Electron crystallographic studies of water channel regulation

水通道调节的电子晶体学研究

• 批准号: 7611345
• 负责人: Stephen Loen Reichow
• 金额: $ 5.01万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7611345
• 关键词: Achievement; Address; Affect; Binding; Binding Proteins; Biological; Blindness; Calmodulin; Cataract; Cells; Cellular biology; Complex; Coupled; Cryoelectron Microscopy; Crystalline Lens; Crystallization; Crystallography; Disease; Drops; Electrons; Electrophysiology (science); Equipment; Histidine; Homeostasis; Human; Ions; Laboratories; Lead; Lesion; Life; Link; Lipids; MIP gene; Malignant Neoplasms; Measurement; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Mutate; Mutation; Nutrient; Organism; Peptides; Permeability; Physiological; Physiology; Proteins; Regulation; Resolution; Rest; Roentgen Rays; Role; Signal Transduction; Structure; System; Vesicle; Vestibule; Waste Products; Water; Work; X-Ray Crystallography; density; electron crystallography; insight; lens; nanometer; particle; porin; professor; response; sensor; structural biology; success; two-dimensional; water channel

DESCRIPTION (provided by applicant): Most of the interior of a cell is water, and life could not exist without circulating water: Nutrients and ions that are vital for our existence are solvated by water, and waste products are removed by circulating currents. Therefore, all three kingdoms of life, from the simplest unicellular organisms to humans, express specialized membrane-embedded proteins that form pores for water conduction called aquaporins. Because of their abundance and their role in maintaining celluar homeostasis, many diseases and cancers are directly related to misregulation or mutation in aquaporins. We study aquaporin 0 (AQPO), a water channel from the eye lens, causing severe lesions in the lens, cataracts and blindness when mutated. AQPO tightly regulates water permeability within the eye lens by responding to changes in pH, as well as by Ca2*/calmodulin. Calmodulin (CaM) is a ubiquitous cytosolic protein commonly coupled with Ca2+ regulation. It is proposed that these two AQPO regulatory signals are separable, and that AQPO exists in two functional states: a low water permeability resting state at pH 7.2; and a high permeability state induced by a drop in pH or mediated by Ca2VCaM. Moreover, we suggest that these regulatory signals lead to major conformational changes in AQPO, causing water channels to open/close and permeability to increase/decrease. The aims of this proposal are to use cryo electron microscopy (cryo EM) and electrophysiology to unravel these two regulatory mechanisms by studying the conformational changes in AQPO as its water channels open/close in response to pH and Ca2+/CaM. Results from this study have wide implications in the fields of water channel regulation, physiology as well as broader implications to the Ca2VCaM signaling system and to membrane and cellular biology in general. Success in achieving the aims set in this proposal would additionally mark a technological achievement in the field of structural biology by providing the first structure of a membrane protein embedded in a membrane and bound to a soluble protein. It is hoped that by gaining a fundamental understanding of how cellular water homeostasis is regulated by aquaporins, we may begin to better understand the disease states and cancers associated with their misregulation.

描述（由申请人提供）：细胞的大部分内部是水，没有循环水就不可能存在生命：对我们生存至关重要的营养和离子是用水溶解的，而废物产物被循环电流清除。因此，从最简单的单细胞生物到人类，所有三个生命的王国都表达了专门的膜包含的蛋白质，这些蛋白质形成了称为水通道蛋白的水传导的孔。由于它们的丰富性及其在维持大地球稳态中的作用，许多疾病和癌症与水通道蛋白中的正调或突变直接相关。我们研究了来自眼镜的水通道Aquaporin 0（AQPO），在突变时会导致晶状体，白内障和失明的严重病变。 AQPO通过响应pH的变化以及Ca2*/钙调蛋白来严格调节眼镜内的水渗透性。钙调蛋白（CAM）是一种无处不在的胞质蛋白，通常与CA2+调节结合。有人提出，这两个AQPO调节信号是可分离的，并且AQPO存在于两个功能状态：pH 7.2处的低水渗透性静止状态；以及pH下降或由Ca2VCAM介导的高渗透率。此外，我们建议这些调节信号导致AQPO的重大构象变化，从而导致水通道开/关闭，并渗透性增加/减少。该提案的目的是使用冷冻电子显微镜（冷冻EM）和电生理学来揭示这两种调节机制，通过研究AQPO中的构象变化，因为其水通道对pH和CA2+/CAM的响应开放/关闭。这项研究的结果在水道调节，生理学以及对CA2VCAM信号系统以及一般的膜和细胞生物学方面具有广泛的影响。通过在结构生物学领域中，通过提供嵌入膜中并与可溶性蛋白结合的膜蛋白的第一个结构，在结构生物学领域取得了成功，这将在结构生物学领域中获得成功。希望通过对蜂窝水稳态如何受到水通道蛋白的调节的基本了解，我们可以开始更好地理解疾病状态和癌症与它们的正常情况相关的疾病。