Molecular Architecture of the Mosquito Carbon Dioxide Receptor Complex

蚊子二氧化碳受体复合物的分子结构

• 批准号: 9808786
• 负责人: Conor James McMeniman
• 金额: $ 19.54万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-23 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808786
• 关键词: Aedes; Agonist; Alcohols; Alleles; Anatomy; Animals; Architecture; Behavior; Behavioral; Bicarbonates; Blood; Butanones; C-terminal; CRISPR/Cas technology; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Cells; Chemicals; Complex; Confocal Microscopy; Cues; Culicidae; Cyclohexanones; Dendrites; Detection; Development; Disease Vectors; Dissociation; Electrophysiology (science); Epitopes; Family; Fluorescence; Gases; Genes; Genomics; Genotype; Goals; Grant; Human; Human body; Immunohistochemistry; In Vitro; Individual; Insecta; Ions; Ketones; Label; Ligand Binding; Ligands; Location; Lymph; Maxilla; Mediating; Membrane; Methods; Molecular; Mosquito-borne infectious disease; N-terminal; Names; Nervous system structure; Neurons; Odors; Olfactory Pathways; Pharmacology; Play; Population; Preparation; Property; Protein Family; Proteins; Protons; Resolution; Role; Sensory; Sensory Process; Signal Transduction; Skin; Smell Perception; Specificity; Spermidine; Structure; Supporting Cell; Surface; System; Testing; Transgenic Organisms; Vector-transmitted infectious disease; Yellow Fever; aqueous; capitate bone; carbon dioxide receptor; carbonate dehydratase; cofactor; combat; disorder control; ethyl pyruvate; feeding; genome editing; homologous recombination; improved; insect disease vector; molecular mechanics; mutant; neuronal cell body; novel; olfactory sensory neurons; pyridine; receptor; receptor function; reconstitution; response; screening; trafficking

Blood feeding mosquitoes use sensitively tuned chemosensory systems to locate their vertebrate hosts. At locations far from a target host, anthropophilic mosquitoes predominantly rely on their sense of smell to follow plumes of volatile host emissions emanating from the human body, which include skin odor and carbon dioxide (CO2). In the yellow fever mosquito Aedes aegypti, CO2 is sensed by specialized populations of olfactory sensory neurons (OSNs) housed within capitate peg sensilla on the ventral surface of the maxillary palps. In each CO2-sensitive neuron, three gustatory receptors (Grs) named Gr1, Gr2 and Gr3, are hypothesized to interact to form a CO2 receptor complex mediating detection of this volatile gas, as well as structurally diverse ligands including certain heterocyclics, ketones and alcohols. Whether these three Gr subunits are all functionally required for CO2 receptor complex formation and the broad tuning responses of this neuron to this gas and other odorants, or whether additional signaling cofactors are involved is unknown. Here, we propose to apply integrative methods to functionally define the molecular architecture of the mosquito CO2 receptor complex. We will initially apply single-sensillum electrophysiology to functionally probe responses of Ae. aegypti Gr1, 2 and 3 null mutants to CO2 and a select panel of volatile agonists and antagonists of this receptor complex. Using CRISPR-Cas9 genome editing, we will additionally insert small-epitope tags onto the N– and C–terminal ends of Gr1, 2 and 3 to evaluate the ability of each of these individual Grs to traffic independently from the neuronal cell bodies to the lamellate cpA dendrite using immunohistochemistry. These approaches will reveal Gr subunits that may influence the odor tuning sensitivity or specificity of the complex, or those that may serve as co-receptors for transport of ligand-binding subunits to the sensory membrane. To evaluate a specific role for carbonic anhydrases (CAHs) in CO2 receptor complex function, which we hypothesize may catalyze the conversion of CO2 inside the aqueous sensillar lymph to bicarbonate ions and protons to act as receptor ligands, we will ablate target CAHs expressed in the maxillary palps using genome- editing and pharmacological manipulations. We will further highlight the anatomy of carbonic anhydrase expressing cells in the mosquito olfactory system using transgenic tracing methods to query their potential role in gas detection. CO2 is one of the most universal and important components of odor-baited traps used for surveillance and control of hematophagous insects including mosquito disease vectors. An improved understanding of the molecular architecture of the broadly conserved CO2 receptor complex may facilitate the development of efficient heterologous expression systems that reconstitute the mosquito CO2 receptor complex in vitro. Such an advance may aid in the rapid identification of novel volatile compounds mimicking or antagonizing the effects of CO2 for applied use in vector-borne disease control strategies.

食血蚊子使用灵敏的化学传感系统来定位它们的脊椎动物宿主。在… 远离目标宿主的地方，嗜人蚊子主要依靠它们的嗅觉跟随 人体散发的挥发性宿主排放物，包括皮肤气味和二氧化碳 (二氧化碳)。在黄热病蚊子埃及伊蚊身上，二氧化碳是由特殊的嗅觉种群感受到的。 感觉神经元位于上颌骨触须腹面的头状钉感受器内。在……里面 每个对二氧化碳敏感的神经元，三个味觉受体(GRs)，分别被命名为Gr1，Gr2和Gr3，被假设为 相互作用形成二氧化碳受体复合体，介导对这种挥发性气体的检测，以及结构上的多样性 配体包括某些杂环、酮和醇。这三个GR亚基是否都是 形成二氧化碳受体复合体所需的功能以及该神经元对此的广泛调节反应 气体和其他气味，或者是否涉及其他信号辅助因素是未知的。在这里，我们建议 应用综合方法从功能上定义蚊子二氧化碳受体的分子结构 很复杂。我们将首先应用单感受器电生理学从功能上探测Ae的反应。 埃及伊蚊对二氧化碳的Gr1、2和3零突变体及其挥发性激动剂和拮抗剂的精选小组 受体复合体。使用CRISPR-Cas9基因组编辑，我们将另外将小表位标签插入到 GR1、2和3的N端和C端，以评估这些单独GR中的每一个对业务的能力 免疫组织化学染色显示，从神经元胞体独立到板层状CPA树突。这些 方法将揭示可能影响复合体的气味调节敏感性或特异性的GR亚基， 或那些可以作为辅助受体将配体结合亚单位运输到感觉膜的受体。至 评估碳酸酐酶(CAHs)在二氧化碳受体复合体功能中的特定作用，我们 假设可能催化房水感受器淋巴中的二氧化碳转化为重碳酸盐离子 质子作为受体配体，我们将利用基因组来去除在上颌骨触须中表达的靶CAH- 编辑和药理操作。我们将进一步强调碳酸酐酶的解剖。 用转基因示踪方法在蚊子嗅觉系统中表达细胞以质疑其潜在作用 在气体检测方面。二氧化碳是气味诱捕器中最普遍和最重要的成分之一，用于 监测和控制食血昆虫，包括蚊子病媒。一种改进的 了解广泛保守的二氧化碳受体复合体的分子结构可能有助于 重组蚊子二氧化碳受体的高效异源表达系统的开发 体外合成复合体。这一进展可能有助于快速识别模拟或 在病媒传播疾病控制策略中的应用中对抗二氧化碳的影响。