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: 9197312
• 负责人: L. Mario Amzel
• 金额: $ 57.43万
• 依托单位: YALE 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 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197312
• 关键词: Active Biological Transport; Address; Affinity; Amino Acid Substitution; Anabolism; Bacterial Proteins; Binding; Biochemical; Biological Assay; Calorimetry; Cell membrane; Cells; Characteristics; Cloning; Collaborations; Computer Analysis; Computing Methodologies; Couples; Cytosol; Data; Defect; Development; Electrophysiology (science); Environmental Pollutants; Extracellular Fluid; Family; Fetus; Gene Transfer; Generations; Goals; Growth; Homologous Gene; Homology Modeling; Hypothyroidism; Individual; Iodides; Iodine; Ion Transport; Ions; Kinetics; Lead; Leucine; Malignant Neoplasms; Malignant neoplasm of thyroid; Measurement; Mediating; Medical; Membrane Potentials; Membrane Proteins; Mental Retardation; Metabolic; Methods; Modeling; Molecular; Molecular Conformation; Movement; Mutation; Na(+)-K(+)-Exchanging ATPase; Neuraxis; Newborn Infant; Pathway interactions; Patients; Perchlorates; Physiological; Play; Predictive Value; Production; Protein Family; Proteins; Public Health; Radiation therapy; Regulation; Research; Role; SLC5A5 gene; Site; Site-Directed Mutagenesis; Sodium Iodide; Structure; Substrate Specificity; Testing; Thermodynamics; Thyroid Gland; Thyroid Hormones; Thyroidectomy; Time; Tissues; Titrations; Triiodothyronine; base; biophysical techniques; cancer therapy; driving force; experimental study; inhibitor/antagonist; innovation; insight; interest; internal radiation; member; molecular dynamics; novel; perrhenate; public health relevance; sodium sulfide; stoichiometry; symporter; three dimensional structure; uptake

 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?