Electrophysiological characterization of a recombinant human Na⁺‐coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes

Abstract

Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na + down its electrochemical gradient. The two-microelectrode voltage-clamp technique was used to measure steady-state and presteady-state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na + currents included the anticancer drugs 5-fluorouridine, 5-fluoro-2 -deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-β-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene. Apparent K m values for 5-fluorouridine, 5-fluoro-2 -deoxyuridine and zidovudine were 18, 15 and 450 µM, respectively. hCNT1 was Na + specific, and the kinetics of steady-state uridineevoked Na + currents were consistent with an ordered simultaneous transport model in which Na + binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na + -nucleoside coupling stoichiometry, determined directly by comparing the uridine-induced inward charge movement to [ 14 C]uridine uptake was 1 : 1. hCNT1 presteady-state currents were used to determine the fraction of the membrane field sensed by Na + (61%), the valency of the movable charge (−0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 × 10 10 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

“…In the case of hfCNT, both sites are specific for Na ϩ (14). hCNT1 and hCNT2, in contrast, have single Na ϩ -specific sites (11,35 (17) and C. elegans CeCNT3 (15). A central question in CNT energetics is the structural, functional, and evolutionary relationship between these various cation-binding sites in different CNT family members.…”

“…In studies of wild-type hCNT1 and hCNT3, we have established that Na ϩ binds to the transporter first, increasing the affinity for nucleoside, which then binds second (19,35). To minimize the potential effects of altered Na ϩ apparent affinity on uridine kinetic parameters, experiments to investigate uridine transport kinetics were undertaken at the maximum possible Na ϩ concentration of 100 mM.…”

“…Electrophysiology Current Measurements-Membrane currents were measured at room temperature (20°C) using the whole-cell, two-electrode voltage clamp technique (GeneClamp 500B, Molecular Devices Corp.), as described previously (19,35). The GeneClamp 500B was interfaced to an IBM-compatible PC via a Digidata 1322A A/D converter and controlled by pCLAMP software (version 9.0, Molecular Devices Corp.).…”

“…ϩ :nucleoside and H ϩ :nucleoside stoichiometries of E343C, E343Q, E343D, E519C, E519D and, as a control, wild-type hCNT3 were determined by simultaneously measuring cation currents and radiolabeled uridine uptake under voltage clamp conditions, as described previously (11,19,35). Na ϩ and H ϩ currents were determined in transport media containing 100 mM NaCl at pH 8.5 and 100 mM ChCl at pH 5.…”

Transport Mechanisms of Nucleosides and Nucleoside Analogues ReverseTranscriptase Inhibitors in the Brain

“…They have been also produced as recombinant proteins in African clawed frog (Xenopus laevis) oocytes, that allowed detailed kinetic studies of transport processes; data obtained mainly by this technique, which describe kinetics of transport of naturally occurring nucleosides and nucleobases towards hENTs are presented in Table 1, while kinetic of transport of synthetic analogues that are used as reverse transcriptase inhibitors drugs by hENTs are presented in Table 2 Griffits et al, 1997a;1997b;Baldwin et al, 2005;Barnes et al, 2006;Ritzel et al, 1997;Smith et al, 2004 Guanosine hENT1-3, hCNT2, hCNT3 0.04 (hCNT3) Griffits et al, 1997a;1997b;Baldwin et al, 2005;Ritzel et al, 1997; , 1997a;1997b;Baldwin et al, 2005;Ritzel et al, 1997;200;Slugoski et al, 2007Slugoski et al, , 2008;…”