Abstract
Background: In order to evaluate the function of transporters in detail, it is required to observe the transporting dynamics in living cells and the rapid evaluation is needed to screen novel inhibitors. The aim of this study is to develop a high-throughput biosensing method for analysis of transporter uptake.
Methods: Urate transporter 1 (URAT1) and organic anion transporter 1 (OAT1) -transfected COS7 cells were added into 0-5 μM fluorescein (FL) containing buffer and the relative fluorescence unit (RFU) per μg protein of the cell lysate was determined with 96 well plate reader. FL transport was observed by fluorescence microscopy when URAT1 or OAT1/COS7 cells were added with FL 0.5 μM.
Results and discussion: The URAT1 or OAT1/COS7 cell lysate showed the significant increase of RFU when 0.5-5 μM of FL was applied. The FL uptake was suppressed to background level with supplement of 100-500 μM of uric acid. Fluorescent bioimaging also indicated intense FL signals in the cytosol of URAT1 or OAT1/COS7. These results suggested that FL was transported via both URAT1 and OAT1. In conclusion, FL-based analyzing method for characterization of urate transporter was developed. The technique described here has the potential for broad application for high throughput screening or bioimaging of transport kinetics.
