Role of a-actinin in Cav1.2 Function

a-肌动蛋白在 Cav1.2 功能中的作用

基本信息

批准号：
9924426
负责人：
JOHANNES W HELL
金额：
$ 50.16万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9924426
关键词：
Actinin Action Potentials Affect Alzheimer&apos s Disease Alzheimer&apos s disease model Back Binding Biotinylation Brain Brain Diseases CREB1 gene Calcium-Activated Potassium Channel Calmodulin Cell Density Cell surface Cells Cellular biology Charge Cognitive Coupled Coupling Data Degradation Pathway Dendritic Spines Docking Electrophysiology (science)Electrostatics Endocytosis Endosomes Estrogen receptor positive Excision Fingers Fostering Functional disorder Future Gene Expression Gene Expression Regulation Genetic Transcription Glutamate Receptor Goals Golgi Apparatus Grant Health Heart Hippocampus (Brain)Hypoglycemia Immunologic Deficiency Syndromes Impairment Individual Internet Ions Kinetics Knock-in Mouse Label Learning Lobe Measures Mediating Memory Molecular Monitor Mus Mutate Mutation Neurons Nuclear Translocation Paper Pathologic Pathway interactions Periodicity Physiological Play Point Mutation Post-Traumatic Stress Disorders Probability Proteins Recycling Regulation Rodent Model Role Running Science Senility Signal Transduction Site Structural Protein Structure Surface Symptoms Synapses Synaptic plasticity System Testing Time Timothy syndrome Toes Toxic effect Transcriptional Activation Transcriptional Regulation Vertebral column Work alpha Actinin autism spectrum disorder autistic behaviour density experimental study glycosylation insight interest knock-down mutant neuron loss neuronal excitability patch clamp postsynaptic premature prevent synaptic function trafficking transcription factor transcription factor NF-AT c3

项目摘要

Role of α-actinin in Cav1.2 Function Abstract L-type Ca2+ channels are tightly coupled to gene transcription, control neuronal excitability, and mediate multiple forms of synaptic plasticity. Yet its cell biology and regulation is remarkably poorly understood. Our long term interest is to determine the molecular mechanisms that regulate the L-type channel Cav1.2 (e.g., Science 293, 98; Science 293, 2205; PNAS 103, 7500; Neuron 78, 483), which is the most prevalent L-type channel in brain and heart. We recently found that α-actinin binds directly to the IQ motif of the central pore- forming Cav1.2 subunit α11.2 and augments its surface localization (Neuron 78, 483). We now identify three point mutations in the IQ motif that individually impair α-actinin binding. Our preliminary electrophysiological and surface labeling data suggest that impairing α-actinin binding to the IQ motif decreases surface expression and, unexpectedly, also channel open probability (Po). Aim 1 is the very first comprehensive analysis of trafficking kinetics of WT and point mutated Cav1.2 through the ER-Golgi-TGN secretory pathway and endocytic recycling and degradation pathway by surface biotinylation, N-glycosylation analysis and colocalization with respective markers like BiP and Rab5. Aim 2 is to determine and compare current density, gating currents, and single channel currents to test whether the α-actinin binding - deficient point mutations of Cav1.2 have decreased Po. Our structural analysis is guiding charge reversal experiments for unequivocal (!) assignment of deficits in surface expression and Po in the Cav1.2 mutants to loss of α-actinin binding. We found that Ca2+ influx specifically through Cav1.2 leads to displacement of α-actinin from the IQ motif by Ca2+/calmodulin and in parallel to run down of Cav1.2 via endocytosis and reduction in Po. Aim 3 is to unravel the interplay between α-actinin and calmodulin at the IQ motif to precisely define the molecular mechanisms of how Ca2+/calmodulin displaces α-actinin and leads to endocytosis and run down of Cav1.2. Aim 4 will test first the role of α-actinin in neuronal Cav1.2 functions including Ca2+ influx into spines and regulation of gene expression via NFAT and then why α-actinin association and, fittingly, surface expression of Cav1.2 is increased in rodent models of senility and Alzheimer’s disease (AD). Increased Cav1.2 channel activity contributes to senile symptoms and AD (e.g., Science 272, 1017; Science 243, 809). Cav1.2 also plays an important role in PTSD and dysregulation of Cav1.2 leads to autism spectrum disorders (Cell 119, 19-31). Thus our work on the functional interplay of Cav1.2 with α-actinin and calmodulin is of high significance for understanding and ultimately treatment of these brain diseases. On a broader perspective it is of physiological importance as it will define important aspects that govern the functional availability of Cav1.2 with its manifold functions in neurons and beyond including learning and memory.

α-肌动蛋白在 Cav1.2 功能中的作用抽象的 L 型 Ca2+ 通道与基因转录紧密耦合，控制神经兴奋性并介导然而，人们对突触可塑性的多种形式却知之甚少。长期兴趣是确定调节 L 型通道 Cav1.2 的分子机制（例如， Science 293, 98；Science 293, 2205；PNAS 103, 7500；Neuron 78, 483)，这是最常见的 L 型我们最近发现 α-肌动蛋白直接与中央孔的 IQ 基序结合。形成 Cav1.2 亚基 α11.2 并增强其表面定位（神经元 78、483）。 IQ 基序中的点突变会单独损害 α-肌动蛋白结合。表面标记数据表明，削弱 α-肌动蛋白与 IQ 基序的结合会降低表面表达式，并且出乎意料的是，通道打开概率 (Po) 是第一个综合值。 WT 和点突变 Cav1.2 通过 ER-Golgi-TGN 分泌途径的运输动力学分析通过表面生物素化、N-糖基化分析和内吞回收和降解途径与 BiP 和 Rab5 等相应标记的共定位是为了确定和比较电流密度，门控电流和单通道电流来测试 α-辅肌动蛋白结合是否存在缺陷点突变 Cav1.2 降低了 Po。我们的结构分析正在指导电荷反转实验，以实现明确的（！）将 Cav1.2 突变体中表面表达和 Po 的缺陷归因于 α-辅肌动蛋白结合的丧失。发现 Ca2+ 专门通过 Cav1.2 流入，导致 α-肌动蛋白从 IQ 基序中移位 Ca2+/钙调蛋白以及通过内吞作用降低 Cav1.2 并减少 Po 的目的是解开谜团。 α-辅肌动蛋白和钙调蛋白在 IQ 基序上的相互作用，以精确定义 Ca2+/钙调蛋白如何取代 α-肌动蛋白并导致胞吞作用和 Cav1.2 的耗尽将首先进行测试。 α-辅肌动蛋白在神经元 Cav1.2 功能中的作用，包括 Ca2+ 流入棘和基因调节通过 NFAT 表达，然后为什么 α-辅肌动蛋白关联以及 Cav1.2 的表面表达是在衰老和阿尔茨海默病 (AD) 的啮齿动物模型中，Cav1.2 通道活性增加。导致老年症状和 AD（例如，Science 272, 1017；Science 243, 809）。 Cav1.2 在 PTSD 中发挥重要作用，而 Cav1.2 失调会导致自闭症谱系障碍（细胞 119、19-31）。我们对 Cav1.2 与 α-肌动蛋白和钙调蛋白功能相互作用的研究对于从更广泛的角度来看，这些脑部疾病的理解和最终治疗是生理学的。重要性，因为它将定义控制 Cav1.2 及其流形的功能可用性的重要方面神经元及其他功能，包括学习和记忆。