Sodium Ions and Calcium Signaling in Neurons and Glia

神经元和神经胶质细胞中的钠离子和钙信号传导

• 批准号: 7545824
• 负责人: MORDECAI P BLAUSTEIN
• 金额: $ 39.54万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-08-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545824
• 关键词: Amino Acid Sequence; Amino Acids; Ankyrins; Astrocytes; Binding; Biological; Brain Hypoxia-Ischemia; Brain Pathology; C-terminal; Calcium; Camping; Cell membrane; Cells; Chimera organism; Complex; Coupled; Coupling; Dendrites; Destinations; Diffusion; Endoplasmic Reticulum; G-substrate; Goals; Homeostasis; Immunoprecipitation; Knockout Mice; Light; Location; Measures; Membrane; Membrane Microdomains; Molecular; Mutant Strains Mice; Mutate; N-terminal; Na(+)-K(+)-Exchanging ATPase; Nerve; Neuroglia; Neurons; Process; Protein Engineering; Protein Isoforms; Regulation; Research; Role; Signal Transduction; Sorting - Cell Movement; Testing; base; novel; sodium ion; tool

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.

本研究的目的是阐明Na+转运影响Ca 2+的基本机制 神经元和神经胶质中的稳态和信号传导。神经元和胶质细胞都表达Na+泵， 催化（a）亚基的同种型和Na+泵与α 2（神经胶质）或α 3（神经元）同种型。Na+ 具有α 2或α 3同种型的泵定位于质膜-内质网（PM-ER）连接处， 在一些实施方案中，磷脂酶复合物（“磷脂酶体”）与PM Na/Ca交换剂（NCX）偶联，并因此与细胞溶质Na/Ca交换剂（NCX）偶联。 ([Ca2+]CYr）和ER Ca ~（2+）浓度及Ca ~（2+）信号调节。关键问题是：如何 a2和a3 Na+泵是否分类并连接到相应的PM目的地？那么， 这一特殊组织的地方和全球功能后果？有四个具体目标：目标1。 为了确定Na+泵α 2和α 3亚基是如何靶向和束缚到它们适当的PM位置的， 亚基嵌合体（例如，部分a2和部分a1，反之亦然）、WT和突变的α截短体，以及锚蛋白B 将使用基因敲除小鼠来检验α 2和α 3亚基靶向PLASMER体的假设 通过特定的N-末端氨基酸（AA）序列连接，并通过锚蛋白B连接。目标二。以确定 PM-ER连接处的亚PM Ca 2+浓度是否独立于“本体”[Ca 2 +]CYT进行控制。 新的近膜Ca 2+指示剂（FFP-18和G-CaMP-2，一种靶向PM的工程蛋白质， ER连接）将用于直接测试PM-ER连接空间Ca 2+受α 2/α 3调节的想法 星形胶质细胞和神经元中的Na+泵和NCX 1。目标3.为了确定Na+泵a2 亚基亚型，NCX 1和锚蛋白B有助于它们在局部Ca 2+调节和全局Ca 2+调节中的中心作用。 星形胶质细胞中的Ca 2+信号。目标4。为了确定Na+泵和NCX在Ca 2+流出中的作用， 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。目标3 和4，无效突变小鼠，以及分子生物学和药理学工具将用于测试 这一假说认为，磷脂酶体中关键的质膜Na+和Ca 2+转运蛋白的结构连接使它们能够 在神经元（目的4）和星形胶质细胞中的Ca 2+调节和信号传导中发挥关键的功能偶联作用 (Aim 3）。这些研究将为调节正常Ca 2+稳态的特定机制提供新的线索 和信号传导，并且在缺氧/缺血和其他脑病理期间可能出错。