Mechanism of anion selectivity and stoichiometry of the Na+/I- symporter (NIS)

Abstract

I(-) uptake in the thyroid, the first step in thyroid hormone biosynthesis, is mediated by the Na(+)/I(-) symporter (NIS) with an electrogenic 2Na(+):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(+) and the environmental pollutant perchlorate electroneutrally, G93T/N/Q/E/D NIS, strikingly, do it electrogenically with a 21 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(+)/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.

Fig. 1.

Analysis of G93 NIS substitutions in COS-7 cells and X. laevis oocytes. (A) Steady-state I^- transport in WT, G93R, or G93K NIS-transfected COS-7 cells. Cells were incubated with 20 μM I^- in the absence (blue bars) or presence (light blue bars) of 80 μM , or with 200 μM I^- in the absence (red bars) or presence (pink bars) of 800 μM . Values shown (pmol I^-/μg DNA ± SD) are from one of at least five different experiments and corrected for transfection efficiency. (B) Flow cytometry under nonpermeabilized conditions with an Ab against the extracellular HA epitope at the NIS N_t, showing WT (19%) and G93R NIS (15%). (C) HA immunostaining of WT and G93R NIS under nonpermeabilized conditions. NIS is stained with Alexa 488 (green) and nuclei with DAPI (blue). Red scale bar = 20 μm. (D and E) Current traces are shown in response to 5 mM I^- in (D) control (water-injected) X. laevis oocytes or oocytes expressing WT, G93R, or G93K NIS, or (E) G93A, N, T, or Q NIS. The evoked inward currents represent NIS-mediated electrogenic Na⁺/I^- cotransport into the cell. V_m = -50 mV. (F) I^- transport as in A in COS-7 cells transfected with WT, G93A, N, Q, S, or T NIS cDNAs. (G) Kinetics of I^- transport by WT and G93K, A, S, T, N, and Q NIS. [Na⁺]_o = 100 mM and V_m = -50 mV. For each mutant, the current values were normalized to the maximum current obtained at saturating [I^-]. The smooth lines are fits of the data to the Michaelis–Menten equation. Values represent the means ± SE from at least four oocytes.

Fig. 2.

Kinetics of I^- transport in X. laevis oocytes and MDCK cells. (A) Kinetics of Na⁺ dependence of I^- transport by WT, G93T, and G93N NIS. [I^-] = 5 mM and V_m = -50 mV. Current values were normalized to the maximum current obtained at saturating [Na⁺]. The smooth lines are fits of the data to the Hill equation. The Hill coefficient values were 1.9 ± 0.2 for WT, 2.0 ± 0.1 for G93T, and 1.9 ± 0.1 for G93N NIS. Values represent the mean ± SE from at least four oocytes. (B) Initial rates (2-min time points) of I^- uptake were determined at the indicated [I^-]s. K_m(I^-)s are indicated as an average ± SE (n = 5 experiments). The graph shown is a representative experiment. (C and D) Na⁺ dependence of I^- uptake. Cells were incubated for 2 min with (C) 250 or (D) 750 μM I^- and the indicated [Na⁺]s. The graph is representative of > 3 experiments. Isotonicity was kept constant with choline-Cl. In all flux experiments, the activity was standardized by expression levels at the cell surface, and background values obtained with nontransfected cells were subtracted (< 5 in B and C; and 12 pmol/μg DNA in D).

Fig. 3.

transport kinetics in MDCK cells and X. laevis oocytes. (A) Initial rates (2 min) of uptake in transduced MDCK cells were determined at the indicated []s. Background in nontransfected cells (< 2 pmol/μg DNA) was subtracted. (B) Na⁺ dependence of transport: Cells were incubated for 2 min with 180 μM and the indicated [Na⁺]s. s are given as an average ± SE of all experiments. The graph is a representative experiment. Background obtained with NT cells (< 2 pmol/μg DNA) was subtracted. (C) Current traces in response to 1 mM (Top Traces) or (Bottom Traces) in oocytes expressing WT, G93R, K, N, or T NIS. V_m = -50 mV. For each mutant, and traces were obtained in the same oocyte. (D) Kinetics of transport by G93T and N NIS in oocytes. [Na⁺]_o = 100 mM and V_m = -50 mV. Data analyzed as in Fig. 1G. (E) Kinetics of Na⁺ dependence of anion transport by G93T and G93N NIS in oocytes with 1 mM V_m = -50 mV. Data were processed as in Fig. 2A. The Hill coefficient values were 1.9 ± 0.2 for G93N, and 2.2 ± 0.2 for G93T NIS.

Fig. 4.

G93E NIS does not transport I^- but transports electrogenically. (A) Steady-state transport assays in nontransfected (NT) or COS-7 cells transfected with WT or G93K, D, or E NIS cDNAs. Cells were incubated for 1 h with 3 μM in the absence (light green bars) or presence (yellow bars) of 120 μM , or with 30 μM in the absence (dark green bars) or presence (olive bars) of 800 μM . (B) Steady-state I^- transport activity was assayed in WT, G93D, or G93E NIS-expressing COS-7 cells as in Fig. 1A. (C) Current traces in response to 5 mM I^- (Left) or 1 mM (Center) or (Right) in oocytes expressing G93D (Top Traces), G93E (Middle Traces), or G93Q NIS (Bottom Traces). V_m = -50 mV. For each mutant, all current traces were obtained in the same oocyte. (D) Kinetics of anion transport by G93D, E, and Q NIS, as in Fig. 1G. (E) Kinetic parameters.

Fig. 5.

NIS model based on the vSGLT structure. The homology model, including NIS residues 50 to 476, was generated as described in SI Text. (A) Overall structure. TMS III–VI^2–5 are colored magenta and TMS VII–XI^6–10 orange. G93 (red) abuts the indole ring of W255 (olive). The rest of the model is colored green. (B) Close-up of the NIS cavity depicting G93 and W255 opposite the unwound portion of TMS VII⁶. N97 and Y259 also face each other at the bottom end of the cavity. M90 forms part of the upper roof of the cavity. Transition between two conformations of NIS: (C) Superposition of the inwardly open and outwardly open conformations showing the movement of the hairpins and the connecting residues. The inwardly open conformation (vSGLT-like) is colored light green and the outwardly open conformation (LeuT-like) red. The green arrow shows the position of TMS III² in the inwardly open conformation and the red arrow the position of the outwardly open conformation. The black arrow indicates the point around which the connecting short helix pivots to allow the rotation of the two helical hairpins. (D) Close-up of boxed region in C. (E) Schematic representation of D; TMS III and IV^2–3 are depicted as cylinders, the short helix as a rectangle, the unstructured connections between TMS III and IV^2–3, and the rectangle as rods. Color scheme as in C and D.