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First published online 20 January 2004
doi: 10.1242/jcs.00898

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The Q267E mutation in the sodium/iodide symporter (NIS) causes congenital iodide transport defect (ITD) by decreasing the NIS turnover number

Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (99K): [in a new window]   Fig. 1. Human NIS (hNIS) secondary structure model and immunohistochemistry of thyroid tissue from the Q267E NIS patient. (A) Current WT hNIS 13-transmembrane-segment secondary structure model; ITD-causing mutations are shown in rectangles by the WT residue involved, indicating its position and the substituted residue. The single-letter amino acid code is used. X, stop codon; fS, frame shift; , deletion. (B-E) NIS expression was analyzed in paraffin-embedded sections of thyroid by immunohistochemistry using anti-carboxyl terminus hNIS Ab. (B) Normal thyroid staining. (C) Thyroid from a patient with Graves' disease and (D,E) thyroid from a patient with the Q267E NIS mutation (kindly provided by Dr Refetoff, Chicago University). Pronounced NIS protein staining was observed in both these cases. No immunoreactivity was detected in the presence of the C-terminus synthetic peptide (not shown). Magnifications: B,D, 250x; E,C, 1000x.

View larger version (40K): [in a new window]   Fig. 2. Characterization of expression and activity of Q267E NIS in transfected COS-7 cells. (A) Immunoblot analysis. Membrane fractions (20 µg) were isolated from non-transfected COS-7 cells (NT) (lanes 1 and 4) or from COS-7 cells transfected either with WT rNIS (lane 2), rQ267E NIS (lane 3), WT hNIS (lane 5) or hQ267E NIS (lane 6). Membrane fractions were then electrophoresed, electrotransferred and immunoblotted with either 2 nM anti-Ct-rNIS Ab (lanes 1-3) or 4 nM anti-Ct-hNIS Ab (lanes 4-6). This is a representative immunoblot. Protein loading was standardized with anti-tubulin Ab (not shown). Expression of both NIS and tubulin was quantified in five different experiments. No statistically significant difference was found in the NIS/tubulin expression ratio of Q267E NIS when compared to WT NIS. (B) I- transport. Assays were performed in the presence of 20 µM I-/140 mM Na+ (colored bars) or 20 µM I-/140 mM Na+ plus 80 µM perchlorate (white bars). Results are expressed as the percentage I- uptake with respect to WT NIS. Values represent the average of at least five different experiments; in each experiment, activity was analyzed in triplicate or sextuplicate. (C) Time-course of I- uptake in cells transfected with either WT rNIS (red squares) or rQ267E NIS (green triangles). Cells were incubated with 20 µM I-/140 mM Na+ for the indicated times. Values obtained with non-transfected cells were subtracted. (D) Effect of increasing concentrations of I- on I- uptake. COS-7 cells not transfected (black circles) or transfected with either WT rNIS (red squares) or rQ267E NIS (green triangles) were assayed at steady-state (60 minutes) in the presence of the indicated I- concentrations. I- uptake values are the mean±s.d. Statistical significance of the data was calculated by t-test analysis using two-tailed P values. *P<0.05. Y-axes have been split into two scales (B-D).

View larger version (30K): [in a new window]   Fig. 3. Flow cytometry analysis of Q267E NIS. Non-permeabilized (A,C) or permeabilized (B,D) COS-7 cells not-transfected (black dotted line) transfected with WT hNIS (red line) or hQ267E NIS (green line) were incubated with 4 nM anti-Ct-hNIS Ab (A,B) or with a 1:50 dilution of anti-hNIS VJ1 Ab (C,D), followed by incubation with fluorescein-conjugated goat anti-rabbit Ab (A,B) or fluorescein-conjugated anti-mouse Ab (C,D). Aliquots containing 2x106 cells were subjected to flow cytometry.

View larger version (60K): [in a new window]   Fig. 4. Surface biotinylation analysis of Q267E NIS. Non-transfected COS-7 cells (NT) or COS-7 cells transfected with WT rNIS, rQ267E NIS, WT hNIS or hQ267E NIS were biotinylated with 1 mg/ml Sulfo-NHS-SS-biotin (A,D) or Sulfo-NHS-LC-biotin (B,C,E,F). Immunoblot analysis of surface biotinylated polypeptides precipitated with streptavidin-agarose beads was performed with either 2 nM anti-Ct-rNIS Ab (A) or 4 nM anti-Ct-hNIS Ab (D). This is a representative immunoblot. Nitrocellulose membranes were stripped and reprobed with anti-Na+/K+-ATPase alpha subunit antibody (not shown). Expression of both NIS and Na+/K+ ATPase alpha subunit was quantified in five different experiments. No statistically significant difference was found between the NIS/ATPase-alpha-subunit expression ratio of Q267E NIS and WT NIS. (B,E) Blot analysis of surface-biotinylated polypeptides immunoprecipitated with anti-Ct-rNIS (B) or anti-Ct-hNIS (E) and probed with streptavidin-HRP. (C,F) Stripped nitrocellulose membranes were reprobed with anti-Ct-rNIS (C) or anti-hNIS VJ1 (F).

View larger version (71K): [in a new window]   Fig. 5. Immunofluorescence analysis of COS-7 cells not transfected or transfected with WT rNIS or rQ267E NIS. Non-permeabilized (A,C) or permeabilized (B,D) transfected COS-7 cells were incubated with 2 nM anti-Ct-rNIS Ab followed by fluorescein-conjugated goat anti-rabbit Ab. The rectangular panels above B and D show xz cross-sectional images obtained using a confocal microscope

View larger version (68K): [in a new window]   Fig. 6. Immunofluorescence analysis of COS-7 cells transfected with WT hNIS or hQ267E NIS. Non-permeabilized (top panels) or permeabilized (bottom panels) transfected COS-7 cells were incubated with 4 nM anti-Ct-hNIS Ab (A-D) or with anti-hNIS VJ1 Ab (1:50) (EH). A second incubation was performed with fluorescein-conjugated goat anti-rabbit Ab (A-D) or fluorescein-conjugated anti-mouse Ab (E-H). The upper rectangular panels show xz cross-sectional images obtained using a confocal microscope.

View larger version (55K): [in a new window]   Fig. 7. Effect of charged side-chain Q267 substitutions on NIS expression. (A) Non-transfected COS-7 cells (NT) or COS-7 cells transfected with either WT rNIS or rQ267D, rQ267K or rQ267R NIS were assayed for I- transport activity at 20 µM I- at steady-state. Assays were performed in the presence of 20 µM I-/140 mM Na+ (colored bars) or 20 µM I-/140 mM Na+ plus 80 µM perchlorate (white bars). Results are expressed as a percentage of WT NIS activity. The y-axis has been split into two scales. (B) Immunoblot. (C) Surface biotinylation. (D) Immunofluorescence analysis. Procedures were performed as described in Figs 3, 5 and 6, and in Materials and Methods.

View larger version (55K): [in a new window]   Fig. 8. Effect of neutral and polar side-chain Q267 substitutions on NIS expression. Non-transfected COS-7 cells (NT) or COS-7 cells transfected either with WT rNIS, rQ267A or rQ267N NIS were assayed for I- transport activity at 20 (A) or 160 (B) µM I-. Assays were performed in the presence of 20 µM or 160 µM I-/140 mM Na+ (colored bars) or 20 µM or 160 µM I-/140 mM Na+ plus 80 µM perchlorate (white bars). Results are expressed in percentages with respect to WT NIS activity. y-Axis has been split into two scales. (C) Immunoblot. (D) Surface biotinylation. (E) Immunofluorescence analysis. All procedures were performed as described in Figs 3, 5 and 6, and in Materials and Methods.

View larger version (24K): [in a new window]   Fig. 9. Kinetic analysis of I- uptake in transfected COS cells. (A) Initial rates (4-minute time points) of I- uptake were determined at the indicated concentrations of I-, as described in Materials and Methods. Calculated curves (smooth lines) were generated using the equation: v([I-])=(Vmaxx[I-])/(Km+[I-])+0.03xI-]+0.37. The terms 0.03x[I-]+0.37 correspond to background adjusted by least squares to the data obtained with non-transfected cells. Vmax-I- and Km-I- values are indicated in Table 1. Symbols: non-transfected cells (black circles), WT NIS (red squares), rQ267E (green triangles), rQ267A (blue diamonds) and rQ267N (gray circles). (B) Na+-dependent kinetic analysis. To assess Na+ dependence of I- uptake, cells were incubated for 4 minutes with the indicated concentrations of Na+; isotonicity was maintained constant with choline chloride. Na+ dependence data were analyzed using the equation v=(Vmaxx[Na+]2)/(Km+[Na+]2)-0.001x[Na+]+0.87. The term 0.001x[Na+]+0.87 corresponds to the background adjusted by lineal regression analysis obtained with non-transfected cells. Data were fitted by non-linear least squares using the Marquard-Levenberg algorithm (Press et al., 1986). Vmax-Na+ and Km-Na+ values are indicated in Table 1.

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