Electron crystallographic studies of water channel regulation

描述(申请人提供)：细胞内部大部分是水，没有循环水，生命就不可能存在：对我们生存至关重要的营养物质和离子被水溶解，废物通过循环电流清除。因此，从最简单的单细胞生物体到人类，生命的三个王国都表达特殊的膜嵌入蛋白，这些蛋白形成了称为水通道蛋白的水孔。由于它们的丰富及其在维持细胞内稳态方面的作用，许多疾病和癌症都与水通道蛋白的调控不当或突变直接相关。我们研究了水通道蛋白0(AQPO)，这是一种来自眼睛晶状体的水通道，当突变时会导致晶状体严重损伤、白内障和失明。AQPO通过对pH的变化以及钙/钙调蛋白的反应来严格调节眼晶状体内的水分渗透性。钙调素(CaM)是一种普遍存在的胞浆蛋白，常与钙调节偶联。结果表明，这两种AQPO调控信号是可分离的，AQPO存在两种功能状态：一种是pH值7.2的低透水性静息状态，另一种是由pH下降或Ca2VCaM介导的高通透性状态。此外，我们认为这些调控信号导致AQPO的主要构象变化，导致水通道的开放/关闭和通透性的增加/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在其水通道开放/关闭时对pH和Ca~(2+)/CaM的响应而发生的构象变化来揭示这两种调控机制。这项研究的结果在水通道调节、生理学以及对Ca2VCaM信号系统和一般的膜和细胞生物学的更广泛的影响方面具有广泛的意义。如果能成功实现这项提案中确定的目标，将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，从而标志着结构生物学领域的一项技术成就。人们希望，通过对水通道蛋白如何调节细胞水稳态的基本了解，我们可能会开始更好地了解与水通道蛋白调控不当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.