陷会导致白内障的形成和尿液浓度的问题。几种水通道蛋白的一个令人兴奋的特点是，它们的透水性是主动门控的。AQP0(以前称为MIP)的水通道活性，发现于眼晶状体纤维细胞中，受pH和钙的调节。AQP4存在于大脑新皮质和眼睫状体，其水通道活性受磷酸化的调节。在肾脏集合管细胞中发现的AQP6的水通道活性也受pH的调节。我们假设，由磷酸化或氨基酸荷电引起的静电变化驱动了负责调节的变构孔道修饰。闭合和开放状态的高分辨率结构数据是检验这一假说的关键。我们之前生长了有序的水通道蛋白的螺旋晶体，α-TIP，它是由磷酸化门控的。在7.7埃的投影图上，分辨率显示AQP0、AQP1和α-TIP的α-螺旋设计是保守的。因此，我们对α-TIP的结构研究为理解AQP0、AQP4和AQP6等水通道蛋白的门控机制提供了一个范例。为了探索水通道蛋白α-TIP的磷酸化依赖门控的分子基础，我们将追求以下5个特定目标：目的1：利用质谱仪确定α-TIP的磷酸化位点。目的2：使用电子冷冻显微镜和图像分析来描绘与磷酸化依赖的门控相关的α螺旋运动。目的3：建立突变水通道蛋白的高效表达系统，用于半胱氨酸的定点自旋标记。目的4：建立突变水通道蛋白水通道活性的功能分析方法。目的5：利用自旋标记水通道蛋白的电子顺磁共振(EPR)谱来确定调节水运输的氨基酸重排。在这一改进的应用中，我们成功地在巴斯德毕赤酵母中表达了他的(10)标记的α-末端，并使用镍亲和层析纯化了毫克量的蛋白质。此外，我们还在酵母球形体中建立了渗透压休克实验，以验证重组蛋白具有水通道活性。我们的分析揭示的结构细节将是磷酸化依赖的通道门控的第一个分子描述。此外，该项目将提供对通道调节的分子基础的洞察，并将有助于控制膜蛋白构象变化的一般原则。

DESCRIPTION (provided by applicant): The MIP family of water-selective transmembrane pore proteins, commonly known as "aquaporins", are responsible for the rapid movement of water across cell membranes, and are thus crucial for solute transport and cellular, tissue, and organismal osmoregulation. Molecular defects in these proteins can result in cataract formation and problems in urine concentration. An exciting feature of several aquaporins is that their water permeability is actively gated. Water channel activity of AQP0 (formerly known as MIP), found in eye lens fiber cells, is regulated by both pH and calcium. Water channel activity of AQP4, found in brain neocortex and ocular ciliary bodies, is regulated by phosphorylation. Water channel activity of AQP6, found in renal collecting duct cells, is also regulated by pH. We hypothesize that electrostatic changes, resulting from either phosphorylation or amino acid charging, drive the allosteric pore modifications responsible for regulation. High resolution structural data of the closed and open states are essential to test this hypothesis. We previously grew well-ordered helical crystals of the aquaporin, alpha-TIP, which is gated by phosphorylation. A projection map at 7.7Angstroms, resolution showed conservation in the alpha-helical design of AQP0, AQP1 and alpha-TIP. Consequently, our structural studies on alpha-TIP serve as a paradigm for understanding gating mechanisms in aquaporins such as AQP0, AQP4, and AQP6. To explore the molecular basis of phosphorylation-dependent gating of the aquaporin alpha-TIP, we will pursue the following 5 specific aims: Aim 1: Use mass spectrometry to determine the phosphorylation sites of alpha-TIP. Aim 2: Use electron cryo-microscopy and image analysis to delineate the alpha-helical movements associated with phosphorylation-dependent gating. Aim 3: Develop an overexpression system to produce mutant aquaporins for site-directed cysteine spin labeling. Aim 4: Develop a functional assay for water channel activity of the mutant aquaporins. Aim 5: Use electron paramagnetic resonance (EPR) spectroscopy of spin-labeled aquaporins to define the amino acid rearrangements that regulate water transport. For this revised application, we have successfully expressed His(10)-tagged alpha-TIP in the methylotrophic yeast Pichia pastoris and used Ni-affinity chromatography to purify milligram quantities of the protein. In addition, we have developed an osmotic-shock assay in yeast spheroplasts to verify that the recombinant protein exhibits water channel activity. The structural details revealed by our analysis will be the first molecular description of phosphorylation-dependent channel gating. In addition, this project will provide insight into the molecular basis for channel regulation and will contribute to the general principles that govern conformational changes of membrane proteins.