Nanobodies for Probing CACNA2D2 and CACNA2D3 Function, Expression, and Therapeutics

总结 CACNA 2D 1-CACNA 2D 4基因编码α 2 delta-1、α 2 delta-2、α 2 delta-3和α 2 delta-4蛋白，这些蛋白是最熟知的高电压激活的Ca 2+通道的辅助亚基。有七种不同的HVA CaV通道类型（CaV1.1 - CaV1.4; CaV2.1 - CaV2.3），并且通常，α 2 δ-1-α 2 δ-4蛋白可以与七种孔形成亚基中的任何一种混杂地相互作用，以增强细胞表面的运输和稳定通道，从而增加全细胞电流。然而，这些蛋白质具有广泛但不完全重叠的分布，并且人类中的功能丧失突变或动物模型中的个体敲除会引起不同的疾病表型，表明一些独特的功能特性。错义突变或α 2 δ-1和α 2 δ-2的敲除与包括癫痫和智力残疾在内的神经系统症状最显著相关; α 2 δ-3的缺失与包括神经胶质瘤和鼻咽癌在内的恶性肿瘤相关; α 2 δ-4的失调导致视网膜营养不良和夜盲症。该蛋白质家族作为治疗疾病的治疗靶点的潜力通过以下偶然发现而提高：临床上用于治疗神经性疼痛和癫痫的加巴喷丁类化合物通过结合α 2 δ-1发挥其作用。加巴喷丁与α 2 δ-1结合如何减轻神经性疼痛还不清楚。提出的候选机制包括CaV 2通道运输的慢性下调; α 2 δ-1与细胞外血小板反应蛋白相互作用的破坏;以及α 2 δ-1介导的NMDA型谷氨酸受体向背角中突触末端的运输的预防。因此，除了它们作为辅助CaV通道亚基的作用之外，α 2 δ亚基还可以与其他蛋白质差异地相互作用，这些蛋白质可能在其独特的生理功能中发挥作用。该建议主要集中在CACNA 2D 2和CACNA 2D 3上，这是IDG合格的待研究蛋白质列表中包含的两个基因。能够与这些蛋白质特异性相互作用并用于原位检测它们或操纵它们的功能的配体对于阐明它们的特定功能作用以及实现它们作为治疗靶点的潜力是必不可少的。在这里，我们建议开发nanobodies到alpha 2delta-2和alpha 2delta-3作为新的试剂来检测和操纵这些蛋白质在体外，原位和体内的功能。我们的实验室最近开发了针对辅助CaV的纳米抗体？亚基，并使用这些遗传编码的分子来调节CaV 1/CaV 2通道的功能表达，具有前所未有的精确度。我们从这些先前研究中获得的专业知识不仅证明了总体可行性，而且还提供了初步数据，支持我们的期望，即本文提出的实验将产生独特的试剂来阐明α 2 δ-2和α 2 δ-3的生物学和治疗潜力。(1)分离抗α 2 δ-2和α 2 δ-3的纳米抗体并表征其结合特征。(2)确定α 2 δ-2和α 2 δ-3纳米抗体对CaV通道功能表达的功能影响。(3)确定α 2 δ-2/α 2 δ-3纳米抗体对α 2 δ亚基的推定非CaV通道功能的影响。

SUMMARY CACNA2D1 – CACNA2D4 genes encode alpha2delta-1, alpha2delta-2, alpha2delta-3, and alpha2delta-4 proteins which are most well known as auxiliary subunits of high-voltage-activated Ca2+ channels. There are seven different HVA CaV channel types (CaV1.1 – CaV1.4; CaV2.1 – CaV2.3), and in general, alpha2delta-1 – alpha2delta-4 proteins can interact promiscuously with any of the seven pore-forming subunits to enhance trafficking and stabilize channels at the cell surface to increase whole-cell current. Nevertheless, these proteins have broad but not completely overlapping distributions, and loss-of-function mutations in humans, or their individual knockout in animal models, gives rise to distinct disease phenotypes, indicating some unique functional properties. Missense mutations or knockout of alpha2delta-1 and alpha2delta-2 are most prominently linked to neurological symptoms including epilepsy and intellectual disability; loss of alpha2delta-3 is associated with malignancies including gliomas and nasopharyngeal carcinoma; and dysregulation of alpha2delta-4 causes retinal dystrophy and night blindness. The potential of this protein family as therapeutic targets to treat disease was elevated by the serendipitous finding that gabapentinoids which are used clinically to treat neuropathic pain and epilepsy exert their actions via binding alpha2delta-1. It is less clear precisely how gabapentin binding to alpha2delta-1 alleviates neuropathic pain. Proposed candidate mechanisms include chronic down-regulation of CaV2 channel trafficking; disruption of alpha2delta-1 interaction with extracellular thrombospondins; and prevention of alpha2delta-1 mediated trafficking of NMDA type glutamate receptors to the synaptic terminus in the dorsal horn. Thus, beyond their role as auxiliary CaV channel subunits, alpha2delta subunits may also differentially interact with other proteins which may play a role in their distinctive physiological functions. This proposal is primarily focused on CACNA2D2 and CACNA2D3 which are two genes included in the list of IDG-eligible understudied proteins. Ligands that can specifically interact with these proteins and be used to either detect them in situ or manipulate their function are essential for illuminating their specific functional roles as well as realizing their potential as therapeutic targets. Here, we propose to develop nanobodies to alpha2delta-2 and alpha2delta-3 as novel reagents to detect and manipulate the functions of these proteins in vitro, in situ, and in vivo. Our lab has recently developed nanobodies against auxiliary CaV? subunits and used these genetically-encoded molecules to regulate functional expression of CaV1/CaV2 channels with unprecedented levels of precision. The expertise we have gained from these previous studies not only demonstrate overall feasibility, but also provides preliminary data that bolsters our expectations that the experiments proposed here will yield unique reagents to illuminate alpha2delta-2 and alpha2delta-3 biology and therapeutic potential. (1) Isolate nanobodies against alpha2delta-2 and alpha2delta-3 and characterize their binding characteristics. (2) Determine functional impact of alpha2delta-2 and alpha2delta-3 nanobodies on CaV channel functional expression. (3) Determine impact of alpha2delta-2/alpha2delta-3 nanobodies on putative non-CaV channel functions of alpha2delta subunits.