I − uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na þ ∕I − symporter (NIS) with an electrogenic 2Na þ : 1I − stoichiometry. We have obtained mechanistic information on NIS by characterizing the congenital I − transport defect-causing NIS mutant G93R. This mutant is targeted to the plasma membrane but is inactive. Substitutions at position 93 show that the longer the side chain of the neutral residue at this position, the higher the K m for the anion substrates. Unlike WT NIS, which mediates symport of Na þ and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 2∶1 stoichiometry. Furthermore, G93E/Q NIS discriminate between anion substrates, a discovery with potential clinical relevance. A 3D homology model of NIS based on the structure of the bacterial Na þ ∕galactose transporter identifies G93 as a critical player in the mechanism of the transporter: the changes from an outwardly to an inwardly open conformation during the transport cycle use G93 as a pivot.iodide transport defect | homology modeling | radioiodide therapy | sodium solute cotransporter family

The aims of this study were to optimize the experimental conditions for labeling extracellularly oriented, solvent-exposed cysteine residues of γ-aminobutyric acid transporter 1 (GAT1) with the membrane-impermeant sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) and to characterize the functional and pharmacological consequences of labeling on transporter steady-state and presteady-state kinetic properties. We expressed human GAT1 in Xenopus laevis oocytes and used radiotracer and electrophysiological methods to assay transporter function before and after sulfhydryl modification with MTSET. In the presence of NaCl, transporter exposure to MTSET (1–2.5 mM for 5–20 min) led to partial inhibition of GAT1-mediated transport, and this loss of function was completely reversed by the reducing reagent dithiothreitol. MTSET treatment had no functional effect on the mutant GAT1 C74A, whereas the membrane-permeant reagents N-ethylmaleimide and tetramethylrhodamine-6-maleimide inhibited GABA transport mediated by GAT1 C74A. Ion replacement experiments indicated that MTSET labeling of GAT1 could be driven to completion when valproate replaced chloride in the labeling buffer, suggesting that valproate induces a GAT1 conformation that significantly increases C74 accessibility to the extracellular fluid. Following partial inhibition by MTSET, there was a proportional reduction in both the presteady-state and steady-state macroscopic signals, and the functional and pharmacological properties of the remaining signals were indistinguishable from those of unlabeled GAT1. Therefore, covalent modification of GAT1 at C74 results in completely nonfunctional as well as electrically silent transporters.

The Na+/I− symporter (NIS) mediates Na+‐dependent active I− uptake in the thyroid. We report the electrophysiological characterization of G93R NIS, a clinically‐identified NIS mutation that causes congenital I− transport defect, and other substitutions at position 93. Wild‐type (WT) NIS and G93N, G93T, and G93R mutants were expressed in X. laevis oocytes and the two‐electrode voltage clamp technique was used to study steady‐state and presteady‐state kinetic properties of the transporters. Similar to WT NIS, the G93N and G93T mutants exhibited electrogenic Na+‐dependent I− transport; however, the mutants displayed reduced apparent affinity for I−(>500 μM vs. 30 μM). In contrast to WT NIS, G93N and G93T NIS mutants exhibited electrogenic transport of perchlorate, an environmental pollutant and well‐known NIS inhibitor. Steady‐state kinetics revealed the apparent affinity of the mutants for perrhenate and perchlorate to be 20–30 μM. Steady‐state Na+ kinetics showed a sigmoidal dose‐response relationship with all substrates examined (I−, perchlorate, perrhenate, and chlorate). The Na+ apparent affinity (~30 mM) and the Na+ Hill coefficient (~2) of the mutants were similar to those of the WT. Presteady‐state currents of the mutants were similar to those of the WT. Although trafficked to the cell surface, G93R NIS was inactive. The data suggest that residue 93 may play a key role in coordinating substrate and/or Na+ in the binding pocket. NIH grants S06 GM53933 to S.E. and DK41544 and CA098390 to N.C.