Targeting and function of presynaptic Ca2+ channels

突触前 Ca2 通道的靶向和功能

• 批准号: 6605805
• 负责人: STEFAN HERLITZE
• 金额: $ 41.29万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-03 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605805
• 关键词: G protein; calcium channel; calcium flux; gene mutation; laboratory mouse; neural transmission; protein localization; tissue /cell culture; voltage gated channel

DESCRIPTION (provided by applicant): Ca2+ channels mediate voltage-dependent Ca2+ influx in subcellular compartments of neurons, triggering such diverse processes such as neurotransmitter release, dendntic action potentials and excitation-transcnption coupling. One of the Ca2+ channels responsible for fast synaptic transmission is the P/Q-type Ca2+ channel. A fundamental question that remains unsolved is how Ca2+ channels and their associated modulatory proteins are targeted to the appropriate cellular compartments like presynaptic terminals to fulfil their designated function. In order to understand this question we will first determine which P/Q-type Ca2+ channel subunits and their intracellular domains are responsible for axonal/dendritic targeting of the Ca2+ channel complexes in hippocampal neurons. We will then correlate the axonal/dendritic targeting of P/Q-type Ca2+ channel complexes with their specific role in synaptic transmission using hippocampal neurons in culture from VQ-type channel knock out mice. These experiments will descnbe how changes in the localization and biophysical properties of wild type and mutated Ca2+ channels responsible for spinocerebellar ataxia 6 (SCA6) phenotypes effect synaptic transmission. Following these experiments we will analyze the specificity of the nodulation of these axonal/dendritic targeted Ca2+ channel complexes by G proteins and relate the specificity n modulation to the structure of the interacting proteins Protein interactions will be analyzed using a new developed two hybrid system and co-expression of Ca2+ channel complexes with G protein constructs in heterologous expression systems. The results will verify whether P/Q-type Ca2+ channels including their mutations have different specificity for G protein subunits and will identify the protein domains of the G protein subunits responsible for modulation of this presynaptic Ca2+ channel type. Elucidating the mechanisms that regulate Ca2+ channel targeting is critical to understanding both the basic physiology of neurons as well as several important neurological diseases. SCA6 appears to be caused by mutations in P/Q-type voltage-gated Ca2+ channels responsible for transmitter release. The identified mutations alter the biophysical properties of Ca2+ channel and change their potency to interact with intracellular modulating proteins like G proteins and Ca2+ channel ancillary subunits. Therefore a better understanding of the molecular epitopes underlying targeting, assembly and regulation of Ca2+ channels in subcellular compartments of neurons will help to design new strategies for treating ataxia and may identify new diseases related to ionic channel targeting.

描述(申请人提供)：钙离子通道介导电压依赖性的钙离子内流进入神经元的亚细胞室，触发不同的过程，如神经递质释放、树突动作电位和兴奋-传递耦合。负责突触快速传递的钙通道之一是P/Q型钙通道。一个尚未解决的基本问题是，如何将钙离子通道及其相关的调制蛋白定位于适当的细胞间隔，如突触前终末，以实现其指定的功能。为了理解这个问题，我们将首先确定哪些P/Q型钙通道亚基及其胞内结构域负责海马神经元中钙通道复合体的轴突/树突靶向。然后，我们将使用VQ型通道敲除小鼠培养的海马神经元，将P/Q型钙通道复合体的轴突/树突靶向与它们在突触传递中的特定作用联系起来。这些实验将描述与脊髓小脑性共济失调6(SCA6)表型有关的野生型和突变钙通道的定位和生物物理性质的变化如何影响突触传递。在这些实验之后，我们将分析G蛋白对这些轴突/树突状钙通道复合体结瘤的特异性，并将这种特异性n调节与相互作用蛋白的结构联系起来。我们将使用新开发的双杂交系统来分析蛋白质的相互作用，并在异源表达系统中将钙通道复合体与G蛋白构建物共表达。这些结果将验证P/Q型钙通道及其突变是否对G蛋白亚基具有不同的特异性，并将确定负责调节这一突触前钙通道类型的G蛋白亚基的蛋白结构域。 阐明调控钙通道靶向的机制对于理解神经元的基本生理学以及几种重要的神经系统疾病都是至关重要的。SCA6似乎是由负责递质释放的P/Q型电压门控钙通道突变引起的。已发现的突变改变了钙通道的生物物理性质，改变了它们与G蛋白和钙通道辅助亚单位等细胞内调节蛋白相互作用的效力。因此，更好地了解神经元亚细胞室中钙离子通道靶向、组装和调控的分子表位，将有助于设计治疗共济失调的新策略，并可能发现与离子通道靶向相关的新疾病。