Membrane Targeting of Calcium-Pumping ATPase

描述(申请人提供)：质膜钙ATPase(PMCA)从所有动物细胞中泵出钙离子。作为钙信号“工具箱”的组成部分，PMCA定位于不同的质膜结构域。适当的膜靶向的重要性通过以膜上缺乏特定PMCA亚型为特征的疾病来说明，例如耳蜗毛细胞立体纤毛中缺乏PMCA2而导致的耳聋。长期的目标是了解PMCA的特定膜靶向的机制和功能影响。其具体目的是(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剪接变异体的特定定位是否改变极化上皮细胞中的跨上皮钙通量和全球钙信号；以及(4)确定PMCA2变异体在海马神经元中的亚细胞分布和神经靶向元件。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被用来分析PMCA2异构体的膜动力学，光学和电子显微镜将被用来确定这些泵在成年大鼠海马神经元和培养神经元中的定位。功能研究将涉及极化MDCK肾细胞的跨上皮钙流量测量和比率钙成像。这项工作将探索一个新的概念，即PMCA的生理作用与它们在膜上的精确定位密切相关。与公众健康相关：PMCA2在神经元中大量存在，并专门定位于这些细胞中。尽管细胞内钙离子运动是神经转导的重要组成部分，但控制钙离子运动的通道和泵的相对作用尚不清楚。这些研究将有助于我们理解PMCA2调节钙离子的机制。了解这些机制将有助于我们理解和抗击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.