Conserved charged amino acid residues in the extracellular region of sodium/iodide symporter are critical for iodide transport activity

Abstract

Background: Sodium/iodide symporter (NIS) mediates the active transport and accumulation of iodide from the blood into the thyroid gland. His-226 located in the extracellular region of NIS has been demonstrated to be critical for iodide transport in our previous study. The conserved charged amino acid residues in the extracellular region of NIS were therefore characterized in this study. Methods: Fourteen charged residues (Arg-9, Glu-79, Arg-82, Lys-86, Asp-163, His-226, Arg-228, Asp-233, Asp-237, Arg-239, Arg-241, Asp-311, Asp-322, and Asp-331) were replaced by alanine. Iodide uptake abilities of mutants were evaluated by steady-state and kinetic analysis. The three-dimensional comparative protein structure of NIS was further modeled using sodium/glucose transporter as the reference protein.

Inter-species variation in monovalent anion substrate selectivity and inhibitor sensitivity in the sodium iodide symporter (NIS)

“…However, since the proteins in this study clearly respond to the tested substrates differently, this indicates the previously described residues are not the sole sites of anion selectivity and ion coordination (Fig 1 and S1 Fig, bold red residues). Of thirteen charged residues located on the extracellular loops of hNIS which were reported to significantly reduce iodide uptake when individually mutated to alanine, zNIS lacks four: R82L, D163N, H226G, and R239Q (Fig 1, red triangles) [71]. zNIS has four additional positively charged residues (Fig 1, open diamonds) and wNIS has three additional negatively charged residues (Fig 1, blue diamonds) on the extracellular face.…”

“…Iodide uptake assays to evaluate K m and V max were performed following the protocol described by [71] with the following modifications: 2x10 4 HeLa-HA-NIS cells were plated in 96 well plates (Corning Falcon #351172) three days prior to the iodide uptake assay. Cells were washed with 200 μl 37˚C uptake buffer.…”

“…Open circles indicate site of a mutation known to cause membrane trafficking defect in humans [22]. Black triangles indicate a charged residue where mutation to alanine significantly reduces iodide uptake in human NIS [71]. Red triangles indicate a charged residue where mutation to alanine significantly reduces iodide uptake in human NIS and this residue is not charged in zebrafish NIS [71].…”

“…Black triangles indicate a charged residue where mutation to alanine significantly reduces iodide uptake in human NIS [71]. Red triangles indicate a charged residue where mutation to alanine significantly reduces iodide uptake in human NIS and this residue is not charged in zebrafish NIS [71]. Open diamonds indicate additional positively charged residues in zebrafish NIS.…”

“…mutation to alanine significantly reduces iodide uptake in human NIS and this residue is not charged in zebrafish NIS [71]. Bold red lettering indicates residue reported to be involved in stoichiometry control and translocation dynamics [52,53].…”