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（由适用提供）：Na+/I-响应蛋白（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-转运是甲状腺激素T3和T4生物合成的第一步。 NIS将I-与其电化学梯度的内向转移到Na+向下的电化学梯度向内运输。 NIS活性是电源的，具有2 Na+：1 i-入学计量。相比之下，我们已经表明，NIS运输perrhenate（REO4-）和环境污染物高氯酸盐（Clo4-）电动。在65年以上，NIS介导的放疗一直是甲状腺甲状腺切除术后甲状腺癌的最成功的靶向内放射癌治疗。自从我们克隆它以来，NIS在甲状腺外癌中通过基因转移而表达了NIS，使它们容易受到放射性碘治疗的影响。 NIS的研究是基本和医疗利益的考虑。我们通过研究患者发现的先天I传输缺陷的NIS突变通过研究NIS中的结构/功能关系有了重大见解：尤其是，我们正确地预测NIS的折叠与李氨酸念珠菌转运蛋白转运蛋白Leut及其家族中的其他蛋白质相同。对这种相似性的认识使我们能够基于具有相同折叠的另一种细菌蛋白的结构来构建NIS同源性模型。使用我们的同源性模型的分子动力学（MD）模拟可以准确预测，该模拟恢复了NIS功能，底物特异性，动力学和化学计量的至关重要的作用。 NIS在分子水平上的所有这些特征使我们提出了NIS I-Entramption的机制。在这个项目中，我们将测试有关运输机制的假设。我们将完成这项任务 一系列的生物物理方法将整个基于细胞的生物化学实验与使用纯化的NIS [闪烁接近度测定（SPA）和等热滴定热量法（ITC）和计算分析（包括统计热力学（ST）和MD模拟）的研究结合了一系列。我们还将检验有关个人保留在运输周期中作用的特定假设。我们在解决NIS如何有效积累I的基本问题方面取得了考虑，鉴于细胞外液在极低（sub-µm）的I-浓度中，该问题比卤化物的蛋白质的预期Kd低得多：我们已经确定了第一个离子（Na+或I-I-）的结合，而不是n. iS n is ai the Is n is AniS ai增加了IS and iS n is and nis nis ai at iS nis ai at iSAI〜———〜赚取〜〜———〜———〜赚取〜〜———〜———〜赚取〜〜———〜———〜将追求以下至关重要的具体目的：1。在存在不同浓度的其他离子的情况下，纯化的NI对每个运输离子的亲和力是什么？在其他站点上，与一个位点结合对离子亲和力的影响是什么？ 2。在我运输的离子内部开放构象中，NIS的亲和力是什么？ 3。哪个结果在Na2位点坐标为Na+和/或参与将Na+释放到细胞质中的途径？ 4。哪个结果在Na1位点坐标了Na+，它们如何确定由单个氨基酸取代带来的Na+/reo4-静态计量的变化？