Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L

摘要

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15 mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5 mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K-m of 35 mu M and a V-max of 55 mu M/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K-m of 2.2 mM and a V-max of 43 mu M/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells. (C) 2007 Elsevier Inc. All rights reserved.