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

描述（由申请人提供）：氨基酸/多胺/组织位（APC）转运蛋白超家族包括人类LAT和CAT氨基酸转运蛋白以及Na-K-Cl、Na-Cl和K-Cl阳离子-氯共转运蛋白（CCCs）。CAT和LAT氨基酸转运蛋白介导了氨基酸跨细胞膜、肾上皮和肠上皮的大部分运动。Na-K-Cl共转运体、Na-Cl和K-Cl共转运体在细胞体积和氯离子稳态中发挥核心作用，在神经元细胞中，通过细胞内氯离子的变化负责调节兴奋性。在分泌上皮中，NKCC1与Cl通道、Na泵和K通道一起发挥作用，带来大部分受调节的盐分泌运动，而在哺乳动物肾脏中，另一种异构体NKCC2介导盐吸收，是环利尿剂速尿和布美他尼的作用部位。由于我们不知道APC家族成员的结构，这些转运蛋白在分子水平上都没有被理解。该项目的目标是获得原核APC超家族成员的第一个高分辨率晶体结构，并了解该结构背景下转运蛋白的功能。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，而且我们可以在细菌中高水平地表达这些蛋白质。强比对确保我们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。这个高回报的项目是范式的转变，因为它将为氨基酸转运体和阳离子-氯共转运体的广泛研究领域打开大门，其中结构信息用于告知机制研究，包括调节和药物相互作用的研究。具体目的是：1)克隆一组不同的原核阳离子氨基酸转运蛋白，并检测其高产、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；3)确定选定的原核转运蛋白的功能，并将其功能与新出现的结构联系起来。包括高血压、脑水肿、多囊性肾病、分泌性腹泻、囊性纤维化和一些神经系统疾病在内的疾病和病症涉及负责盐和氨基酸跨细胞膜运动的细胞机制的缺陷或过度活动。这项研究的目的是了解细胞机制的一部分，一组被称为阳离子-氯共转运蛋白和氨基酸转运蛋白的相关蛋白质，它们直接负责协调钠、钾和氯的运动，或氨基酸的运动。通过了解这些蛋白质的分子结构，我们将能够了解它们的作用机制和调节机制，从而能够更好地设计诊断和治疗药物并治疗疾病状态。

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.