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-M) 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?

Public Health Relevance

The purpose of this project is to carry out a detailed study of a key protein, the sodium/iodide symporter (NIS), a molecule present most notably in the thyroid gland, where it is responsible for the crucial first step in the production of the thyroid hormones One of our research groups was the first to clone and characterize NIS, and both our groups have since collaborated in investigating detailed characteristics of the molecule, including the manner in which it handles its substrates (e.g., sodium and iodide). In this project, our two groups will use a highly innovative approach that combines experiments using whole cells and purified NIS with computational analysis to uncover structure/function and mechanistic information on NIS that will likely enhance considerably our understanding of this crucial protein and of other transporters

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM114250-03
Application #
9416154
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ainsztein, Alexandra M
Project Start
2016-01-01
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Ravera, Silvia; Reyna-Neyra, Andrea; Ferrandino, Giuseppe et al. (2017) The Sodium/Iodide Symporter (NIS): Molecular Physiology and Preclinical and Clinical Applications. Annu Rev Physiol 79:261-289
Ferrandino, Giuseppe; Nicola, Juan Pablo; Sánchez, Yuly E et al. (2016) Na+ coordination at the Na2 site of the Na+/I- symporter. Proc Natl Acad Sci U S A 113:E5379-88