Novel electrogenic cation-chloride cotransporters in Drosophila melanogaster

果蝇中新型产电阳离子-氯离子协同转运蛋白

• 批准号: 10046786
• 负责人: Christopher M Gillen
• 金额: $ 31.37万
• 依托单位: KENYON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046786
• 关键词: Aedes; Affect; Affinity; Amino Acid Substitution; Antiepileptic Agents; Binding; Biological Assay; Biological Models; Blood Pressure; Cations; Cell Volumes; Cell membrane; Cells; Chlorides; Chromatography; Clinical; Collaborations; Coupled; Cryoelectron Microscopy; Culicidae; Data; Distal convoluted renal tubule structure; Diuretics; Drosophila genus; Drosophila melanogaster; Drug Targeting; Electrodes; Environment; Epithelial; Epithelial Cells; Epithelium; Exhibits; Exposure to; Family; Fluid Balance; Fluids and Secretions; Functional disorder; Genetic; Glycine; Goals; Hemolymph; Hindgut; Homeostasis; Human; Impairment; Insecta; Insecticides; Institution; Intestines; Ions; Kidney; Light; Limb structure; Liquid substance; Lithium; Lung; Malpighian Tubules; Mammals; Measures; Medical; Modeling; Movement; Mutation; Neurons; Neurotransmitters; Oocytes; Orthologous Gene; Pathology; Pharmaceutical Preparations; Physiological; Play; Potassium; Property; Publishing; Renal tubule structure; Research; Resolution; Role; Rubidium; SLC12A3 gene; Sampling; Seizures; Sensory; Sf9 cell line; Site; Site-Directed Mutagenesis; Sodium; Sodium Channel; Sodium Chloride; Sodium-Potassium-Chloride Symporters; Structure; Structure-Activity Relationship; Students; Sweat Glands; System; Testing; Thick; Tissues; Tracer; Transmembrane Domain; Uniport; Urine; Work; Xenopus laevis; Xenopus oocyte; absorption; apical membrane; base; basolateral membrane; blood pressure regulation; cell type; college; experience; experimental study; extracellular; gamma-Aminobutyric Acid; inhibitor/antagonist; insect disease vector; knock-down; neuronal excitability; novel; prevent; response; sodium-potassium-chloride cotransporter 1 protein; thiazide; tool; undergraduate research; undergraduate student; voltage clamp; wasting

7. Project Summary Cation-chloride cotransporters (CCCs) regulate cell volume and intracellular chloride concentration in many different cell types. They contribute to salt secretion by epithelial cells in the lung, intestine, and sweat glands and salt absorption by epithelial cells in the kidney. They influence excitability of neurons in response to neurotransmitters such as gamma-aminobutyric acid and glycine by regulating the electrochemical gradient for chloride movement across the cell membrane. In mammals, the sodium-dependent CCCs include a potassium- independent sodium-chloride cotransporter (NCC) and two sodium-potassium-chloride cotransporters (NKCC1 and NKCC2). NCC and NKCC2 are targets of the clinically important thiazide and loop diuretics, whereas NKCC1 is a promising target for anti-epileptic drugs. Despite their physiological and medical importance, our understanding of the structure-function relationships of these transporters is incomplete. In this R15-AREA project, an undergraduate research group will evaluate the structure-function relationships of the NaCCC2s, a group of sodium-dependent CCCs that is specific to insects. The work will focus on Ncc83, a NaCCC2 from the fruit fly Drosophila melanogaster. According to preliminary data and published work, Ncc83 and its ortholog from the mosquito Aedes aegypti induce a chloride-independent sodium current when expressed in Xenopus oocytes. This activity contrasts with the electroneutral, chloride-dependent cation transport of other sodium- dependent cation-chloride cotransporters, including Drosophila Ncc69. The first aim characterizes the fundamental transport properties of Ncc83 such as ion and inhibitor affinities, testing the hypothesis that Ncc83 is a sodium transporter rather than a sodium channel. Ncc83 will be expressed in Xenopus oocytes and the insect Sf9 cell line and its activity assessed by two-electrode voltage clamp and tracer flux assays. Cation chromatography will be used to evaluate tracer levels of non-radioactive lithium (a sodium tracer) and rubidium (a potassium tracer). The second aim explores structure-function relationships through domain swap and site- directed mutagenesis experiments, testing the hypothesis that amino acid substitutions between Ncc83 and Ncc69 determine electrogenic versus electroneutral transport activity. Finally, the third aim tests the hypothesis that Ncc83 contributes to salt secretion and/or absorption by renal (Malpighian) tubules and hindgut. Using the genetic tools available for Drosophila, tissue-specific knockdowns of Ncc83 will be produced. Physiological effects will be assessed by sampling hemolymph following exposure to conditions that challenge ion homeostasis and by fluid secretion assays of tubules. Cation chromatography will be used to evaluate ion concentrations in fluids collected during these experiments. This project will uncover new information about the structure-function of CCCs, a medically important family of transporters. It will also determine the physiological role of Ncc83, setting the stage for work in mosquitoes or other insect disease vectors. Since the NaCCC2s are insect-specific, they may be good targets for insect-specific control agents.

7。项目摘要 阳离子 - 氯化物共转运蛋白（CCCS）调节许多细胞体积和细胞内氯化物浓度 不同的细胞类型。它们导致肺，肠和汗腺中上皮细胞的盐分泌 肾脏中上皮细胞吸收盐。它们会影响神经元的兴奋性 通过调节电化学梯度的神经递质，例如γ-氨基丁酸和甘氨酸 氯化物的运动穿过细胞膜。在哺乳动物中，依赖钠的CCC包括钾 独立的氯化钠共转运蛋白（NCC）和两个 - 氯化钠 - 氯化钠（NKCC1） 和NKCC2）。 NCC和NKCC2是临床上重要的噻嗪和循环利尿剂的靶标，而 NKCC1是抗癫痫药的有前途的靶标。尽管它们的生理和医学重要性，但我们 了解这些转运蛋白的结构 - 功能关系是不完整的。在这个R15区 项目，一个本科研究小组将评估NACCC2S的结构功能关系 一组依赖昆虫的钠依赖性CCC。这项工作将集中于NCC83，这是来自 果蝇果蝇黑甲烷戈斯特。根据初步数据和发布的工作，NCC83及其直系同源 从蚊子埃及埃及埃及在Xenopus中表达时诱导氯化物独立的钠电流 卵母细胞。该活性与其他钠的电反性，氯化物依赖性阳离子转运形成对比 依赖性阳离子 - 氯化物共转运蛋白，包括果蝇NCC69。第一个目的是 NCC83的基本运输特性，例如离子和抑制剂亲和力，检验了NCC83的假设 是钠转运蛋白而不是钠通道。 NCC83将以武士卵母细胞和 昆虫SF9细胞系及其活性通过两电极电压夹和示踪剂通量测定法进行了评估。阳离子 色谱法将用于评估非放射性锂（钠示踪剂）和rubidium的示踪剂水平 （钾示踪剂）。第二个目标通过域交换和站点 - 定向诱变实验，检验了以下假设：NCC83和 NCC69确定了电源性转运活性。最后，第三个目标检验了假设 NCC83有助于肾（Malpighian）小管和后肠吸收盐分泌和/或吸收。使用 将生产用于果蝇的遗传工具，将生产NCC83的组织特异性敲低。生理 暴露于挑战离子的疾病后，将通过抽样血淋巴来评估效果 稳态和小管的流体分泌测定法。阳离子色谱法将用于评估离子 在这些实验过程中收集的流体的浓度。该项目将发现有关 CCC的结构功能，是医学上重要的转运蛋白家族。它还将决定生理 NCC83的作用，为蚊子或其他昆虫疾病媒介的工作奠定了基础。自NACCC2S以来 是特定于昆虫的，它们可能是昆虫特异性对照剂的良好目标。