Membrane Targeting of Calcium-Pumping ATPase

描述（由申请人提供）：质膜钙atp酶（PMCAs）将Ca2+泵出所有动物细胞。作为Ca2+信号“工具箱”的组成部分，PMCAs定位于不同的质膜结构域。以膜中缺乏特异性PMCA同工异构体为特征的疾病，如耳蜗毛细胞静纤毛中缺乏PMCA2引起的耳聋，说明了适当的膜靶向的重要性。长期目标是了解特异性膜靶向PMCAs的机制和功能影响。具体目的是：(1)确定PMCA2剪接变异体中的顶端靶向元件；(2)确定PDZ蛋白相互作用是否以及如何稳定顶膜中的PMCA2；(3)确定PMCA2剪接变体的特异性定位是否会改变极化上皮细胞中跨上皮Ca2+通量和全局Ca2+信号传导；(4)确定海马神经元中PMCA2变异的亚细胞分布并鉴定神经元靶向元件。这些研究将涉及共聚焦荧光显微镜和双杂交相互作用分析，以确定PMCA2剪接变体的特定靶向元件。光漂白后的荧光恢复和半衰期研究将用于分析PMCA2异构体的膜动力学，光镜和电镜将用于确定这些泵在成年大鼠海马和培养神经元中的定位。功能研究将涉及跨上皮Ca2+通量测量和比例Ca2+成像极化MDCK肾细胞。这项工作将探索PMCAs的生理作用与其在膜中的精确定位密切相关的新概念。与公共卫生相关：PMCA2在神经元中含量丰富，并特异性定位于这些细胞内。虽然细胞内Ca2+运动是神经转导的重要组成部分，但控制Ca2+运动的通道和泵的相对作用尚未得到很好的理解。这些研究将帮助我们了解PMCA2有助于控制Ca2+的机制。了解这些机制将有助于我们理解和对抗由局部钙调节缺陷引起的疾病，如听力损失和衰老中的神经元变性。

DESCRIPTION (provided by applicant): Plasma membrane calcium ATPases (PMCAs) pump Ca2+ out of all animal cells. As components of the Ca2+ signaling "toolbox", the PMCAs are localized in distinct plasma membrane domains. The importance of proper membrane targeting is illustrated by diseases characterized by the absence of specific PMCA isoforms in the membrane, such as deafness caused by a lack of PMCA2 in cochlear hair cell stereocilia. The long-term goal is to understand the mechanism and functional impact of specific membrane targeting of the PMCAs. The specific aims are (1) to identify the apical targeting elements in PMCA2 splice variants; (2) to determine if and how PDZ protein interactions stabilize PMCA2 in the apical membrane; (3) to determine if specific localization of PMCA2 splice variants alters trans-epithelial Ca2+ flux and global Ca2+ signaling in polarized epithelial cells; and (4) to determine the subcellular distribution and identify neuronal targeting elements of PMCA2 variants in hippocampal neurons. The studies will involve confocal fluorescence microscopy and two-hybrid interaction analyses to identify specific targeting elements of PMCA2 splice variants. Fluorescence recovery after photobleaching and half-life studies will be employed to analyze the membrane dynamics of PMCA2 isoforms, and light and electron microscopy will be used to determine the localization of these pumps in adult rat hippocampus and cultured neurons. Functional studies will involve trans-epithelial Ca2+ flux measurements and ratiometric Ca2+ imaging in polarized MDCK kidney cells. This work will explore the novel concept that the physiological role of the PMCAs is tightly linked to their precise localization in the membrane. Relevance to public health: PMCA2 is abundant in neurons and is specifically localized within these cells. Although intracellular Ca2+ movements are an essential part of neurotransduction, the relative roles of the channels and pumps which control Ca2+ movements are not well understood. These studies will help us understand the mechanisms by which PMCA2 contributes to control of Ca2+. Understanding these mechanisms will help us understand and fight diseases caused by defects in local calcium regulation, such as hearing loss and neuronal degeneration in aging.