Function and High-Resolution of an APC superfamily amino acid transporter

APC 超家族氨基酸转运蛋白的功能和高分辨率

• 批准号: 7450594
• 负责人: BLISS FORBUSH
• 金额: $ 20.67万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7450594
• 关键词: Address; Amino Acid Transporter; Amino Acids; Bacteria; Basic Amino Acid Transport Systems; Bumetanide; Carrier Proteins; Cations; Cell membrane; Cells; Cerebral Edema; Chloride Ion; Chlorides; Collaborations; Condition; Crystal Formation; Crystallization; Cystic Fibrosis; Defect; Detergents; Diagnostic; Diarrhea; Disease; Diuretics; Drug Interactions; Drug Regulations; Epithelium; Family; Family member; Furosemide; Goals; Homeostasis; Human; Hypertension; Investigation; Ion Cotransport; Kidney; Laboratories; Mechanics; Mediating; Membrane Transport Proteins; Molecular; Molecular Conformation; Molecular Structure; Movement; Neurons; Numbers; Pharmaceutical Preparations; Play; Polyamines; Polycystic Kidney Diseases; Potassium Channel; Potassium Chloride; Protein Isoforms; Proteins; Pump; Range; Rate; Regulation; Research; Resolution; Role; Site; Sodium; Sodium Chloride; Structure; Substrate Specificity; Techniques; Therapeutic Agents; Work; absorption; base; chloride-cotransporter potassium; design; intestinal epithelium; member; nervous system disorder; research study; stoichiometry

DESCRIPTION (provided by applicant): The aminoacid/ polyamine/organocation (APC) superfamily of transporters includes human LAT and CAT amino acid transporters as well the Na-K-Cl, Na-Cl and K-Cl cation-chloride cotransporters (CCCs). The CAT and LAT amino acid transporters mediate a major fraction of amino acid movement across cell membranes and across renal and intestinal epithelia. The Na-K-Cl cotransporter, Na-Cl, and K-Cl cotransporters play a central role in cellular volume and chloride homeostasis, and in neuronal cells are responsible for regulation of excitability through changes in intracellular chloride. In secretory epithelia, NKCC1 functions together with Cl channels, the Na pump, and K channels to bring about the major fraction of regulated salt secretion movement, while in the mammalian kidney another isoform, NKCC2, mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. None of these transport proteins is understood at the molecular level because we do not know the structure of any of the APC family members. The goal of this project is to obtain the first high-resolution crystal structure of a prokaryotic APC superfamily member, and to understand the function of the transporter in the context of the structure. We have chosen prokaryotic APCs because we can select among a large number of potential candidates, and because we can express these proteins at high levels in bacteria. Strong alignment assures that we will be able to apply the structural information to high-resolution predictions for the structure of the eukaryotic transporters. This high-payoff project is paradigm-shifting in that it will open the door to a broad field of investigation in amino acid transporters and cation-chloride cotransporters in work in which structural information is used to inform mechanistic studies, including studies of regulation and drug interactions. The specific aims are 1) to clone a diverse set of prokaryotic cationic amino acid transporters and examine their ability to be produced in high yield and with high stability, 2) to obtain the high resolution crystal structure of an APC transporter, and 3) to determine the function of selected prokaryotic transporters, relating the function to the emerging structure. Diseases and disease conditions including hypertension, cerebral edema, polycystic kidney disease, secretory diarrhea, cystic fibrosis, and some diseases of the nervous system involve defects or over activity of the cellular machinery that is responsible for salt and amino acid movements across cell membranes. This research is directed to understanding one part of that cellular machinery, a set of related proteins called cation- chloride cotransporters and amino acid transporters that are responsible for directly handling coordinated sodium, potassium and chloride movements, or amino acid movements. By understanding the molecular structure of these proteins, we will be able to understand the mechanics of their action, and the mechanism of their regulation, thus being better able to design diagnostic and therapeutic agents and treat the disease states.

描述（由申请人提供）：转运蛋白的氨基酸/多胺/有机化（APC）包括人LAT和CAT氨基酸转运蛋白以及Na-K-CL，Na-CL，Na-CL和K-CL阳离子 - 氯化物 - 氯化物共转运蛋白（CCCS）。 CAT和LAT氨基酸转运蛋白介导跨细胞膜以及跨肾上皮和肠上皮的主要部分氨基酸运动。 Na-K-CL共转运蛋白，Na-CL和K-CL共转运蛋白在细胞体积和氯化物稳态中起着核心作用，而在神经元细胞中，通过细胞内氯化物的变化来调节兴奋性。在分泌性上皮中，NKCC1与CL通道，Na泵和K通道一起发挥作用，以带来受调节的盐分分泌运动的主要部分，而在哺乳动物肾脏中，另一种同工型NKCC2介导了盐的吸收，是循环的loop loop loop furop furops furosemide anf furosemide and bumetanide and Bumetanide的作用。这些转运蛋白都在分子水平上都没有理解，因为我们不知道任何APC家族成员的结构。该项目的目的是获得原核APC超家族成员的第一个高分辨率晶体结构，并了解在结构上下的转运蛋白的功能。我们之所以选择原核APC，是因为我们可以在大量潜在的候选物中进行选择，并且因为我们可以在细菌中以高水平表达这些蛋白质。强有力的一线确保我们将能够将结构信息应用于真核转运蛋白结构的高分辨率预测。这个高额付款项目是范式转移的，它将为氨基酸转运蛋白和阳离子 - 氯化物共转运蛋白的广泛研究打开大门，其中使用结构信息来为机械研究提供信息，包括调节和药物相互作用的研究。具体目的是1）克隆一组多样的原用阳离子氨基酸转运蛋白，并检查其以高产量和高稳定性生产的能力，2）获得APC转运蛋白的高分辨率晶体结构，以及3）确定所选原核转运蛋白的功能，将功能与出现的结构相关联。疾病和疾病疾病，包括高血压，脑水肿，多囊性肾脏疾病，分泌性腹泻，囊性纤维化以及神经系统的某些疾病涉及导致细胞和氨基酸运动的细胞机制的缺陷或过度活性。这项研究旨在理解该细胞机械的一部分，该细胞机械是一组称为阳离子 - 氯基共转运蛋白和氨基酸转运蛋白，这些蛋白质负责直接处理配位钠，钾和氯化物运动或氨基酸运动。通过了解这些蛋白质的分子结构，我们将能够理解其作用的机制及其调节机制，从而更好地设计诊断和治疗剂并治疗疾病状态。