Fig. 1.

Characterization of expression and activity of R124H Na⁺/I⁻ symporter. (A) Na⁺/I⁻ symporter (NIS) secondary structure model. Cylinders represent the 13 transmembrane segments (TMS), in Roman numerals; branches indicate N-linked glycosylation sites (N225, N489 and N502). The N-terminus faces the extracellular milieu and the C-terminus the cytosol. The iodide transport defect (ITD)-causing NIS mutant R124H is indicated in intracellular loop (IL)-2. IL-6 is also labeled. (B) Steady-state I⁻ transport assays in non-transfected (NT), wild-type (WT) or R124H NIS cDNA transiently transfected COS-7 cells. Cells were incubated with 20 µM I⁻ in the absence or presence of 80 µM ClO₄⁻. The results are expressed in pmol I⁻/µg of DNA ± s.d. Values are representative of at least five different experiments; each experiment was performed in triplicate. (C) Immunofluorescence analysis of permeabilized WT and R124H NIS-transfected COS-7 cells. Immunostaining was performed with anti-human NIS and anti-Na⁺/K⁺ ATPase antibodies (Abs), followed by anti-rabbit Alexa 488- and anti-mouse Alexa 555-conjugated Abs. Nuclei were stained with DRAQ5 dye (blue). The overlay of the two images is shown (Merge). Scale bars, 20 µm. (D) Flow cytometry analysis of total and plasma membrane (PM) NIS expression in permeabilized (P) and non-permeabilized (NP) COS-7 cells non-transfected or transfected with WT or R124H NIS. Cells were stained with anti-NIS VJ1 Ab, followed by Alexa-488-conjugated anti-mouse Ab. (E) Immunoblot analysis of cell surface-biotinylated proteins from non-transfected and WT- or R124H NIS-transfected COS-7 cells, performed with anti-human NIS Ab (upper panel). The plasma membrane marker Na⁺/K⁺ ATPase was used as a positive control (lower panel). NS, non-specific. (F) Immunoblot of membrane fractions from COS-7 cells transfected with WT or R124H NIS cDNA with anti-human NIS Ab. Letters on the right side of the blot indicate the relative electrophoretic mobilities of the corresponding NIS bands [A: non-glycosylated, B: partially glycosylated, C: fully glycosylated (mature), BB: dimer of the partially glycosylated, and CC: dimer of the mature polypeptide]. α-Tubulin was used as a loading control.