Chemical biological dissection of Ca2+ entry through Ca2+ channels

描述(申请人提供)：一种类型的电压激活的钙离子通道，称为CaV1.3，是进入位于脑节律性和神经退行性疾病震中的神经元的一种重要的钙进入途径。开放CaV1.3所需的较低跨膜电压允许这些通道对起搏和阈值以下电压波动做出重要贡献。因此，CaV1.3通道构成了许多经历振荡和阈值下活动的神经元的主要钙进入模块。这种钙进入功能在黑质神经元中最为显著，CaV1.3通道提供了钙离子进入的最大份额，同时驱动着对运动控制至关重要的快速起搏。值得注意的是，黑质神经元的变性是帕金森病(PD)的中心，细胞内钙调节失调和过载是PD发病的关键。因此，寻找选择性抑制CaV1.3通道开放的小分子是新的PD治疗方法的一个非常有希望的途径。然而，对CaV1.3通道开放概率PO的控制机制却知之甚少。因此，正在进行的小分子筛选依赖于等级经验主义，在很大程度上失去了药物结合可能改变开放的已知通道界面。使挑战倍增的是最近的发现，CaV1.3通道不是单一的，而是由许多RNA编辑和剪接变体组成，每个变体对通道的开放概率PO都有潜在的不同影响。变异体相关PO调控的机制目前尚不清楚。此外，GPCR介导的血浆膜脂PIP2的变化有力地调节PO，但尚不清楚这种变化是如何发生的，以及它如何与编辑/剪接变异相关。总之，与这两个系统相关的机制空白阻碍了对钙离子如何通过这些通道进入致病机制的定量理解，并掩盖了通往CaV1.3调节剂的合理小分子筛选的途径。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的化学-生物和活细胞FRET工具相结合来阐明CaV1.3 PO的调节。总体而言，这项建议承诺优雅地澄清、简化、统一CaV1.3 PO调制的看似不同的机制；识别可能被用于发现小分子PO调节器的通道接口；以及广泛适用的新的基于化学-生物和FRET的工具。

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.