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：
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
项目类别：
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&apos s Disease Alzheimer&apos 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

Actinin Action Potentials Affect Alzheimer&apos s Disease Alzheimer&apos 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

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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，控制神经元和培养基紧密耦合多种形式的突触可塑性。然而，其细胞生物学和调节知之甚少。我们的长期关注是确定调节L型通道CAV1.2的分子机制（例如，科学293，98;科学293，2205; PNAS 103，7500;神经元78，483），这是最普遍的L型大脑和心脏的渠道。我们最近发现α-肌动蛋白直接与中央孔的智商基序结合形成CAV1.2亚基α11.2并增强其表面定位（Neuron 78，483）。我们现在确定三个智商基序中的点突变会损害α-肌动蛋白结合。我们的初步电生理学表面标记数据表明，损害α-肌动蛋白与智商基序的结合会下降表面表达，并且出乎意料地引导开放概率（PO）。 AIM 1是第一个综合通过ER-Golgi-TGN秘密途径分析WT和点突变Cav1.2的贩运动力学通过表面生物素化，N-糖基化分析和与BIP和RAB5等相对标记共定位。目标2是确定和比较电流密度，门控电流和单通道电流，以测试α-肌动蛋白结合 - 缺陷的点突变 Cav1.2已提前PO。我们的结构分析是指导电荷反向实验（！）（！） CAV1.2突变体中表面表达和PO的定义分配对α-肌动蛋白结合的丧失。我们发现Ca2+通过CAV1.2专门影响会导致α-肌动蛋白从IQ基序中位移 Ca2+/钙调蛋白，并平行于Cav1.2通过内吞作用和PO减少。目标3是解开 IQ基序处的α-肌动蛋白和钙调蛋白之间的相互作用，以精确定义分子机制 Ca2+/钙调蛋白如何置换α-肌动蛋白并导致内吞作用并降低Cav1.2。 AIM 4将首先测试 α-肌动蛋白在神经元CAV1.2功能中的作用，包括Ca2+影响脊柱的作用和基因调节通过NFAT的表达，然后是为什么α-肌动蛋白关联以及恰当地表达Cav1.2的表面表达啮齿动物敏感性和阿尔茨海默氏病（AD）的啮齿动物模型增加。 CAV1.2通道活动增加有助于老年符号和广告（例如科学272，1017; Science 243，809）。 cav1.2也扮演 CAV1.2的PTSD和失调的重要作用导致自闭症谱系障碍（细胞119，19-31）。那我们在CAV1.2与α-肌动蛋白和钙调蛋白的功能相互作用方面的工作对了解并最终治疗这些脑部疾病。从更广泛的角度来看，它是生理的重要的是，它将定义重要的方面，以控制Cav1.2的功能可用性神经元和以后的功能，包括学习和记忆。