Sodium Ions and Calcium Signaling in Neurons and Glia

本研究的目的是阐明钠离子转运影响钙离子的基本机制 神经元和神经胶质细胞的动态平衡和信号传递。神经元和神经胶质细胞都表达A1的Na+泵 催化(A)亚单位的异构体和Na+泵与ot2(神经胶质细胞)或A3(神经元)异构体。NA+ 具有A2或A3异构体的泵定位于质膜-内质网(PM-ER)连接 复合体(“PLasmERosome”)，并与PM Na/Ca交换器(NCX)偶联，从而与胞浆偶联 ([Ca~(2+)]Cyr)和内质网钙离子浓度及细胞内钙信号调节。关键问题是：如何 A2和OC3钠离子泵是否已分类并系在其适当的PM目的地？那么，什么是 这个特殊组织在当地和全球的职能后果是什么？有四个具体目标：目标1。 为了确定Na+泵A2和A3亚单位如何被靶向并拴在它们适当的PM位置， A亚单位嵌合体(例如，a2和a1部分，反之亦然)，WT和突变的a截断，以及锚蛋白B 基因敲除小鼠将被用来检验Ot2和A3亚基针对PLasmERosome的假设 通过特定的N-末端氨基酸(AA)序列，并被锚蛋白B拴住。目的2.确定 质膜-内质网连接处的亚质膜钙离子浓度是否独立于“块状”[Ca~(2+)]Cyt- 新型近膜钙指示剂(FFP-18和G-cAMP-2，一种针对PM的工程蛋白- ER连接)将被用来直接测试PM-ER连接空间受oc2/a3调节的想法 星形胶质细胞和神经元的Na+泵和NCX1。目的3.确定Na+泵A2的连接如何 亚基异构体NCX1和Ankyrin B在局部钙调节和全球 星形胶质细胞中的钙信号。目的4.确定Na+泵和NCX在钙离子外流中的作用。 神经元树突和神经末梢以及这些过程如何影响神经元功能。AIMS 3 和4，零突变小鼠，以及分子生物学和药理学工具将被用于测试 假设质膜上关键的Na+和Ca2+转运体的结构连接使他们能够 在神经元(目标4)和星形胶质细胞的钙调节和信号传递中发挥关键的功能偶联作用 (目标3)。这些研究将为调节正常钙稳态的具体机制提供新的线索。 和信号，这可能会在缺氧/缺血和其他大脑病理过程中出错。

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 ot2 (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+]CYr) and ER Ca2+ concentration and Ca2+ signal regulation in these cells. Critical questions are: How are the a2 and oc3 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 ot2and 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 oc2/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.