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

新型基因编码抑制剂探索 Cav-beta 分子多样性的功能逻辑

• 批准号: 10581282
• 负责人: Henry M. Colecraft
• 金额: $ 44.98万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581282
• 关键词: Action Potentials; Animal Model; Binding; Biological Assay; Calcium Channel; Cell membrane; Cell surface; Cells; Chemicals; Closure by clamp; Complex; Coupling; Dependence; Electrophysiology (science); Engineering; Flow Cytometry; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Genes; Helping to End Addiction Long-term; Heterogeneity; Image; Inflammation; Kinetics; Knock-out; Knockout Mice; Libraries; Link; Logic; Macromolecular Complexes; Mediating; Molecular; Neurons; Pain; Pain management; Pathology; Pathway interactions; Phage Display; Pharmacology; Physiological; Population; Positioning Attribute; Probability; Protein Engineering; Protein Isoforms; Proteins; Proteome; Role; Signal Transduction; Speed; Spinal Ganglia; Toxin; Translational Repression; Ubiquitination; United States National Institutes of Health; Work; Yeasts; addiction; base; clinical pain; deep sequencing; inhibitor; knock-down; knockout gene; mechanical stimulus; nanobodies; nerve injury; neuronal excitability; neurotransmitter release; novel; opioid use disorder; painful neuropathy; patch clamp; polypeptide; response; small hairpin RNA; small molecule; somatosensory; tool; trafficking; ubiquitin-protein ligase; voltage; voltage clamp

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.

通过高压门控的Ca2+通道（HVGCC）的CA2+影响对于将电信号转换为出色的细胞中的物理反应，并介导了包括控制神经递质释放，调整神经递质兴奋的多样性功能，并将神经递质兴奋以及将电活动耦合到基因表达的电活活性。从结构上讲，HVGCC是与辅助（β，α2δ以及在某些情况下γ）亚基组装的孔形成的α1多肽的杂多物质大分子复合物。有四个CAVβ亚基同工型（β1-β4）由不同的基因-CACNB1 – CACNB4编码。 CAVβ是胞质蛋白，对于将α1亚基运输到质膜所需，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；开放概率； PO）。体感神经元表达多个HVGCC孔形成α1（Cav1.2，cav2.1，cav2.2和cav2.2）和β（β1-β4）亚基同工型。即使CAVα1亚基的表达保持不变，神经损伤也会显着降低HVGCC电流。相比之下，神经损伤后的某些背侧神经节神经元（DRG）神经元的某些种群的β3和β4（但β1和β2）的表达下降，有可能导致整个细胞Ca2+电流（ICA）降低并导致病理学。虽然使用可用的小分子和毒素阻滞剂很容易研究不同CAVα1亚基的功能作用，但由于缺乏能够基于与之相关的CAVβ亚型的身份，CAVβ分子多样性的功能逻辑被理解并被低估了HVGCC。在先前的工作中，我们开创了一种独特的靶向泛素化方法，该方法通过连接纳米机构（NB）有效地消除了HVGCC络合物，该方法将所有辅助CAVβ与E3泛素连接酶的催化Hect Hect域（NEDD4L（NEDD4L），NEDD4L（NEDD4L）（termeD cav-apapace-abaβlalator））。与ShRNA敲低或基因敲除方法相反，CAV-Aβlator译者后，跨化抑制了整个通道复合物（而不是仅除去靶向的β亚基），从而限制了由于分子改组或重叠的不同CAVβ同工型而引起的混杂解释。 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.我们提出了两个具体的目的：（1）开发一般编码的CAVβ-异型特异性HVGCC抑制剂（凿子）。 （2）利用工程化的CAVβ靶向纳米剂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的一部分，有助于长期（HEL）倡议结束成瘾，以加快科学解决方案，以了解疼痛的基础并增强临床疼痛管理。