The Na+/I- symporter (NIS) is the key plasma membrane protein that mediates active I- transport into the thyroid, the first step in thyroid hormone biosynthesis. NIS couples the inward translocation of I- against its elec- trochemical gradient to the inward transport of Na+ down its electrochemical gradient. NIS activity is electrogenic, with a 2Na+:1I- stoichiometry. We have shown that NIS also transports the environmental pollutant perchlorate (ClO4-, an inhibitor of I- transport), but electroneutrally (1Na+:1ClO4-). How does NIS translocate different sub- strates with different stoichiometries? We discovered a high-affinity non-transport oxyanion binding site in NIS that, when occupied by ClO4-, allosterically prevents one of the two Na+ ions from binding, changing the stoi- chiometry of I- transport from 2Na+:1I- to 1Na+:1I-. This reduces the driving force for I- transport, markedly de- creasing I- uptake. Thus, drinking water contaminated with ClO4- is more deleterious to human health than pre- viously thought. A crucial question about NIS is: how can NIS efficiently transport I-, given the extremely low [I-]s in the extracellular milieu? We have made significant progress toward solving this puzzle. Nevertheless, fully elucidating the mechanism of transport by NIS will require extensive further computation and experimentation. For these studies, we built NIS homology models based on the structures of two proteins with the same fold: vSGLT and SiaT. MD simulations using these models have accurately predicted which residues play key roles in NIS function; all predictions have been experimentally confirmed. The NIS transport cycle involves 16 species: 1 empty NIS, 3 NIS species with one ion, 3 NIS species with two ions, and 1 NIS with three ions, in the outwardly (8 states) and the inwardly open conformation (8 states). The 16 species can be considered to be very close to equilibrium. Therefore, the NIS mechanism can be described by determining the populations and free energies of the 16 species. We propose to identify the residues making up the ClO4- allosteric site using MD simulations of WT NIS and mutant NIS proteins we have shown to transport oxyanions electrogenically, not electroneutrally. The residues suggested by the simulations to make up the allosteric site will be investigated experimentally. We will search computationally for endogenous compounds that may bind to the allosteric site, and experimentally test their effects on NIS activity. Having identified NIS residues that likely interact with I- in our simulations, we will investigate them experimentally. We will use site-directed mutagenesis, transport assays and kinetics in whole cells and in proteoliposomes reconstituted with purified NIS, electrophysiological experiments, scintillation proximity assays, isothermal titration calorimetry, statistical thermodynamics modeling, and computational meth- ods (MD simulations, including metadynamics) to answer the following questions (S...