Chemical biological dissection of Ca2+ entry through Ca2+ channels

Ca2+通过Ca2+通道进入的化学生物学解剖

• 批准号: 9322758
• 负责人: Ivy E Dick
• 金额: $ 27.83万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9322758
• 关键词: Address; Affect; Affinity; Automobile Driving; Binding; Biological; Biological Assay; Brain; Calcium Channel; Calmodulin; Cells; Chemicals; Confidential Information; Dimensions; Dissection; Electrophysiology (science); Empiricism; Employee Strikes; Fluorescence Resonance Energy Transfer; Formulation; G-Protein-Coupled Receptors; Health; Ion Channel; Kinetics; Learning; Left; Life; Lipids; Mediating; Monitor; Movement; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenesis; Pathway interactions; Periodicity; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phosphoric Monoester Hydrolases; Physiological; Probability; Process; RNA Editing; RNA Splicing; Role; System; Therapeutic; Variant; base; drug discovery; novel; novel therapeutics; residence; sensor; small molecule; tool; voltage

DESCRIPTION (provided by applicant): One type of voltage-activated Ca2+-permeable ion channel, known as CaV1.3, is emerging as a preeminent Ca2+ entry pathway into neurons residing at the epicenter of brain rhythmicity and neurodegenerative disease. The lower transmembrane voltages required to open CaV1.3 allow these channels to contribute importantly to pacemaking and subthreshold voltage fluctuations. CaV1.3 channels thus constitute a dominant Ca2+ entry module into many neurons undergoing oscillatory and subthreshold activity. Nowhere is this Ca2+ entry function more salient than in substantia nigral neurons, where CaV1.3 channels furnish the lion's share of Ca2+ entry, while driving rapid pacemaking essential for movement control. Notably, degeneration of substantia nigral neurons is central to Parkinson's disease (PD), and intracellular Ca2+ dysregulation and overload are crucial to PD pathogenesis. Accordingly, a highly promising avenue for novel PD therapeutics involves the burgeoning search for small molecules that selectively inhibit the opening of CaV1.3 channels. Yet, comparatively little is known about the mechanisms controlling the open probability PO of CaV1.3 channels. Ongoing small-molecule screens thereby rely on rank empiricism, largely bereft of known channel interfaces to which drug binding would likely alter opening. Multiplying the challenge is the recent discovery that CaV1.3 channels are not monolithic, but comprised of numerous RNA-edited and splice variants, each with potentially distinct effects on the open probability PO of channels. The mechanism underlying variant-related PO modulation is currently obscure. Additionally, GPCR-mediated changes in the plasmalemmal lipid PIP2 powerfully regulates PO, but it is unknown how this occurs, and how it relates to edited/splice variation. Together, the mechanistic void relating to these two systems precludes quantitative understanding of how Ca2+ entry through these channels contributes to pathogenesis, and obscures the path to rational small-molecule screens for CaV1.3 modulators. Yet, forward progress has proven difficult by traditional means alone. This project thus proposes to clarify CaV1.3 PO modulation by melding electrophysiology with novel chemical-biological and live-cell FRET tools. Overall, this proposal promises elegant clarification, simplification, an unification of seemingly diverse mechanisms of CaV1.3 PO modulation; identification of channel interfaces that could be targeted for discovery of small-molecule PO modulators; and new chemical-biological and FRET-based tools of wide applicability.

描述（由申请人提供）：一种电压激活的 Ca2+ 渗透性离子通道，称为 CaV1.3，正在作为一种卓越的 Ca2+ 进入神经元的通道出现，该神经元位于大脑节律性和神经退行性疾病的中心。打开 CaV1.3 所需的较低跨膜电压使这些通道对起搏和阈下电压波动发挥重要作用。因此，CaV1.3 通道构成了进入许多经历振荡和阈下活动的神经元的主要 Ca2+ 进入模块。这种 Ca2+ 进入功能在黑质神经元中最为突出，其中 CaV1.3 通道提供了大部分 Ca2+ 进入，同时驱动对运动控制至关重要的快速起搏。值得注意的是，黑质神经元变性是帕金森病 (PD) 的核心，而细胞内 Ca2+ 失调和超载对于 PD 发病机制至关重要。因此，新型 PD 疗法的一个非常有前途的途径是快速寻找选择性抑制 CaV1.3 通道开放的小分子。然而，人们对 CaV1.3 通道开放概率 PO 的控制机制知之甚少。因此，正在进行的小分子筛选依赖于经验主义，很大程度上缺乏药物结合可能会改变开放的已知通道界面。最近发现 CaV1.3 通道不是整体的，而是由许多 RNA 编辑和剪接变体组成，每个变体对通道的开放概率 PO 都有潜在的不同影响，这使挑战更加严峻。与变异相关的 PO 调节的潜在机制目前尚不清楚。此外，GPCR 介导的质膜脂质 PIP2 的变化可以强有力地调节 PO，但尚不清楚这是如何发生的，以及它与编辑/剪接变异的关系。总之，与这两个系统相关的机制空白阻碍了对 Ca2+ 通过这些通道进入如何促进发病机制的定量理解，并模糊了 CaV1.3 调节剂的合理小分子筛选的路径。然而事实证明，仅靠传统手段很难取得进展。因此，该项目建议通过将电生理学与新型化学生物和活细胞 FRET 工具相结合来阐明 CaV1.3 PO 调节。总体而言，该提案承诺对 CaV1.3 PO 调制看似不同的机制进行优雅的澄清、简化和统一；识别可用于发现小分子 PO 调节剂的通道接口；以及具有广泛适用性的新型化学生物和基于 FRET 的工具。