Targeting and function of presynaptic Ca2+ channels

描述（由申请人提供）：Ca 2+通道介导神经元亚细胞区室中的电压依赖性Ca 2+内流，触发诸如神经递质释放、树突状动作电位和兴奋-转运偶联等多种过程。负责快速突触传递的Ca 2+通道之一是P/Q型Ca 2+通道。 一个尚未解决的基本问题是Ca 2+通道及其相关的调节蛋白如何靶向合适的细胞隔室，如突触前末梢，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Ca 2+通道亚基及其胞内结构域负责海马神经元中Ca 2+通道复合物的轴突/树突靶向。然后，我们将P/Q型Ca 2+通道复合物的轴突/树突靶向与它们在突触传递中的特定作用相关，使用来自VQ型通道敲除小鼠的培养物中的海马神经元。这些实验将描述负责脊髓小脑共济失调6（SCA 6）表型的野生型和突变型Ca 2+通道的定位和生物物理特性的变化如何影响突触传递。在这些实验之后，我们将分析这些轴突/树突靶向的Ca 2+通道复合物通过G蛋白的调节的特异性，并将特异性与相互作用蛋白的结构调节相关。将使用新开发的双杂交系统和Ca 2+通道复合物与G蛋白构建体在异源表达系统中的共表达来分析蛋白质相互作用。结果将验证P/Q型Ca 2+通道（包括其突变）是否对G蛋白亚基具有不同的特异性，并将鉴定负责调节这种突触前Ca 2+通道类型的G蛋白亚基的蛋白结构域。 阐明调节Ca 2+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至关重要。SCA 6似乎是由负责递质释放的P/Q型电压门控Ca 2+通道突变引起的。所鉴定的突变改变了Ca 2+通道的生物物理性质，并改变了它们与细胞内调节蛋白如G蛋白和Ca 2+通道辅助亚基相互作用的效力。因此，更好地了解分子表位的基础上的靶向，组装和调节的钙离子通道在亚细胞区室的神经元将有助于设计新的策略治疗共济失调，并可能确定新的疾病相关的离子通道靶向。

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.