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

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 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.

Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors

“…YFP-based probes have been successfully used for quantitative screening of chloride transport in different cells (Galietta et al 2001b;Rhoden et al 2007) and for high-throughput screening of Cl-selective glycine and GABA A receptor channels (Kruger et al 2005). Also transgenic mice expressing YFP were generated and used for the analysis of glutamate-induced changes in intracellular Cl and pH (Metzger et al 2002;Slemmer et al 2004).…”

Anoctamin 1 is Apically Expressed on Thyroid Follicular Cells and Contributes to ATP- and Calcium-Activated Iodide Efflux

“…We have previously demonstrated that FRTL-5/YFP cells cultured in the presence of TSH, respond to 100 µM NaI with a perchlorate-sensitive decrease in YFP fluorescence due to NIS-mediated iodide uptake [31]. Rinsing iodide-loaded cells with iodide-free BSS results in a slow recovery of resting fluorescence due to iodide efflux, complete within 15-20 min (Fig.…”

“…A cell line with stable expression of the iodide-sensitive yellow fluorescent protein YFP-H148Q/I152L (FRTL-5/YFP) was used to monitor iodide fluxes [31]. ANO1 gene silencing in FRTL-5/YFP cells was carried out with siRNA directed against rat ANO1.…”

“…Cell lines with persistent and uniform fluorescence were then transfected with pcDNA3.1/Zeo containing the full length human NIS cDNA (S. Jhiang, Ohio State University), and selected with 250 µg/ml zeocin. Cell lines were screened for NIS-mediated iodide uptake using the YFP-based fluorescence assay, as previously described [31]. ANO1 was transiently expressed in CHO/YFP/NIS cells by transfection with pcDNA 3.1 containing cDNA for human ANO1 abc isoform (LJV Galietta, Genova, Italy) using Lipofectamine.…”

“…Iodide fluxes were monitored as iodide-induced changes in fluorescence in cells expressing YFP-H148/I152L, as described previously [31]. Cells were cultured on glass coverslips, mounted in a thermostatically-controlled imaging chamber (Warner Instruments, Hamden, CT, USA), and perfused at 37°C with a phosphate-buffered balanced salt solution (BSS) composed of 137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl 2 , 1.1 mM MgCl 2 , 1.5 mM KH 2 PO4, 8.1 mM Na 2 HPO4 and 10 mM glucose (pH 7.4).…”