Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L.
Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na+-dependent, consistent with the Na+-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K(m) 4.6 microM for perchlorate and 34.8 muM for iodide) and lower maximal velocity (V(max) 6.8 microM/s for perchlorate and 39.5 microM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 muM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.
Cianchetta
Stefano
S
Medical Genetics Unit, Department of Gynecologic, Obstetric and Pediatric Sciences, University of Bologna, Bologna, Italy.
di Bernardo
Julie
J
Romeo
Giovanni
G
Rhoden
Kerry J
KJ
D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D007700:Kinetics; D008164:Luminescent Proteins; D010472:Perchlorates / Q000378:metabolism* / Q000633:toxicity*; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D000099209:Sodium-Iodide Symporters
D008164:Luminescent Proteins; D010472:Perchlorates; D011994:Recombinant Proteins; D027981:Symporters; D000099209:Sodium-Iodide Symporters