Mechanism of I- transport by the Na+/I- symporter (NIS)

Na /I- 同向转运体 (NIS) 的 I- 转运机制

• 批准号: 10047846
• 负责人: L. Mario Amzel
• 金额: $ 9.67万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10047846
• 关键词: Mechanism; transport; Na+; symporter; NIS

 DESCRIPTION (provided by applicant): The Na+/I- symporter (NIS) is the key plasma membrane protein that mediates I- transport in the thyroid and other tissues. In the thyroid, I- transport is the first step in the biosynthesis of the thyroid hormones T3 and T4. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. NIS activity is electrogenic, with a 2 Na+:1 I- stoichiometry. By contrast, we have shown that NIS transports perrhenate (ReO4-) and the environmental pollutant perchlorate (ClO4-) electroneutrally. For over 65 years, NIS-mediated radiotherapy has been the most successful targeted internal radiation cancer treatment available, as administered in thyroid cancer post-thyroidectomy. Since we cloned it, NIS has been ectopically expressed by gene transfer in extra-thyroidal cancers, rendering them susceptible to radioiodide treatment. The study of NIS is of considerable basic and medical interest. We have gained significant insights into structure/function relations in NIS by studying congenital I- transport defect-causing NIS mutations found in patients: in particular, we correctly predicted that NIS would have the same fold as the bacterial leucine transporter LeuT and the other proteins in its family. The recognition of this similarity enabled us to build a NIS homology model based on the structure of another bacterial protein with the same fold, vSGLT. Molecular dynamics (MD) simulations using our homology model have accurately predicted which residues play crucial roles in NIS function, substrate specificity, kinetics, and stoichiometry. All this characterizatio of NIS at the molecular level has allowed us to propose a mechanism for I- transport by NIS. In this project, we will test our hypothesis about the transport mechanism. We will bring to this task a battery of biophysical approaches combining whole cell-based biochemical experiments with studies using purified NIS [scintillation proximity assays (SPA) and isothermal titration calorimetry (ITC)] and computational analysis, including statistical thermodynamics (ST) and MD simulations. We will also test specific hypotheses about the role of individual residues in the transport cycle. We have made considerable progress in addressing the fundamental question of how NIS can efficiently accumulate I- given the extremely low (sub-µM) I- concentrations in the extracellular fluids, which are much lower than the expected Kd of a protein for a halide: we have determined that binding of the first ion (either Na+ or I-) increases the affinity of NIS for he other ion by a factor of ~10. The following crucially important Specific Aims will be pursued: 1. What is the affinity of purified NIS for each of the transported ions in the presence of varying concentrations of the other ions? What is the effect of binding to one site on the ion affinities o the other sites? 2. What are the affinities of NIS in the inwardly open conformation for the ions i transports? 3. Which residues coordinate Na+ at the Na2 site and/or participate in the pathway that releases Na+ into the cytosol? 4. Which residues coordinate Na+ at the Na1 site, and how do they determine the change in Na+/ReO4- stoichiometry brought about by single amino acid substitutions?

 描述（由申请人提供）：Na+/I-同向转运蛋白（NIS）是介导甲状腺和其他组织中 I- 转运的关键质膜蛋白。在甲状腺中，I- 转运是 I- 生物合成的第一步。甲状腺激素 T3 和 T4 将 I- 逆其电化学梯度向内转运与 Na+ 沿其电化学梯度向内转运耦合，其活性为 2。相比之下，我们已经证明 NIS 可以电中性地传输高铼酸盐 (ReO4-) 和环境污染物高氯酸盐 (ClO4-) 65 年来，NIS 介导的放射治疗一直是最成功的靶向内放射治疗。自从我们克隆NIS以来，NIS已经通过基因转移在甲状腺外癌症中异位表达，从而使它们成为可用的治疗方法。 NIS 的研究具有相当大的基础和医学意义，通过研究患者中发现的先天性 I 转运缺陷引起的 NIS 突变，我们对 NIS 的结构/功能关系有了重要的了解：特别是，我们正确预测了 NIS。 NIS 与细菌亮氨酸转运蛋白 LeuT 及其家族中的其他蛋白质具有相同的折叠。对这种相似性的认识使我们能够基于另一种具有相同折叠的细菌蛋白质 vSGLT 的结构构建 NIS 同源模型。使用我们的同源模型进行的分子动力学 (MD) 模拟准确预测了哪些残基在 NIS 功能、底物特异性、动力学和化学计量中起关键作用。NIS 在分子水平上的所有这些表征使我们能够提出一种 I- 转运机制。在这个项目中，我们将测试我们对这项任务的传输机制的假设。 我们还将测试一系列生物物理方法，将基于全细胞的生化实验与使用纯化 NIS [闪烁邻近分析 (SPA) 和等温滴定量热法 (ITC)] 的研究和计算分析相结合，包括统计热力学 (ST) 和 MD 模拟。关于单个残基在运输循环中的作用的具体假设，我们在解决 NIS 如何有效积累 I- 的基本问题方面取得了相当大的进展，因为细胞外的 I- 浓度极低（亚微米）。液体，其远低于卤化物蛋白质的预期 Kd：我们确定第一个离子（Na+ 或 I-）的结合可将 NIS 对其他离子的亲和力增加约 10 倍。至关重要的具体目标是： 1. 在存在不同浓度的其他离子的情况下，纯化的 NIS 对每种转运离子的亲和力是多少？ 2. 向内开放构象的 NIS 对离子 i 转运的亲和力是什么？ 3. 哪些残基在 Na2 位点上协调 Na+ 和/或参与将 Na+ 释放到细胞质中的途径？残基在Na1位点协调Na+，它们如何确定单个氨基酸取代带来的Na+/ReO4-化学计量的变化？