Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells - ENT2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine

We stably transfected the cloned human equilibrative nucleoside transporter s 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD c ells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 r uns as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosyla te hENT1 to 37 kDa and hENT2 to 45 kDa, With hENT1 being more sensitive, th ere is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthio inosine (NBMPR) (IC50, 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 mu M) and dipyrida mole (IC50, 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [H-3]NBMPR binds to ENT1 cells with a high affinity K-d of 0.377 +/- 0.098 nM, and eac h ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/ s for uridine, Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7.7-, and 1 9.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1, The nucleobase hypoxanthine inhibits [H-3]uridine uptake by hENT2 but has minimal effect on hENT1, Taken together, these res ults suggest that hENT2 might be important in transporting adenosine and it s metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.