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 (i) high affinity for extracellular I(-) (35 microM), (ii) inhibition by the NIS inhibitor perchlorate, (iii) extracellular Na(+)-dependence and (iv) 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 single cell differences 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 non-thyroidal cells following gene transfer, and suggest that fluorescence detection of cellular I(-) may be a useful tool to study the pathophysiology and pharmacology of NIS. Key words: thyroid, iodide, yellow fluorescent protein, imaging.

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L.

RHODEN, KERRY JANE;ROMEO, GIOVANNI
2007

Abstract

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 (i) high affinity for extracellular I(-) (35 microM), (ii) inhibition by the NIS inhibitor perchlorate, (iii) extracellular Na(+)-dependence and (iv) 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 single cell differences 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 non-thyroidal cells following gene transfer, and suggest that fluorescence detection of cellular I(-) may be a useful tool to study the pathophysiology and pharmacology of NIS. Key words: thyroid, iodide, yellow fluorescent protein, imaging.
Rhoden KJ.; Cianchetta S.; Stivani V.; Portulano C.; Galietta L.; Romeo G.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/35169
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