Role of a-actinin in Cav1.2 Function

a-肌动蛋白在 Cav1.2 功能中的作用

• 批准号: 9333166
• 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
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333166
• 关键词: Actinin; Action Potentials; Affect; Alzheimer' s Disease; Alzheimer' s disease model; Autistic Disorder; Back; Behavior; 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; 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; 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; 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

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-辅肌动蛋白在Cav1.2功能中的作用 摘要 L型Ca 2+通道与基因转录紧密耦合，控制神经元兴奋性，并介导 多种形式的突触可塑性。然而，它的细胞生物学和调控却知之甚少。我们 长期的兴趣是确定调节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），其是最普遍的L型 大脑和心脏的通道。我们最近发现α-辅肌动蛋白直接结合到中央孔的IQ基序上， 形成Cav1.2亚基α11.2并增强其表面定位（Neuron 78，483）。我们现在确定三个 IQ基序中单独损害α-辅肌动蛋白结合的点突变。我们初步的电生理检查 表面标记数据表明，削弱α-辅肌动蛋白与IQ基序的结合， 表达式，并且出乎意料地，还包括通道开放概率（Po）。目标1是第一个全面的 WT和点突变Cav1.2通过ER-高尔基体-TGN分泌途径的运输动力学分析 和内吞再循环和降解途径，通过表面生物素化，N-糖基化分析， 与各自的标记如BiP和Rab 5共定位。目的2是确定和比较电流密度， 门控电流和单通道电流，以测试是否α-辅肌动蛋白结合缺陷的点突变， Cav1.2已经降低了Po。我们的结构分析是指导明确的（！） 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。我们 发现Ca 2+通过Cav1.2特异性流入导致α-辅肌动蛋白从IQ基序中被置换， Ca 2 +/钙调素和平行运行下来的Cav1.2通过内吞作用和减少Po。目标3：解开 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 Ca 2 +/钙调蛋白如何取代α-辅肌动蛋白并导致Cav1.2的内吞和流失。Aim 4将首先进行测试 α-辅肌动蛋白在神经元Cav1.2功能中作用，包括Ca ~（2+）内流和基因调控 通过NFAT表达，然后为什么α-辅肌动蛋白协会，适当地，Cav1.2的表面表达， 在衰老和阿尔茨海默病（AD）的啮齿动物模型中增加。增加Cav1.2通道活性 导致老年症状和AD（例如，Science 272，1017; Science 243，809）。Cav1.2也扮演着 Cav1.2在PTSD中的重要作用和失调导致自闭症谱系障碍（Cell 119，19-31）。因此 我们对Cav1.2与α-辅肌动蛋白和钙调蛋白的功能相互作用的研究对于 理解并最终治疗这些脑部疾病。从更广泛的角度来看， 重要性，因为它将定义管理Cav1.2功能可用性的重要方面， 包括学习和记忆在内的神经元功能。