Sodium Ions and Calcium Signaling in Neurons and Glia

描述（由申请人提供）：本研究的目的是阐明Na+转运影响神经元和神经胶质细胞中Ca 2+稳态和信号传导的基本机制。神经元和神经胶质细胞都表达具有催化（a）亚基的a1同种型的Na+泵和具有a2（神经胶质细胞）或a3（神经元）同种型的Na+泵。具有α 2或α 3同种型的Na+泵定位于质膜-内质网（PM-ER）连接复合物（“PLAsmER体”），并与PM Na/Ca交换剂（NCX）偶联，从而与这些细胞中的胞质（[Ca 2 +]CYT）和ER Ca 2+浓度和Ca 2+信号调节偶联。关键问题是：a2和a3 Na+泵如何分类并连接到其适当的PM目的地？这种特殊组织的地方和全球功能后果是什么？有四个具体目标：目标1。为了确定Na+泵α 2和α 3亚基如何被靶向并拴系到其适当的PM位置，亚基嵌合体（例如，部分a2和部分a1，反之亦然）、WT和突变的α截短，以及锚蛋白B敲除小鼠将被用于检验以下假设：α 2和α 3亚基通过特定的N-末端氨基酸（AA）序列靶向PLASMA受体体，并被锚蛋白B栓系。目标二。为了确定PM-ER连接处的亚PM Ca 2+浓度是否独立于“本体”[Ca 2 +]CYT而被控制。新型近膜Ca 2+指示剂（FFP-18和G-CaMP-2，一种靶向PM-ER连接的工程蛋白）将用于直接测试PM-ER连接空间Ca 2+由星形胶质细胞和神经元中的α 2/α 3 Na+泵和NCX 1调节的想法。目标3：确定Na+泵α 2亚基亚型、NCX 1和锚蛋白B的连接如何在星形胶质细胞的局部Ca 2+调节和整体Ca 2+信号传导中发挥中心作用。目标4。确定Na+泵和NCX在神经元树突和神经终末Ca 2+流出中的作用以及这些过程如何影响神经元功能。对于目标3和4，将使用无效突变小鼠以及分子生物学和药理学工具来检验以下假设：PLasmER体中关键PM Na+和Ca 2+转运蛋白的结构连接使其能够在神经元（目标4）和星形胶质细胞（目标3）中的Ca 2+调节和信号传导中发挥关键功能偶联作用。这些研究将揭示调节正常Ca 2+稳态和信号传导的特定机制，以及在缺氧/缺血和其他脑病理过程中可能出错的机制。

DESCRIPTION (provided by applicant): The goal of this research is to elucidate the fundamental mechanisms by which Na+ transport influences Ca2+ homeostasis and signaling, in neurons and glia. Neurons and glia both express Na+ pumps with the a1 isoform of the catalytic (a) subunit and Na+ pumps with either the a2 (glia) or a3 (neurons) isoform. Na+ pumps with a2 or a3 isoforms are localized to plasma membrane-endoplasmic reticulum (PM-ER) junctional complexes ("PLasmERosomes") and are coupled to PM Na/Ca exchangers (NCX) and, thus, to cytosolic ([Ca2+]CYT) and ER Ca2+ concentration and Ca2+ signal regulation in these cells. Critical questions are: How are the a2 and a3 Na+ pumps sorted and tethered to their appropriate PM destinations? And, what are the local and global functional consequences of this special organization? There are four Specific Aims: Aim 1. To determine how Na+ pump a2 and a3 subunits are targeted and tethered to their appropriate PM locations, a subunit chimeras (e.g., part a2 and part a1, and vice-versa), WT and mutated a truncations, and ankyrin B knockout mice will be used to test the hypothesis that a2 and a3 subunits are targeted to PLasmERosomes by specific N-terminal amino acid (AA) sequences and are tethered by ankyrin B. Aim 2. To determine whether the sub-PM Ca2+ concentration at PM-ER junctions is controlled independently of "bulk" [Ca2+]CYT- Novel near-membrane Ca2+ indicators (FFP-18 and G-CaMP-2, an engineered protein targeted to the PM- ER junction) will be used to test, directly, the idea that PM-ER junctional space Ca2+ is regulated by a2/ a3 Na+ pumps and NCX1 in astrocytes and neurons. Aim 3. To determine how linkage of the Na+ pump a2 subunit isoform, NCX1, and ankyrin B contributes to their central roles in local Ca2+ regulation and global Ca2+ signaling in astrocytes. Aim 4. To determine the roles of the Na+ pump and NCX in Ca2+ efflux from neuronal dendrites and nerve terminals and how these processes influence neuronal function. For Aims 3 and 4, null mutant mice, and molecular biological and pharmacological tools will be used to test the hypothesis that structural linkage of key PM Na+ and Ca2+ transporters in PLasmERosomes enables them to serve critical functionally-coupled roles in Ca2+ regulation and signaling in neurons (Aim 4) and in astrocytes (Aim 3). These studies will shed new light on specific mechanisms that regulate normal Ca2+ homeostasis and signaling, and that may go awry during hypoxia/ischemia and other brain pathologies.