Abstract

6-Mercaptopurine (6-MP) is a nucleobase analog used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disorders. However, the mechanisms underlying its transport into target cells have remained elusive. The protein encoded by SLC43A3_1 (ENBT1) has recently been shown to transport endogenous nucleobases. A splice variant (SLC43A3_2), encoding a protein with 13 additional amino acids in the first extracellular loop, is also expressed but its function is unknown. We hypothesized that 6-MP is a substrate for both variants of ENBT1. HEK293 cells (lacking endogenous ENBT1 activity) were transfected with each of the coding region variants of SLC43A3. ENBT1 function was assessed via the rate of flux of [³H]adenine and [¹⁴C]6-MP across the plasma membrane. Both SLC43A3 variants encoded proteins with similar functional properties. [¹⁴C]6-MP and [³H]adenine had K_m values (±SD) of 163 ± 126 μM and 37±26 μM, respectively, for this system. Decynium-22, 6-thioguanine and 6-methylmercaptopurine inhibited 6-MP uptake with K_i values of 1.0±0.4, 67±30, and 73±20 μM, respectively. ENBT1 also mediated adenine-sensitive efflux of 6-MP from the SLC43A3-HEK293 cells. MRP4 also contributed to the efflux of 6-MP in this model, but was less efficient than ENBT1 in this regard. Furthermore, transfection of HEK293 cells with SLC43A3 increased the sensitivity of the cells to the cytotoxic effects of 6-MP by more than 7-fold. Thus, both variants of ENBT1 are key players in the transfer of 6-MP into and out of cells, and changes in SLC43A3 expression impacts 6-MP cytotoxicity.