Failure of membrane targeting causes the functional defect of two mutant sodium iodide symporters

Molecular cloning of the sodium/iodide symporter (NIS) allowed identificati on of NIS gene mutations in patients with iodide trapping defect. Whereas v arious mutant human (h) NIS molecules display loss of function when express ed by transfection in mammalian cells, the precise mechanism(s) responsible for the functional abnormality of these proteins remains unknown. With the aim to explore these mechanisms in three natural hNIS mutants identified p reviously in patients with iodide trapping defect (Q267E, S515X, and C272X) , we have prepared tools allowing direct measurement of the protein at its normal location in the plasma membrane. A COS-7 cell line was made by transfection that stably expressed high level s of wild-type hNIS. It was used to screen by flow cytometry monoclonal ant ibodies (mAbs) prepared from mice immunized against hNIS. Genetic immunizat ion was performed by im injection of a wild-type hNIS complementary DNA con struct, because this procedure has demonstrated the ability to produce anti bodies recognizing native membrane proteins. One mAb that recognized an epi tope ofhNIS exposed on the extracellular side of the plasma membrane was se lected for further studies. The epitope was localized on the sixth putative extracellular loop of the protein on the basis that the mAb did not recogn ize rat NIS, which exhibits major sequence differences in this segment. When this mAb was used to test by flow cytometry the expression of the thre e mutant hNIS proteins in transfected COS-7 cells, it detected similar amou nts of wild-type, Q267E, and the S515X hNIS molecules in permeabilized cell s. In contrast, only the wild-type hNIS was detected at the surface of nonp ermeabilized cells. The C272X hNIS truncation mutant was not detected in in tact or permeabilized cells. This is consistent with the absence of the mAb epitope from this mutant, which is expected to lack the sixth extracellula r loop. Our data demonstrate that faulty membrane targeting is implicated i n the mechanisms causing iodide trapping defect in the Q267E and S515X natu ral hNIS mutants.