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-氯共转运蛋白（CCCs）在许多细胞中调节细胞体积和细胞内氯离子浓度。 不同的细胞类型它们有助于肺、肠和汗腺上皮细胞分泌盐 以及肾脏上皮细胞对盐的吸收。它们会影响神经元的兴奋性 神经递质，如γ-氨基丁酸和甘氨酸，通过调节电化学梯度， 氯离子穿过细胞膜的运动。在哺乳动物中，钠依赖性CCC包括钾- 独立钠-氯共转运蛋白（NCC）和两种钠-钾-氯共转运蛋白（NKCC 1 NKCC2）。NCC和NKCC 2是临床上重要的噻嗪类和髓袢利尿剂的靶点，而 NKCC 1是一个很有前途的抗癫痫药物靶点。尽管它们在生理和医学上很重要， 对这些转运蛋白的结构-功能关系的理解是不完整的。在此R15区域 项目，一个本科生研究小组将评估NaCCC 2的结构-功能关系， 昆虫特有的钠依赖性CCC。这项工作将集中在Ncc 83上，这是一个来自 果蝇Drosophila melanogaster根据初步数据和已发表的工作，Ncc 83及其直系同源物 埃及伊蚊的DNA在非洲爪蟾中表达时诱导了一种不依赖氯离子的钠电流 卵母细胞这种活性与其他钠离子的电中性、氯离子依赖性阳离子转运形成对比。 依赖阳离子-氯共转运蛋白，包括果蝇Ncc 69。第一个目标的特点是 Ncc 83的基本转运性质，如离子和抑制剂亲和力，测试Ncc 83 是钠转运体而不是钠通道Ncc 83将在非洲爪蟾卵母细胞中表达， 昆虫Sf 9细胞系及其活性通过双电极电压钳和示踪剂通量测定来评估。阳离子 色谱法将用于评估非放射性锂（钠示踪剂）和铷的示踪剂水平 （钾示踪剂）。第二个目标是通过结构域交换和位点交换来探索结构-功能关系， 定向诱变实验，测试Ncc 83和Ncc 84之间的氨基酸取代是可能的假设。 ncc 69决定产电与电中性转运活性。最后，第三个目标检验了假设 Ncc 83有助于盐分泌和/或肾（马氏管）小管和后肠的吸收。使用 利用果蝇可用的遗传工具，将产生Ncc 83的组织特异性敲低。生理 将通过在暴露于挑战离子的条件后采集血淋巴样品来评估效应。 稳态和小管的液体分泌测定。将使用阳离子色谱法评价离子 在这些实验期间收集的流体中的浓度。该项目将揭示有关 CCCs是一个重要的医学转运蛋白家族。它也将决定生理 Ncc 83的作用，为蚊子或其他昆虫疾病媒介的工作奠定了基础。自NaCCC 2以来 是昆虫特异性的，它们可能是昆虫特异性控制剂的良好靶标。