Modulators of CaV1.3 Ca2+ regulation

描述（由申请人提供）：CaV1.3通道是低阈值，二氢吡啶敏感的l型Ca2+通道，介导全身低压信号和节律性。它们对于带状突触（如耳蜗毛细胞中的突触）的神经递质释放至关重要；它们调节心脏的起搏；它们调节整个大脑的振荡行为，比如交叉上核（昼夜节律节律回路）和黑质核（帕金森氏症的原发性损伤位点）的反复破裂。因此，这些通道的过度活动可能会导致Ca2+超载，从而诱发帕金森病，而这些通道的向下调节可能会增强积极的情绪和情绪。显然，选择性抑制或增强CaV1.3通道的小分子化合物，而不是其他CaV1 l型通道，将对CaV1.3作用的基础研究具有巨大的实用性，并可能改善许多CaV1.3相关的病理。然而，虽然已经发现了优秀的l型通道拮抗剂和激动剂，但没有一种可以真正选择l型通道亚型。在这里，为了寻找选择性调节剂，我们将利用CaV1.3通道的ICDI和IQ结构域之间独特的分子相互作用，其中这种相互作用调节这些通道的Ca2+反馈抑制（CDI）的强度。这一前景光明的屏幕将根据三个具体目标进行起诉。1)利用35 -50万个化合物的MLSMR文库，对破坏或增强CaV1.3通道IQ和ICDI结构域之间功能关键相互作用的小分子进行初步筛选。2)使用基于显微镜的单个活细胞FRET分析来确认和鉴定Aim 1中的候选命中点。3)利用膜片钳电生理学方法测试候选化合物对CaV1.3 Ca2+调节的调节作用。总体而言，该项目有望为CaV1.3与其他CaV1 l型钙通道的选择性调节剂提供领先的候选物质。

DESCRIPTION (provided by applicant): CaV1.3 channels are low-threshold, dihydropyridine-sensitive L-type Ca2+ channels which mediate low-voltage signaling and rhythmicity throughout the body. They are essential for neurotransmitter release at ribbon synapses such as found in cochlear hair cells; they mediate pacemaking in the heart; and they modulate oscillatory behavior throughout the brain, such as the repetitive bursting in supra-chiasmatic (circadian pacemaking circuitry) and substantia nigra (locus of primary damage in Parkinson's) nuclei. As such, overactivity of these channels may predispose for Ca2+ overload precipitating Parkinson's, and downward modulation of these channels may enhance positive mood and affect. Clearly, small-molecule compounds that selectively inhibit or enhance CaV1.3 channels, rather than the other CaV1 L-type channels would be of enormous utility for basic studies of CaV1.3 roles, and for potentially amerliorating a number of CaV1.3-related pathologies. However, though excellent L-type channels antagonists and agonists have been discovered, none can truly select among the L-type channel subtypes. Here, in the search for selective modulators, we will exploit a unique molecular interaction between ICDI and IQ domains of CaV1.3 channels, where this interaction modulates the strength Ca2+ feedback inhibition (CDI) of these channels. This promising screen will be prosecuted according to three specific aims. 1) To perform a primary screen for small molecules that disrupt or enhance a functionally critical interaction between IQ and ICDI domains of CaV1.3 channels, using the MLSMR library of 350,000-500,00 compounds. 2) To confirm and identify candidate hits from Aim 1 using a microscope-based FRET analysis of single living cells. 3) To test candidate compounds for modulation of CaV1.3 Ca2+ regulation, using patch-clamp electrophysiology. Overall, this project promises lead candidates for selective modulators of CaV1.3 versus other CaV1 L-type calcium channels.