Novel genetically-encoded inhibitors to probe functional logic of Cav-beta molecular diversity

通过高压门控Ca 2+通道（HVGCC）的Ca 2+内流对于将电信号转化为可兴奋细胞中的生理反应是必需的，并且介导多种功能，包括控制神经递质释放、调节神经元兴奋性以及将电活动耦合到基因表达调控。在结构上，HVGCC是异源多聚体大分子复合物，由成孔α1多肽与辅助（β、α2δ，在某些情况下还有γ）亚基组装而成。CaVβ亚基有四种亚型（β1-β4），由不同的基因编码-CACNB 1-CACNB 4。CaVβs是将α1亚基运输到质膜所必需的胞质蛋白，并且还调节通道门控的各个方面（电压依赖性和激活和失活的动力学;以及开放概率，Po）。躯体感觉神经元表达多种HVGCC孔形成α1（CaV1.2、CaV2.1、CaV2.2和CaV2.3）和β（β1-β4）亚单位亚型。神经损伤导致HVGCC电流显著降低，即使CaVα1亚基的表达保持不变。相比之下，神经损伤后，在背根神经节（DRG）神经元的某些群体中，β3和β4（而不是β1和β2）表达下降，这可能是全细胞Ca 2+电流（伊卡）减少的基础，并有助于病理学。虽然不同CaVα1亚基的功能作用很容易使用可用的小分子和毒素阻断剂进行研究，但由于缺乏能够基于与它们相关的CaVβ同种型的身份来抑制HVGCC的工具，CaVβ分子多样性的功能逻辑未得到充分研究和充分评价。在以前的工作中，我们开创了一种独特的靶向泛素化方法，通过将非选择性结合所有辅助CaVβ的纳米抗体（nb）连接到E3泛素连接酶NEDD 4L（称为CaV-aβlator）的催化HECT结构域，有效地从细胞表面去除HVGCC复合物。与shRNA敲除或基因敲除方法相比，CaV-α βlator在后处理时抑制整个通道复合物（而不是仅去除靶向β亚基），从而限制了由于不同CaVβ同种型的分子重排或重叠功能而导致的混淆解释。该提案的总体目标是：1）基于相关CaVβ-同种型的身份，利用靶向泛素化原理，开发用于HVGCC复合物翻译后抑制的蛋白质工程方法，以及2）利用新创建的遗传编码抑制剂来探测DRG神经元中CaVβ分子多样性的功能逻辑。我们提出了两个具体的目标：（1）开发基因编码的CaVβ-亚型特异性HVGCC抑制剂（Chisels）。(2)利用工程化CaVβ靶向纳米抗体来探测体感神经元中CaVβ分子多样性的功能逻辑。这项研究是NIH帮助结束长期成瘾（HEAL）计划的一部分，旨在加速科学解决方案，以了解疼痛的基础并加强临床疼痛管理。

Ca2+ influx through high-voltage-gated Ca2+ channels (HVGCCs) is necessary for converting electrical signals into physiological responses in excitable cells, and mediates diverse functions including controlling neurotransmitter release, tuning neuronal excitability, and coupling electrical activity to regulation of gene expression. Structurally, HVGCCs are hetero-multimeric macromolecular complexes comprised of a pore-forming α1 polypeptide assembled with auxiliary (β, α2δ, and in some cases γ) subunits. There are four CaVβ subunit isoforms (β1-β4) encoded by distinct genes – CACNB1–CACNB4. CaVβs are cytosolic proteins that are necessary for trafficking α1 subunits to the plasma membrane and also regulate various aspects of channel gating (voltage-dependence and kinetics of activation and inactivation; and open probability, Po). Somatosensory neurons express multiple HVGCC pore-forming α1 (CaV1.2, CaV2.1, CaV2.2, and CaV2.3) and β (β1-β4) subunit isoforms. Nerve injury leads to significantly decreased HVGCC currents even though expression of CaVα1 subunits remain unchanged. By contrast, β3 and β4 (but not β1 and β2) expression declines in some populations of dorsal root ganglion (DRG) neurons after nerve injury, potentially underlying the diminished whole-cell Ca2+ current (ICa) and contributing to pathology. While the functional roles of distinct CaVα1 subunits are readily studied using available small molecule and toxin blockers, the functional logic of CaVβ molecular diversity is understudied and underappreciated due to a lack of tools capable of post-translationally inhibiting HVGCCs based on the identity of the CaVβ isoform associated with them. In previous work, we pioneered a unique targeted ubiquitination approach that effectively removes HVGCC complexes from the cell surface by linking a nanobody (nb) that non-selectively binds all auxiliary CaVβs to the catalytic HECT domain of the E3 ubiquitin ligase, NEDD4L (termed CaV-aβlator). By contrast to shRNA knockdown or gene knockout approaches, CaV-aβlator post-translationally inhibits the entire channel complex (rather than remove just the targeted β subunit) limiting confounding interpretations due to molecular reshuffling or overlapping functions of distinct CaVβ isoforms. The overall objectives of this proposal are to: 1) develop a protein engineering approach for the post-translational inhibition of HVGCC complexes based on the identity of the associated CaVβ-isoform, utilizing the principle of targeted ubiquitination, and 2) exploit newly created genetically-encoded inhibitors to probe the functional logic of CaVβ molecular diversity in DRG neurons. We propose two specific Aims: (1) Develop genetically-encoded CaVβ-isoform specific HVGCC inhibitors (Chisels). (2) Utilize engineered CaVβ-targeted nanobodies to probe the functional logic of CaVβ molecular diversity in somatosensory neurons. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to understand the basis of pain and enhance clinical pain management.