Effects of Na+ and H+ on steady-state and presteady-state currents of the human concentrative nucleoside transporter 3 (hCNT3)

Human concentrative nucleoside transporter 3 (hCNT3) uses the electrochemical gradient of Na+ and H+ to drive the transport of nucleosides and therapeutic nucleoside analogs into the cells. We employed the two-electrode voltage clamp technique to compare the steady-state and presteady-state kinetics of hCNT3 in the presence of Na+ and H+. We found that H+ supported a higher maximal rate of uridine transport than Na+, but the efficiency of transport was lower. For both cations, maximal uridine-induced current increased with hyperpolarizing potentials and did not saturate within the voltage range tested. Apparent affinity of hCNT3 for uridine in H+ was insensitive to membrane voltage at negative potentials, and decreased with depolarization. In contrast, apparent affinity for uridine in Na+ decreased with hyperpolarization and was independent of voltage at depolarizing potentials. H+-coupled hCNT3 exhibited lower affinity for all natural nucleosides and different substrate selectivity compared to Na+-coupled hCNT3. In H+, lack of the hydroxyl groups at 2' and 5' decreased the affinity, while lack of the nitrogen N-7 or inversion of the configuration of the hydroxyl group at 2' prevented transport. Presteady-state charge movements of hCNT3 did not decrease when extracellular cation concentration (Na+ or H+) was reduced, but the tau (ON)-V and Q-V curves were shifted to more negative potentials. The different effects of uridine and inosine on presteady-state currents in H+ indicated a change in rate-limiting step for the transport of these substrates by H+-coupled hCNT3.