Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L.
The sodium iodide symporter (NIS) mediates iodide (I(-)) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I(-)-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I(-) induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I(-) (35 muM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na(+) dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I(-) influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I(-) influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I(-) also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I(-) uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I(-) may be a useful tool by which to study the pathophysiology and pharmacology of NIS.
Rhoden
Kerry J
KJ
U. O. Genetica Medica, Padiglione 11, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy. kerry.rhoden@med.unibo.it
Cianchetta
Stefano
S
Stivani
Valeria
V
Portulano
Carla
C
Galietta
Luis J V
LJ
Romeo
Giovanni
G
D000818:Animals; D015374:Biosensing Techniques; D002460:Cell Line; D002522:Chlorocebus aethiops; D007454:Iodides / Q000378:metabolism; D017136:Ion Transport; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010472:Perchlorates / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D012964:Sodium / Q000378:metabolism*; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology; D013972:Thyrotropin / Q000502:physiology; D000099209:Sodium-Iodide Symporters
D007454:Iodides; D008164:Luminescent Proteins; D010472:Perchlorates; D012964:Sodium; D027981:Symporters; D013972:Thyrotropin; D000099209:Sodium-Iodide Symporters; C494474:perchlorate