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: 8890901
• 负责人: Ivy E Dick
• 金额: $ 35.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8890901
• 关键词: Address; Affect; Affinity; Automobile Driving; Binding; Biological; Biological Assay; Brain; Calcium Channel; Calmodulin; Cells; Chemicals; Confidential Information; Dimensions; Dissection; Drug Formulations; Electrophysiology (science); Empiricism; Employee Strikes; Fluorescence Resonance Energy Transfer; G Protein-Coupled Receptor Genes; 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，正在成为一种卓越的Ca2+进入位于脑节律性和神经退行性疾病中心的神经元的途径。打开CaV1.3所需的较低跨膜电压允许这些通道对起搏和阈下电压波动做出重要贡献。因此，CaV1.3通道构成Ca2+进入许多经历振荡和阈下活动的神经元的主要模块。这种Ca2+进入功能在黑质神经元中最为突出，其中CaV1.3通道提供了最大份额的Ca2+进入，同时驱动运动控制所必需的快速起搏器。值得注意的是，黑质神经元的退化是帕金森病（PD）的核心，细胞内Ca2+失调和超载是PD发病的关键。因此，一种非常有前途的新型PD治疗方法涉及到对选择性抑制CaV1.3通道开放的小分子的快速搜索。然而，对CaV1.3通道打开概率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等级经验主义，在很大程度上缺乏药物结合可能改变开放的已知通道界面。更大的挑战是最近发现CaV1.3通道不是单一的，而是由许多rna编辑和剪接变体组成，每个变体对通道的打开概率PO都有潜在的不同影响。变异相关的PO调制机制目前尚不清楚。此外，gpcr介导的质脂PIP2的变化有力地调节了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妨碍了对Ca2+如何通过这些通道进入促进发病机制的定量理解，并模糊了对CaV1.3调节剂进行合理小分子筛选的途径。然而，单靠传统手段取得进展已被证明是困难的。因此，该项目提出通过将电生理学与新的化学生物和活细胞FRET工具相结合来阐明CaV1.3 PO调制。总的来说，该提案承诺澄清、简化和统一CaV1.3 PO调制的看似不同的机制；确定可能用于发现小分子PO调节剂的通道界面；广泛应用的新型化学-生物和基于fret的工具。