Structural and Functional Insights into Na+/I− Symporter (NIS) Transport Mechanisms and Their Implications for NIS-Mediated Internal Radiation Therapy

Abstract

The Na+/I- symporter (NIS) is a plasma membrane protein crucial for human health, and a central player in both physiology and targeted internal radiation therapy. In the thyroid gland, NIS mediates the active, electrogenic transport of iodide (I⁻) with a 2 Na⁺ : 1 I⁻ stoichiometry—and this is the first, and the rate-limiting, step in the biosynthesis of the thyroid hormones (THs). These hormones, triiodothyronine (T₃) and thyroxine (T₄), are essential regulators of metabolism, influencing growth, development (especially that of the central nervous system), and energy balance. Deficiencies in I⁻ uptake or TH production can lead to severe disorders, including hypothyroidism, goiter, and developmental delays, underscoring how critical it is that the delicate homeostatic balance of circulating TH levels be maintained. Beyond its indispensable role in physiology, NIS underpins the highly successful treatment for thyroid cancer based on radioiodide. Because many thyroid malignancies retain functional NIS expression, metastatic or residual thyroid cancer cells can be selectively targeted using radioactive 131I⁻ isotopes administered systemically post-thyroidectomy. This strategy exploits the ability of NIS-expressing cells to concentrate radioiodide, delivering cytotoxic radiation specifically to malignant tissue while largely sparing healthy organs. Because NIS is naturally expressed predominantly in thyroid tissue, radioiodide therapy remains one of the most elegantly selective anticancer therapies ever devised. Interestingly, NIS not only transports I⁻ with a 2 Na⁺ : 1 I⁻ stoichiometry, but also transports oxyanions, such as pertechnetate (⁹⁹ᵐTcO₄⁻), perrhenate (ReO₄⁻), and the environmental pollutant perchlorate (ClO₄⁻). Strikingly, these oxyanions are transported with a different, electroneutral stoichiometry of 1 Na⁺ : 1 XO₄⁻. Chapter 2 of this dissertation investigates how the same transporter can switch between a 2 Na⁺ : 1 I⁻ (electrogenic) and a 1 Na⁺ : 1 XO₄⁻ (electroneutral) transport stoichiometry. We conducted transport assays and made electrophysiological measurements, and analyzed the data using a statistical thermodynamics-based equation. Our results show that ClO₄⁻ not only competes directly with I⁻ at the substrate binding site, but also binds at a high-affinity non-transport allosteric site. In so doing, ClO₄⁻ prevents one of the two Na⁺ ions from binding to NIS, effectively shifting I⁻ transport toward a 1 Na⁺ : 1 I⁻ stoichiometry and thus substantially diminishing the driving force for I⁻ accumulation. This finding implies that even low levels of ClO₄⁻ in drinking water may be more detrimental to thyroid physiology than previously believed, indicating that environmental safety standards for perchlorate pollution need to be reassessed. Chapter 3 lays out how we determined the first high-resolution cryo-electron microscopy (cryo-EM) structure of a mutant NIS protein, Q72A NIS. Replacing Glu at position 72 with Ala disrupts Na⁺ binding at the Na1 and Na2 sites, and alters the conformation of transmembrane segment 2. Although this mutant does not transport I⁻, it still transports ReO₄⁻—and, crucially, it does so electrogenically, with a 2 Na⁺ : 1 ReO₄⁻ transport stoichiometry (rather than a 1 Na⁺ : 1 ReO₄⁻ stoichiometry). Detailed structural analysis reveals two newly formed Na⁺ sites (Na3 and Na4) along the translocation pathway leading to the cytoplasm, showing how even single amino acid substitutions can drastically change substrate selectivity and stoichiometry. These findings deepen our understanding of the structural plasticity of NIS and of how it coordinates ions. Finally, chapter 4 investigates how NIS could be used to develop innovative cancer therapies, particularly ones for non-thyroidal cancers. By introducing point mutations within the π-helix of transmembrane segment 7, we engineered a variant (the L253P/V254F double mutant, dubbed PF NIS) that efficiently transports oxyanions but transports only a negligible amount of I⁻. To uncover the structural basis for this phenomenon, we determined the cryo-EM structure of PF NIS at 2.58 Å, the highest resolution achieved for any NIS structure to date. Crucially, this structure explains how NIS binds and transports 2 Na+ ions. We propose that this selectively reprogrammed PF NIS protein could be expressed in non-thyroidal cancers by gene transfer, and the malignancies then treated with radioactive 188ReO4- while protecting the thyroid by simultaneously administering non-radioactive I-. The non-radioactive I- would not prevent the cancer cells from accumulating 188ReO4-, but it would prevent healthy thyroid cells from doing so (by saturating WT NIS). As a proof of concept, we tested this hypothesis in in vitro experiments. This idea paves the way for a new approach to treating cancer that relies on the genetic transfer of engineered NIS molecules into non-thyroidal tumors, with fewer off-target side effects. All in all, then, this work combines biochemical, functional, and structural strategies to reveal how the mechanism by which NIS couples ions changes depending on the substrates; identifies residues critical for determining substrate selectivity and transport stoichiometry; and proposes new ways to exploit NIS to treat non-thyroidal cancers in a targeted fashion. These findings not only deepen our fundamental understanding of an essential transporter involved in both normal endocrinology and cancer treatment but also have direct implications for how water pollution should be tackled and what strategies should be deployed in developing some next-generation of anticancer therapies.