Intracellular anion fluorescence assay for sodium/iodide symporter substrates

Abstract

The sodium/iodide symporter (NIS) is primarily responsible for iodide accumulation in the thyroid gland for the synthesis of thyroid hormones; however, it can also transport other lyotropic anions in the thyroid gland and nonthyroid tissues. Some NIS substrates have important physiological or clinical roles, and others are environmental contaminants with health-related consequences. The aim of this study was to assess the utility of a yellow fluorescent protein variant, YFP–H148Q/I152L, as a biosensor to monitor the cellular uptake of NIS substrates, including thiocyanate (SCN −), nitrate ( NO 3 - ), chlorate ( ClO 3 - ), perchlorate ( ClO 4 - ), and perrhenate ( ReO 4 - ). The fluorescence of purified YFP–H148Q/I152L was suppressed by anions with an order of potency of ReO 4 - > ClO 4 - = I − = SCN − = ClO 3 - > NO 3 - ≫ Cl −. Anions also suppressed the fluorescence of YFP–H148Q/I152L expressed in FRTL-5, a thyroid cell line with high NIS expression. Quantitation of intracellular concentrations revealed differences among anions in the affinity and maximal velocity of NIS-mediated uptake as well as in the rate constant for passive efflux. These results suggest that YFP–H148Q/I152L can serve as an intracellular biosensor of NIS-transported anions and may be useful to study the physiology of endogenous anions as well as the health-related consequences of environmental anions.