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

Abstract

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.

Role of the Transporter Regulator Protein (RS1) in the Modulation of Concentrative Nucleoside Transporters (CNTs) in Epithelia

“…Human CNT1 was cloned from human fetal liver (Lostao et al, 2000), and hCNT3 (Errasti-Murugarren et al, 2007) and hCNT2 (this study) were cloned from human kidney. All three were inserted into the oocyte expression vector pBSII(KS), which contained a poly(A) tail of 54 bp (Lostao et al, 2000;Gorraitz et al, 2010). To prepare sense cRNA from hRS1 (Lambotte et al, 1996), hCNT1, hCNT2, or hCNT3, the respective purified plasmids were linearized with XbaI (hCNT1), SalI (hCNT2), SacII (hCNT3), or MluI (hRS1).…”

Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets

“…The sodium/nucleoside coupling ratio of hCNT1 and hCNT2 is 1:1, while hCNT3 shows a 2:1 stoichiometry ( Smith et al, 2004 , 2007 ). Interestingly, hCNT3 is the only transporter in this family able to accept protons as driving force ( Smith et al, 2005 ; Gorraitz et al, 2010 ). Based upon its broad selectivity and highly potential concentrative activity, hCNT3 is considered to be a major player in nucleoside-derived drug uptake.…”

Functional characterization of the human facilitative glucose transporter 12 (GLUT12) by electrophysiological methods

“…Stage VI oocytes from X. laevis (Xenopus Express, France) were obtained as previously described (15). They were microinjected with 50 ng of cRNA coding for human GLUT12 or GLUT1 and maintained at 18 ºC in Barth´s medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO 3 ) 2 , 0.41 mM CaCl 2 , 0.82 mM MgSO 4 , 2.4 mM NaHCO 3 and 10 mM HEPES-Tris, pH 7.4) supplemented with gentamycin (50 mg/L), ciprofloxacin (50 mg/L) and amikacin (50 mg/L).…”

“…In the presence of Na + , protons further decreases sugar uptake, indicating that H + could also interact with GLUT12. In relation to this, Wilson-O´Brien et al The use of Na + or H + gradients has been described for human transporters such as SGLT1 (16), CNT3 (15), and the EAATs (21). For GLUT12, the inhibition of glucose uptake observed in the presence of both Na + and H + could be the result of competition of both ions for a common binding site, which would alter the conformation and function of the transporter; nevertheless, further experiments are needed to address this question.…”

“…At high external K + concentrations, the cell resting membrane potential is depolarized to a low value. Voltage-dependence has been described for different Na + -driven cotransporters (15,44,49), but has not been reported for facilitative transporters. The blockade of glucose uptake in the absence of Na + (in choline buffer) under high external K + , suggests that the facilitative transport of glucose by GLUT12 is voltage dependent.…”