Electron crystallographic studies of water channel regulation

描述（由申请人提供）：细胞内部大部分是水，没有循环水生命就无法存在：对我们生存至关重要的营养物质和离子被水溶解，废物被循环电流除去。因此，从最简单的单细胞生物到人类，所有三种生命体都表达一种特殊的膜内蛋白质，这种蛋白质可以形成水传导的孔，称为水通道蛋白。由于水通道蛋白的丰度及其在维持细胞稳态中的作用，许多疾病和癌症与水通道蛋白的失调或突变直接相关。我们研究了水通道蛋白0 (AQPO)，一种来自眼睛晶状体的水通道，当它发生突变时，会导致严重的晶状体病变、白内障和失明。AQPO通过响应pH值的变化以及Ca2*/钙调蛋白紧密调节晶状体内的水分通透性。钙调蛋白（CaM）是一种普遍存在的细胞质蛋白，通常与Ca2+调节偶联。我们认为这两种AQPO调控信号是可分离的，并且AQPO存在两种功能状态：pH值为7.2时的低透水性静息状态；pH降低或Ca2VCaM介导的高通透性状态。此外，我们认为这些调节信号导致AQPO的主要构象变化，导致水通道的打开/关闭和渗透率的增加/减少。本研究的目的是利用低温电子显微镜（cryo EM）和电生理学，通过研究AQPO的水通道在pH和Ca2+/CaM的作用下打开/关闭时的构象变化，来揭示这两种调节机制。本研究的结果在水通道调控、生理学以及Ca2VCaM信号系统以及膜和细胞生物学等领域具有广泛的意义。通过提供嵌入膜并与可溶性蛋白结合的膜蛋白的第一个结构，成功实现本提案中设定的目标将进一步标志着结构生物学领域的技术成就。希望通过对水通道蛋白如何调节细胞水稳态的基本理解，我们可以开始更好地了解与水通道蛋白失调相关的疾病状态和癌症。

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.