Chemical biological dissection of Ca2+ entry through Ca2+ channels

描述（由申请人提供）：一种称为CaV1.3的电压激活Ca 2+渗透性离子通道正在成为进入位于脑节律和神经退行性疾病中心的神经元的卓越Ca 2+进入途径。打开CaV1.3所需的较低跨膜电压允许这些通道对起搏和亚阈值电压波动做出重要贡献。CaV1.3通道，从而构成了一个占主导地位的Ca 2+进入模块，许多神经元进行振荡和阈下活动。这种Ca 2+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Ca 2+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，黑质神经元的变性是帕金森病（PD）的中心，细胞内Ca 2+失调和过载是PD发病机制的关键。因此，一个非常有前途的途径，为新的PD治疗涉及新兴的搜索小分子，选择性地抑制CaV1.3通道的开放。然而，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秩相关性，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。倍增的挑战是最近的发现，CaV1.3通道不是单一的，而是由许多RNA编辑和剪接变体组成，每种变体对通道的开放概率PO具有潜在的不同影响。变异相关PO调节的机制目前尚不清楚。此外，GPCR介导的质膜脂质PIP 2的变化有力地调节PO，但尚不清楚这是如何发生的，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械空白排除了对Ca 2+通过这些通道进入如何有助于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途径。然而，事实证明，仅凭传统手段难以取得进展。因此，该项目建议通过将电生理学与新型化学生物学和活细胞FRET工具相结合来阐明CaV1.3 PO调节。总的来说，这一建议承诺优雅的澄清，简化，统一的CaV1.3 PO调制的看似不同的机制;识别通道接口，可以有针对性地发现小分子PO调节剂;和新的化学生物学和FRET为基础的工具，具有广泛的适用性。

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.