Characterization of two novel transporters, BOCT1 and BOCT2

Our objective is to identify transporters that are involved in choline transport. Low‐affinity choline uptake (LACU) in cell lines was previously shown to be sensitive to organic cations in a manner similar but not identical to known members of the SLC22 transporter family. We selected two orphan gene members of the organic cation transporter (OCT) family to assay for choline uptake activity. The novel genes, BOCT1 and BOCT2, were expressed as fusion proteins and transfected into MC‐IX‐C and NIH3T3 cells. Transfected cells were assayed for LACU, but did not show an increase in uptake. As technical controls, two known OCTs were expressed as fusion proteins: OCT1 and OCTN2. OCT1 transports tetraethylammonium (TEA) and OCTN2 transports carnitine. After transfection, OCT1 and OCTN2 caused a significant increase in TEA and carnitine uptake, respectively. It was suspected that the lack of functional expression of BOCT1 and BOCT2 may be due to a deficiency in cell surface expression. Fluorescent confocal microscopic analysis indicated that the majority of expressed protein is retained intracellularly. In the N‐terminal region, the sequences of BOCT1 and BOCT2 diverge significantly from those of other OCTs, and we speculated that these novel regions were responsible for the lack of cell surface expression. We replaced the N‐terminal ends of BOCT1 and BOCT2 with the N‐terminal end of OCTN2. Preliminary results indicate that the BOCT1 chimeric protein may transport TEA, which is a typical substrate of OCTs. Further experiments include optimization of protein expression and functional characterization of BOCT1 and BOCT2. This research is supported by the National Institute of General Medical Sciences, R15 GM070578.