Membrane Targeting of Calcium-Pumping ATPase

描述（由申请方提供）：质膜钙ATP酶（PMCA）将Ca 2+泵出所有动物细胞。作为Ca 2+信号“工具箱”的组成部分，PMCA位于不同的质膜结构域中。适当的膜靶向的重要性通过以膜中缺乏特异性PMCA同种型为特征的疾病来说明，例如由耳蜗毛细胞静纤毛中缺乏PMCA 2引起的耳聋。长期目标是了解PMCAs特异性膜靶向的机制和功能影响。具体目的是（1）鉴定PMCA 2剪接变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端膜中的PMCA 2;（3）确定PMCA 2剪接变体的特异性定位是否改变极化上皮细胞中的跨上皮Ca 2+通量和整体Ca 2+信号传导;以及（4）确定海马神经元中PMCA 2变体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PMCA 2剪接变体的特定靶向元件。将采用光漂白和半衰期研究后的荧光恢复来分析PMCA 2亚型的膜动力学，并使用光学和电子显微镜来确定这些泵在成年大鼠海马和培养的神经元中的定位。功能研究将涉及跨上皮Ca 2+通量测量和极化MDCK肾细胞中的比率Ca 2+成像。这项工作将探索新的概念，即PMCAs的生理作用与它们在膜中的精确定位紧密相关。与公共卫生的相关性：PMCA 2在神经元中含量丰富，并特异性地定位于这些细胞中。虽然细胞内Ca 2+运动是神经转导的重要组成部分，但控制Ca 2+运动的通道和泵的相对作用还不清楚。这些研究将有助于我们了解PMCA 2有助于控制Ca 2+的机制。了解这些机制将有助于我们了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.