Targeting and function of presynaptic Ca2+ channels

描述（由申请人提供）：Ca2+通道介导神经元亚细胞区室中电压依赖性Ca2+流入，触发多种过程，例如神经递质释放、树突动作电位和兴奋转录耦合。负责快速突触传递的 Ca2 通道之一是 P/Q 型 Ca2 通道。 尚未解决的一个基本问题是 Ca2 通道及其相关调节蛋白如何靶向适当的细胞区室（如突触前末梢）以实现其指定功能。为了理解这个问题，我们首先确定哪些 P/Q 型 Ca2 通道亚基及其细胞内结构域负责海马神经元中 Ca2 通道复合物的轴突/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 Ca2 通道复合物的轴突/树突靶向与其在突触传递中的特定作用相关联。这些实验将描述负责脊髓小脑共济失调 6 (SCA6) 表型的野生型和突变 Ca2 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 G 蛋白对这些轴突/树突靶向 Ca2 通道复合物结节的特异性，并将特异性调节与相互作用蛋白的结构联系起来。将使用新开发的两个混合系统以及异源表达系统中 Ca2 通道复合物与 G 蛋白构建体的共表达来分析蛋白质相互作用。结果将验证 P/Q 型 Ca2 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 Ca2 通道类型的 G 蛋白亚基的蛋白结构域。 阐明调节 Ca2 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。 SCA6 似乎是由负责递质释放的 P/Q 型电压门控 Ca2 通道突变引起的。已发现的突变改变了 Ca2 通道的生物物理特性，并改变了其与 G 蛋白和 Ca2 通道辅助亚基等细胞内调节蛋白相互作用的能力。因此，更好地了解神经元亚细胞区室中 Ca2+ 通道的靶向、组装和调节的分子表位将有助于设计治疗共济失调的新策略，并可能识别与离子通道靶向相关的新疾病。

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.