Structure Analysis of Aquaporin Membrane Channels

描述（由申请人提供）：MIP家族的水选择性跨膜孔蛋白，通常称为“水通道蛋白”，负责水快速穿过细胞膜，因此对于溶质转运和细胞、组织和生物体的代谢调节至关重要。这些蛋白质的分子缺陷会导致白内障的形成和尿液浓缩的问题。几种水通道蛋白的一个令人兴奋的特征是它们的水渗透性是主动门控的。在眼透镜纤维细胞中发现的AQP 0（以前称为MIP）的水通道活性受pH和钙调节。在大脑新皮层和眼睫状体中发现的AQP 4的水通道活性受磷酸化调节。水通道活性的AQP 6，发现在肾集合管细胞，也调节pH值。我们假设，静电变化，导致磷酸化或氨基酸充电，驱动负责调节变构孔的修改。封闭态和开放态的高分辨率结构数据对验证这一假设至关重要。我们以前生长有序的螺旋晶体的水通道蛋白，α-TIP，这是门控磷酸化。在7.7埃分辨率下的投影图显示了AQP 0、AQP 1和α-TIP的α-螺旋设计的保守性。因此，我们对α-TIP的结构研究可作为理解水通道蛋白（如AQP 0、AQP 4和AQP 6）门控机制的范例。为了探索磷酸化依赖性门控的水通道蛋白α-TIP的分子基础，我们将追求以下5个具体目标：目的1：使用质谱来确定α-TIP的磷酸化位点。目的2：使用冷冻电镜和图像分析描绘与磷酸化依赖性门控相关的α-螺旋运动。 目的3：建立一个过表达系统，产生突变型水通道蛋白，用于定点半胱氨酸自旋标记。目的4：建立突变型水通道蛋白水通道活性的功能检测方法。 目标5：使用自旋标记水通道蛋白的电子顺磁共振（EPR）光谱来定义调节水转运的氨基酸重排。对于这一修改后的应用，我们已经成功地表达了His（10）-标记的α-TIP在甲醇营养型酵母巴斯德毕赤酵母，并使用镍亲和层析纯化毫克量的蛋白质。此外，我们已经开发了一种酵母原生质球中的免疫休克试验，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第一个磷酸化依赖性通道门控的分子描述。此外，该项目将提供深入了解通道调节的分子基础，并将有助于管理膜蛋白的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.