Targeting and function of presynaptic Ca2+ channels

描述（由申请人提供）：Ca2+通道介导电压依赖性Ca2+内流在神经元的亚细胞区室，触发多种过程，如神经递质释放，树突动作电位和兴奋-转录耦联。负责快速突触传递的Ca2+通道之一是P/ q型Ca2+通道。一个尚未解决的基本问题是Ca2+通道及其相关的调节蛋白如何靶向适当的细胞室，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Ca2+通道亚基及其胞内结构域负责海马神经元轴突/树突Ca2+通道复合物的靶向。然后，我们将利用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/树突靶向与它们在突触传递中的特定作用联系起来。这些实验将描述负责脊髓小脑性共济失调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+通道复合物结瘤的特异性，并将特异性调节与相互作用蛋白的结构联系起来。蛋白质相互作用将使用新开发的两种杂交系统和在异源表达系统中与G蛋白构建的Ca2+通道复合物共表达进行分析。结果将验证P/ q型Ca2+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种突触前Ca2+通道类型的G蛋白亚基的蛋白质结构域。

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.