The sodium/iodide symporter is an intrinsic membrane protein that actively transports iodide into thyroid follicular cells. It is a key element in thyroid hormone biosynthesis and in the radiotherapy of thyroid tumours and their metastases. Sodium/iodide symporter is a very hydrophobic protein that belongs to the family of sodium/solute symporters. As for many other membrane proteins, particularly mammalian ones, little is known about its biochemistry and structure. It is predicted to contain 13 transmembrane helices, with an N-terminus oriented extracellularly. The C-terminal, cytosolic domain contains approximately one hundred amino acid residues and bears most of the transporter's putative regulatory sites (phosphorylation, sumoylation, di-acide, di-leucine or PDZ-binding motifs). In this study, we report the establishment of eukaryotic cell lines stably expressing various human sodium/iodide symporter recombinant proteins, and the development of a purification protocol which allowed us to purify milligram quantities of the human transporter. The quaternary structure of membrane transporters is considered to be essential for their function and regulation. Here, the oligomeric state of human sodium/iodide symporter was analysed for the first time using purified protein, by size exclusion chromatography and light scattering spectroscopy, revealing that the protein exists mainly as a dimer which is stabilised by a disulfide bridge. In addition, the existence of a sodium/iodide symporter C-terminal fragment interacting with the protein was also highlighted. We have shown that this fragment exists in various species and cell types, and demonstrated that it contains the amino-acids [512-643] from the human sodium/iodide symporter protein and, therefore, the last predicted transmembrane helix. Expression of either the [1-512] truncated domain or the [512-643] domain alone, as well as co-expression of the two fragments, was performed, and revealed that co-expression of [1-512] with [512-643] allowed the reconstitution of a functional protein. These findings constitute an important step towards an understanding of some of the post-translational mechanisms that finely tune iodide accumulation through human sodium/iodide symporter regulation.

Absorption of dietary iodide, presumably in the small intestine, is the first step in iodide (I(-)) utilization. From the bloodstream, I(-) is actively taken up via the Na(+)/I(-) symporter (NIS) in the thyroid for thyroid hormone biosynthesis and in such other tissues as lactating breast, which supplies I(-) to the newborn in the milk. The molecular basis for intestinal I(-) absorption is unknown. We sought to determine whether I(-) is actively accumulated by enterocytes and, if so, whether this process is mediated by NIS and regulated by I(-) itself. NIS expression was localized exclusively at the apical surface of rat and mouse enterocytes. In vivo intestine-to-blood transport of pertechnetate, a NIS substrate, was sensitive to the NIS inhibitor perchlorate. Brush border membrane vesicles accumulated I(-) in a sodium-dependent, perchlorate-sensitive manner with kinetic parameters similar to those of thyroid cells. NIS was expressed in intestinal epithelial cell line 6, and I(-) uptake in these cells was also kinetically similar to that in thyrocytes. I(-) downregulated NIS protein expression and its own NIS-mediated transport both in vitro and in vivo. We conclude that NIS is functionally expressed on the apical surface of enterocytes, where it mediates active I(-) accumulation. Therefore, NIS is a significant and possibly central component of the I(-) absorption system in the small intestine, a system of key importance for thyroid hormone biosynthesis and thus systemic intermediary metabolism.

The active transport of iodide from the bloodstream into thyroid follicular cells is mediated by the Na+/I− symporter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference, we compared 125I uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the Vmax for mNIS was four times higher than that for hNIS, and that the iodide transport constant (Km) was 2.5-fold lower for hNIS than mNIS. We also performed immunocytolocalization studies and observed that the subcellular distribution of the two orthologs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortholog was intracellular in ∼40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2.5-fold higher than that of the human protein, and therefore cannot alone account for the difference in the obtained Vmax values. We reasoned that the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein.

The Na+/I− symporter (NIS) is a key plasma membrane protein that mediates active I− uptake in the thyroid, lactating breast, and other tissues with an electrogenic stoichiometry of 2 Na+ per I−. In the thyroid, NIS-mediated I− uptake is the first step in the biosynthesis of the iodine-containing thyroid hormones, which are essential early in life for proper CNS development. In the lactating breast, NIS mediates the translocation of I− to the milk, thus supplying this essential anion to the nursing newborn. Perchlorate (ClO4−) is a well known competitive inhibitor of NIS. Exposure to food and water contaminated with ClO4− is common in the U.S. population, and the public health impact of such exposure is currently being debated. To date, it is still uncertain whether ClO4− is a NIS blocker or a transported substrate of NIS. Here we show in vitro and in vivo that NIS actively transports ClO4−, including ClO4− translocation to the milk. A simple mathematical fluxes model accurately predicts the effect of ClO4− transport on the rate and extent of I− accumulation. Strikingly, the Na+/ ClO4− transport stoichiometry is electroneutral, uncovering that NIS translocates different substrates with different stoichiometries. That NIS actively concentrates ClO4− in maternal milk suggests that exposure of newborns to high levels of ClO4− may pose a greater health risk than previously acknowledged because ClO4− would thus directly inhibit the newborns' thyroidal I− uptake.

Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.

Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.