Molecular Architecture of the Mosquito Carbon Dioxide Receptor Complex

吸血蚊子使用敏感的化学感应系统来定位它们的脊椎动物宿主。在 在远离目标宿主的地方，嗜热蚊子主要依靠它们的嗅觉来跟随 人体散发的挥发性宿主排放物，包括皮肤气味和二氧化碳 （CO2）。在黄热病蚊子Aedes aegypti中，CO2是由嗅觉器官的特殊群体感知的。 感觉神经元（OSNs）内的头状钉感器的腹面上的下颚须。在 每个CO2敏感神经元，三个味觉受体（Grs）命名为Gr 1，Gr 2和Gr 3，被假设为 相互作用以形成CO2受体复合物，介导对这种挥发性气体的检测，以及结构多样性 配体包括某些杂环、酮和醇。这三个Gr亚基是否都是 CO2受体复合物的形成和这种神经元对此的广泛调谐反应所需的功能 气体和其他气味，或是否涉及额外的信号传导辅因子是未知的。在这里，我们建议 应用整合方法来功能性地定义蚊子CO2受体的分子结构 复杂.我们将首先应用单感器电生理功能探测响应。 埃及Gr 1、2和3对CO2的无效突变体，以及一组选择的该突变体的挥发性激动剂和拮抗剂， 受体复合物使用CRISPR-Cas9基因组编辑，我们将另外将小表位标签插入到 Gr 1、2和3的N-和C-末端，以评估这些单独Grs中每一个的运输能力 独立地从神经元细胞体到片状cpA树突使用免疫组织化学。这些 这些方法将揭示可能影响复合物的气味调节敏感性或特异性的Gr亚基， 或可作为将配体结合亚单位转运至感觉膜的共受体的那些。到 评估碳酸酐酶（CAH）在CO2受体复合物功能中的特定作用，我们 假设可以催化水感觉淋巴内的CO2转化为碳酸氢根离子， 质子作为受体配体，我们将使用基因组消融在上颌须中表达的靶CAH， 编辑和药理学操作。我们将进一步强调碳酸酐酶的解剖 利用转基因追踪方法在蚊子嗅觉系统中表达细胞以查询它们的潜在作用 气体检测CO2是气味诱捕器中最普遍和最重要的组成部分之一， 监测和控制吸血昆虫，包括蚊虫病媒。一种改进 了解广泛保守的CO2受体复合物的分子结构可能有助于 开发重组蚊子CO2受体的有效异源表达系统 复合物体外培养。这种进展可能有助于快速鉴定新型挥发性化合物， 拮抗CO2的作用，用于病媒传播疾病的控制策略。

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.