Modulators of CaV1.3 Ca2+ regulation

说明(申请人提供)：CaV1.3通道是低阈值、二氢吡啶敏感的L型钙通道，调节全身的低电压信号和节律性。它们对带状突触的神经递质释放是必不可少的，如在耳蜗毛细胞中发现的；它们调节心脏的起搏；它们调节整个大脑的振荡行为，例如交叉上(昼夜节律起搏回路)和黑质(帕金森病的初级损害部位)核的重复爆发。因此，这些通道的过度活跃可能导致钙超载导致帕金森氏症，而这些通道的下调可能会增强积极的情绪和影响。显然，选择性地抑制或增强CaV1.3通道的小分子化合物，而不是其他CaV1 L类型的通道，将对CaV1.3作用的基础研究以及潜在地缓解许多与CaV1.3相关的病理具有巨大的实用价值。然而，虽然已经发现了优秀的L型经络拮抗剂和激动剂，但还没有人能真正从L型经络亚型中挑选出来。在寻找选择性调节剂的过程中，我们将利用CaV1.3通道ICDI和IQ结构域之间的一种独特的分子相互作用，这种相互作用调节这些通道的钙反馈抑制(CDI)强度。这一前景看好的筛查将根据三个具体目标进行起诉。1)使用350,000-500，00种化合物的MLSMR文库，对破坏或增强CaV1.3通道的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的FRET分析来确认和鉴定来自AIM 1的候选命中。3)应用膜片钳电生理学方法检测CaV1.3钙调控的候选化合物。总体而言，该项目有望成为CaV1.3与其他CaV1 L型钙通道选择性调节剂的主要候选者。

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.