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

通过高压门控Ca2+通道（HVGCCs）的Ca2+内流是将电信号转化为可兴奋细胞生理反应所必需的，并介导多种功能，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节。从结构上看，hvgc是一种杂聚大分子复合物，由形成孔隙的α1多肽和辅助亚基（β、α2δ，有时还有γ）组成。CaVβ亚基有四种亚型（β1-β4），分别由不同的CACNB1-CACNB4基因编码。CaVβs是将α1亚基运输到质膜所必需的细胞质蛋白，也调节通道门控的各个方面（激活和失活的电压依赖性和动力学；以及打开概率，Po）。体感神经元表达多种HVGCC成孔α1 （CaV1.2、CaV2.1、CaV2.2和CaV2.3）和β (β1-β4)亚基亚型。尽管CaVα1亚基的表达保持不变，但神经损伤导致HVGCC电流显著降低。相比之下，β3和β4（而不是β1和β2）在神经损伤后背根神经节（DRG）神经元的一些群体中表达下降，可能是全细胞Ca2+电流（ICa）减少的原因，并导致了病理变化。虽然不同的CaVα1亚基的功能作用很容易使用可用的小分子和毒素阻滞剂进行研究，但由于缺乏基于与CaVβ相关的同种异构体的身份能够在翻译后抑制hvgcc的工具，CaVβ分子多样性的功能逻辑尚未得到充分研究和重视。在之前的工作中，我们开创了一种独特的靶向泛素化方法，通过将纳米体（nb）连接到E3泛素连接酶NEDD4L（称为cav -a - βlator）的催化HECT区域，非选择性地结合所有辅助CaVβs，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 -a - β后期翻译抑制整个通道复合物（而不是仅去除目标β亚基），限制了由于不同CaVβ亚型的分子重组或重叠功能而导致的混淆解释。本研究的总体目标是：1)利用靶向泛素化原理，基于相关CaVβ-异构体的特性，开发一种蛋白质工程方法，用于翻译后抑制HVGCC复合物；2)利用新创建的遗传编码抑制剂来探索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目标：(1)开发基因编码的cav β-异构体特异性HVGCC抑制剂（Chisels）。(2)利用工程化的CaVβ靶向纳米体探索CaVβ分子多样性在体感觉神经元中的功能逻辑。这项研究是美国国立卫生研究院帮助结束长期成瘾（HEAL）倡议的一部分，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.