Physiological Sodium Concentrations Enhance the Iodide Affinity

ARTICLE Received 18 Oct 2013 | Accepted 24 Apr 2014 | Published 3 Jun 2014 DOI: 10.1038/ncomms4948 Physiological sodium concentrations enhance the iodide affinity of the Na þ /I À symporter Juan P. Nicola1,w, Nancy Carrasco1 & L. Mario Amzel2 The Na þ /I À symporter (NIS) mediates active I À transport—the first step in thyroid hormonogenesis—with a 2Na þ :1I À stoichiometry. NIS-mediated 131I À treatment of thyroid cancer post-thyroidectomy is the most effective targeted internal radiation cancer treatment available. Here to uncover mechanistic information on NIS, we use statistical thermodynamics to obtain Kds and estimate the relative populations of the different NIS species during Na þ /anion binding and transport. We show that, although the affinity of NIS for I À is þ low (Kd ¼ 224 mM), it increases when Na is bound (Kd ¼ 22.4 mM). However, this Kd is still much higher than the submicromolar physiological I À concentration. To overcome this, NIS takes advantage of the extracellular Na þ concentration and the pronounced increase in its own affinity for I À and for the second Na þ elicited by binding of the first. Thus, at physiological Na þ concentrations, B79% of NIS molecules are occupied by two Na þ ions and ready to bind and transport I À . 1 Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA. 2 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA. w Present address: Departamento de Bioquı´mica Clıńica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Co´rdoba 5000, Argentina. Correspondence and requests for materials should be addressed to N.C. (email: [email protected]) or to L.M.A. (email: [email protected]). NATURE COMMUNICATIONS | 5:3948 | DOI: 10.1038/ncomms4948 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4948 he thyroid hormones—T3 and T4—are required for the Results development and maturation of the central nervous Transport by NIS. Initial rates of transport were assessed for WT Tsystem, skeletal muscle, and lungs and for the regulation NIS in the intestinal epithelial cell line IEC-6 (refs 2,29) using 1 125 À 186 À of intermediate metabolism in all cells throughout life . Iodine, a either I or ReO4 . IEC-6 cells were used because, as we scarce element, is an essential constituent of these hormones. The have reported previously, they express NIS at higher levels than Na þ /I À symporter (NIS) is the key plasma membrane protein FRTL-5 cells, the rat thyroid cells from which NIS was originally that mediates active I À uptake in the thyroid and other tissues, cloned9. In addition, NIS in IEC-6 cells has the same apparent such as lactating breast, salivary glands, stomach and intestine2–4. affinity for its substrates as NIS in FRTL-5 cells2,29. NIS is also at the centre of the highly effective thyroid cancer I À transport was measured in five experiments, three varying treatment involving administration of radioiodide after [Na þ ] at three or four [I À ]s and two varying [I À ] at five thyroidectomy, a strategy that has been used for over 65 [Na þ ]s (one example of each is shown in Supplementary Fig. 1a). 5–8 À years . Despite the obvious pathophysiological significance of Initial rates of ReO4 transport were measured in four different 9 þ À NIS, it was not until 1996 that its cDNA was cloned , making it experiments, two varying [Na ] at four different [ReO4 ]s, and À þ possible to use gene transfer protocols in pre-clinical and clinical two varying [ReO4 ] at four different [Na ]s (one example of studies to extend NIS-mediated radioiodide treatment to each is shown in Supplementary Fig. 2a). The individual curves extrathyroidal cancers10,11. were fitted by least squares to the Michaelis–Menten equation. In À Active accumulation of I by NIS is electrogenic, with a all cases, the KM for the ion being varied depends strongly on the stoichiometry of two Na þ ions per transported I À (2:1), and concentration of the other ion, indicating that binding of either uses as its driving forces the Na þ gradient generated by the Na þ / ion to empty NIS increases the protein’s affinity for the other ion. K þ ATPase and the electrical potential across the plasma Interpretation of these observations requires a theoretical context membrane9,12. The total driving force provided by the beyond the Michaelis–Menten formulation. downhill movement of Na þ ions under typical conditions þ þ ([Na ]extracellular ¼ 140 mM; [Na ]intracellular ¼ 14 mM; mem- À ¼ ° Theoretical background. Transporters such as NIS are expected brane potential nearly 60 mV; temperature 37 C) is 30–33 B4.3 kcal mol À 1. Under these conditions, given the cost of to exist in at least two conformations : one in which they are transporting I À against its concentration gradient and against open to the extracellular milieu and bind the ions to be the membrane potential, the maximum attainable I À gradient transported, and one in which they are open to the cytoplasm À À and release the ions. Since NIS carries out coupled transport {[I ]i/[I ]e}max is r1,000. However, the maximum amount þ À of I À that can be accumulated does not depend only on the involving three ions (2 Na and 1 I ), it must have three driving force: all the individual steps in the process (binding of binding sites—one for each of the transported ions. In principle, extracellular ions, translocation of the ions across the membrane each site may be occupied at any time, by itself or in combination and release of the ions into the cytoplasm) have to be with occupation of the other sites. The eight different molecular species of the outwardly open conformation (no ions bound, one thermodynamically feasible and kinetically competent. þ þ Significant advances have been made in the molecular Na site occupied, the other Na site occupied, and so on) and characterization of NIS—for example, in elucidating its trans- the relations between them are shown in Fig. 1, where each of the criptional and posttranscriptional regulation and in uncovering eight states is a corner of the cube and each edge represents a its structure–function relations13–19. NIS has been demon- reaction involving binding an additional ion. These states À represent not only the addition of the ion but also the strated to transport both perrhenate (ReO4 ) and the À conformational changes that result from binding the ion. (In environmental pollutant perchlorate (ClO4 ) electroneutrally, principle, these conformations exist even in the absence of the indicating that it transports different substrates with different 34,35 stoichiometries20. However, little is known about how its ions , but in the present analysis they are considered of substrates are bound and released—a fundamental mechanistic too-low occupancy to influence the results). question. To address this gap in our knowledge, we devised a new approach. We analysed kinetic data using statistical thermodynamics, and determined the affinity of the symporter E E for the transported ions as well as the relative populations of the different NIS species present during the transport cycle. Whereas a similar approach has been used to study E E E repressor regulation using binding data21–24 and to analyse E protein-folding data25–27, the results presented here show that this approach can also be employed to obtain mechanistic information on transporters and channels and could complement E E energy landscape studies, such as those that have been carried out on ion-translocating ATPases28. À We show that the low affinity of NIS for I (Kd ¼ 224 mM) þ E E increases (Kd ¼ 22.4 mM) when Na is bound, and that at þ À À physiological extracellular Na concentrations, as many as Figure 1 | States contributing to I and ReO4 transport. E represents B79% of NIS molecules are occupied by two Na þ ions NIS. The upper yellow circle is the bound Na þ to site NaA; the lower one, À À À and thus poised to bind and transport I . The statistical bound NaB. The mauve square is bound I or ReO4 . The blue, orange and thermodynamics approach presented here promises to be grey arrows represent ion binding. The cyan arrow represents the switch applicable to other transporters and channels, including from the outwardly to the inwardly facing conformation for I À transport; À those of medical importance. Remarkably, this approach the magenta arrow, the same switch for ReO4 transport. The states makes it possible to obtain quantitative mechanistic represented in grey were shown not to be significantly occupied during the information about these proteins in their native environment— transport cycle. The Na þ sites designated A and B in the text refer only to whole cells—without the need for membrane protein the order of binding and do not necessarily correspond to the purification. crystallographically identified ‘site 1’ and ‘site 2’ of LeuT30. 2 NATURE COMMUNICATIONS | 5:3948 | DOI: 10.1038/ncomms4948 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4948 ARTICLE Assuming that only the triply occupied NIS can switch to the The fraction of protein molecules in state i can be calculated by inwardly open conformation, and that this conformational dividing the statistical weight of state i by the partition function change is slow or much less frequent than the binding and (fi ¼ xi/Q).

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