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

基本信息

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
项目类别：
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

项目摘要

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.

七、项目概要阳离子-氯离子协同转运蛋白 (CCC) 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、肠和汗腺的上皮细胞分泌盐以及肾脏上皮细胞对盐的吸收。它们影响神经元响应的兴奋性通过调节电化学梯度来调节γ-氨基丁酸和甘氨酸等神经递质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- 独立的钠-氯化物协同转运蛋白 (NCC) 和两个钠-钾-氯化物协同转运蛋白 (NKCC1) 和 NKCC2)。 NCC 和 NKCC2 是临床上重要的噻嗪类和袢利尿剂的靶标，而 NKCC1 是抗癫痫药物的一个有前景的靶点。尽管它们具有生理和医学重要性，但我们对这些转运蛋白的结构-功能关系的理解并不完整。在这个 R15 区域项目中，一个本科生研究小组将评估 NaCCC2 的结构与功能关系，一组昆虫特有的钠依赖性 CCC。这项工作将重点关注 Ncc83，这是来自果蝇黑腹果蝇。根据初步数据和已发表的工作，Ncc83 及其直系同源物当埃及伊蚊在非洲爪蟾中表达时，会诱导出不依赖氯化物的钠电流卵母细胞。这种活性与其他钠离子的电中性、氯化物依赖性阳离子传输形成鲜明对比。依赖的阳离子-氯离子协同转运蛋白，包括果蝇 Ncc69。第一个目标的特点是 Ncc83 的基本传输特性，例如离子和抑制剂亲和力，检验 Ncc83 的假设是钠转运蛋白而不是钠通道。 Ncc83 将在非洲爪蟾卵母细胞中表达昆虫 Sf9 细胞系及其通过两电极电压钳和示踪通量测定评估的活性。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）。第二个目标通过域交换和位点探索结构功能关系定向诱变实验，检验 Ncc83 和 Ncc83 之间氨基酸替换的假设 Ncc69 确定生电与电中性运输活性。最后，第三个目标检验假设 Ncc83 有助于肾（马尔皮基）小管和后肠的盐分泌和/或吸收。使用可用于果蝇的遗传工具，将产生 Ncc83 的组织特异性敲除。生理将通过暴露于挑战离子的条件后对血淋巴取样来评估效果体内平衡和小管液体分泌测定。阳离子色谱法将用于评估离子这些实验期间收集的液体中的浓度。该项目将揭示有关 CCC 的结构-功能，是医学上重要的转运蛋白家族。也决定了生理 Ncc83 的作用，为蚊子或其他昆虫疾病媒介的工作奠定了基础。自从NaCCC2s 由于它们是昆虫特异性的，因此它们可能是昆虫特异性控制剂的良好目标。