Role of a-actinin in Cav1.2 Function

α-Actinin在Cav1.2功能中的作用 摘要 L类钙离子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，人们对它的细胞生物学和调控知之甚少。我们的 长期的兴趣是确定调节L类型的通道Cav1.2的分子机制(例如， 理科293，98；理科293,2205；PNA103,7500；神经元78,483)，这是最普遍的L类型 大脑和心脏的经络。我们最近发现，α-肌动蛋白直接与中央毛孔的智商基序结合- 形成Cav1.2亚基α11.2并增强其表面定位(神经元78,483)。我们现在确定了三个 IQ基序中的点突变会单独损害α-肌动蛋白结合。我们初步的电生理学 表面标记数据表明，破坏α-肌动蛋白与IQ基序的结合会降低表面 表达式，并且意外地还包括信道开放概率(Po)。目标1是第一个全面的 WT和点突变Cav1.2通过ER-Golgi-TgN分泌途径的转运动力学分析 通过表面生物素化，N-糖基化分析和细胞内循环和降解途径 与Bip和Rab5等各自的标记进行共定位。目标2是确定和比较电流密度， 门控电流和单通道电流来测试α-肌动蛋白结合缺陷点突变是否 骑士队1.2降低了PO。我们的结构分析正在指导明确的(！) Cav1.2突变体表面表达缺陷和Po缺失与α-肌动蛋白结合缺失的关系。我们 发现钙离子通过Cav1.2特异性地内流导致α-肌动蛋白从IQ基序置换 Ca~(2+)/Ca~(2+)/CaM通过内吞作用和Po的还原作用同时下调Cav1.2的表达。目标3是解开 α-肌动蛋白和钙调蛋白在IQ基序上的相互作用以精确定义糖尿病的分子机制 钙/钙调蛋白如何取代α-肌动蛋白，导致细胞内吞和Cav1.2衰竭。AIM 4将首先进行测试 α-Actinin在神经细胞Cav1.2功能中的作用，包括钙离子内流和基因调控 以及为什么α与肌动蛋白的关联以及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。Cav1.2渠道活跃度增加 导致老年症状和阿尔茨海默病(例如，科学272,1017；科学243,809)。Cav1.2还扮演着一个 在创伤后应激障碍和Cav1.2调节失调中的重要作用会导致自闭症谱系障碍(细胞119，19-31)。因此， 我们关于Cav1.2与α-肌动蛋白和钙调蛋白功能相互作用的工作对 了解并最终治疗这些脑部疾病。从更广泛的角度来看，它是生理学的 重要，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 神经元及其以外的功能，包括学习和记忆。

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.