A multipronged mechanistic study of the sodium iodide symporter (NIS): Subcellular localization, stoichiometry, transport of the environmental pollutant perchlorate, and purification for structural analysis
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Iodide (I-) transport in the thyroid, the rate-limiting step in thyroid hormone biosynthesis, is mediated by the Na+/I - symporter (NIS). NIS also mediates I- transport in other polarized epithelia, where NIS is localized basolaterally. NIS is also the basis for radioiodide therapy in thyroid diseases. Surprisingly, the decrease in radioiodide uptake that is observed in 70% of thyroid cancers is caused by impaired targeting of NIS to the plasma membrane (PM).;In Part I we investigated the signal that determines NIS polarized PM targeting. Deletions of the NIS C-terminus (Ct) cause a decrease in PM expression. In a polarized cell system the full-length protein is expressed at the BL surface, and three amino acids are required for interpretation of the BL sorting signal. Future studies are needed to define NIS partners that may regulate NIS PM targeting and polarized localization in healthy and tumor tissue.;In Part II, we show that, in addition to I-, NIS also actively transports other substrates, including perchlorate (ClO4 -). The transport of non-physiological substrates has public health and medical relevance. ReO4-, for example, is also available as 188ReO4-, and is an excellent candidate drug for targeting tumors with endogenous or exogenous NIS expression. ClO4- is a NIS-mediated I - transport inhibitor and a widespread environmental pollutant. NIS ability to translocate ClO4-, including its transport into maternal milk, reveals that ClO4- water contamination may pose a greater health risk than previously acknowledged. Interestingly, NIS transports these anions with different stoichiometries: an electrogenic 2 Na+: 1 I- stoichiometry and an electroneutral 1 Na+: 1 ClO4-/ReO 4- one.;Detailed molecular information is essential for rational design of NIS molecules optimized for radioisotope therapy after gene transfer. For these reasons, we established a protocol for the purification of NIS, as described in Part III. We expressed eukaryotic NIS constructs in chimera with bacterial proteins, and expressed three NIS bacterial analogues. We also used two eukaryotic expression systems for mammalian NIS. Rat NIS was purified to homogeneity as assessed by Coomassie gel staining, with yields that are suitable for functional studies on purified protein and for preliminary crystallization screenings.