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.

译文

:碘化钠共转运蛋白(NIS)介导碘化物(I(-))在甲状腺和其他组织中的运输,并作为治疗靶标和核成像报告者越来越重要。目前,使用放射性示踪剂和电生理技术测量了NIS的体外活性。我们报告了使用I(-)敏感和遗传可编码的黄色荧光蛋白(YFP)变体YFP-H148Q / I152L对NIS活性进行新型活细胞成像测定的发展报告。在稳定表达YFP-H148Q / I152L的FRTL-5甲状腺细胞中,I(-)诱导了细胞荧光的快速和可逆下降,其特征在于1)对细胞外I(-)(35 muM)的高度亲和力,2)NIS抑制剂的抑制作用高氯酸盐,3)细胞外Na()依赖性和4)TSH依赖性,表明荧光变化是由于I(-)通过NIS流入所致。 FRTL-5细胞群中的单个细胞在NIS介导的I(-)流入速率中表现出3.5倍的变化,说明了YFP-H148Q / I152L可用于检测NIS活性中的细胞间差异。 I(-)还在表达NIS的COS-7细胞中引起高氯酸盐敏感的YFP-H148Q / I152L荧光下降,但在缺乏NIS的细胞中不引起高氯酸盐敏感性下降。这些结果表明,YFP-H148Q / I152L是基因转移后甲状腺细胞和非甲状腺细胞中NIS介导的I(-)摄取的灵敏生物传感器,并表明细胞I(-)的荧光检测可能是一种有用的工具,研究NIS的病理生理学和药理学。

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