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-beta-d-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-beta-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 microm, respectively. hCNT1 was Na(+) specific, and the kinetics of steady-state uridine-evoked 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 x 10(10) per cell). The hCNT1 turnover rate at -50 mV was 9.6 molecules of uridine transported per second.

Figure 1. Nucleoside specificity of hCNT1

The permeant selectivity of hCNT1 was investigated in Na⁺-containing transport medium by measuring the currents evoked by a variety of pyrimidine (100 μm) and purine (100 μm and 1 mm) nucleosides. The nucleobases uracil and hypoxanthine (100 μm and 1 mm) were also tested. hCNT1-mediated currents are expressed as the mean ± s.e.m. of 3–4 different oocytes. The expression vector was pGEM-HE.

Figure 2. Transport of nucleoside analogues and nucleoside drugs by hCNT1

Current responses generated by perfusing hCNT1-producing oocytes with various pyrimidine and purine nucleoside analogues and nucleoside drugs (100 μm and 1 mm) in Na⁺-containing medium (A and B). Values are means ± s.e.m. for 5–6 different oocytes. The same experiment was also performed in control water-injected oocytes (data not shown); no inward currents were generated. The expression vector was pGEM-HE.

Figure 3. Transport of thymidine mimetics by hCNT1

A, structure of β-DFP-5m (1-(2-deoxy-β-d-ribofuranosyl)-2,4-difluoro-5-methylbenzene). B, structure of β-DFP-5I (1-(2-deoxy-β-d-ribofuranosyl)-2,4-difluoro-5-iodobenzene). C, oocytes were injected with 10 nl of water without (control) or with 10 ng of hCNT1 RNA transcript. The expression vector was pGEM-HE. Current responses were generated by perfusing individual hCNT1-producing oocytes with either 100 μmβ-DFP-5m or β-DFP-5I in Na⁺- or choline-containing transport medium (top panel). The current produced by 100 μm uridine in Na⁺-containing medium is shown for comparison. The same experiment was performed in a control water-injected oocyte (bottom panel). D, a comparison of hCNT1-mediated currents following addition of 100 μm uridine, β-DFP-5m or β-DFP-5I in Na⁺-containing medium. Values are means ± s.e.m. for 3 different oocytes.

Figure 4. Steady-state hCNT1 kinetics and the order of solute binding

A, the dependence of hCNT1-mediated currents on the external concentration of uridine (0–1000 μm) was examined at three different concentrations of Na⁺ (5, 25 and 100 mm). hCNT1-mediated currents are expressed as the mean ± s.e.m. of 5–6 different oocytes. B, the dependence of hCNT1-mediated currents on the external concentration of Na⁺ (0–100 mm) was examined at 25 and 100 μm uridine. hCNT1-mediated currents are expressed as the mean ± s.e.m. of 4–5 different oocytes. The expression vector was pGEM-T.

Figure 5. Effect of phloridzin on hCNT1 steady-state kinetics

A, uridine-induced currents (0–1000 μm) in hCNT1-producing oocytes were measured in Na⁺-containing transport medium (100 mm NaCl) before and after incubation with 5 mm phloridzin. Currents are expressed as the mean ± s.e.m. of 5–6 different oocytes. B, uridine-induced currents (100 μm) in hCNT1-producing oocytes were measured in the presence of increasing concentrations of external Na⁺ (0–100 mm) before and after incubation with 5 mm phloridzin. Currents are expressed as the mean ± s.e.m. of 5–6 different oocytes. The expression vector was pGEM-HE.

Figure 6. Stoichiometry of hCNT1

A, representative example of the current generated during application of 200 μm [¹⁴C]uridine to an hCNT1-producing oocyte (V_h = −50 mV). B, hCNT1-producing oocytes were clamped at V_h=−30 mV and perfused with 200 μm [¹⁴C]uridine. Integration of the uridine-evoked current over the uptake period (3 min) yielded the charge moved which was converted to pmoles and plotted against radiolabelled uridine uptake (pmol) in the same oocyte. The experiment was performed in 9 different oocytes. The slope (± s.e.) of the linear fit (Na⁺/nucleoside ratio) is indicated by the continuous line. The dashed line indicates a slope of 1. C, a corresponding experiment at V_h = −50 mV(n =7). D, a corresponding experiment at V_h = −90 mV (n =9). Linear fits were not forced through zero. The expression vector was pGEM-HE.

Figure 7. Dependence of hCNT1 presteady-state currents on external Na⁺

A, representative charge-voltage (Q/V) plot for an hCNT1-producing oocyte in the presence of 25 mm external Na⁺ (pH 7.5). At each clamped voltage, integration of the hCNT1 current (OFF response) with time yielded the charge (Q) moved within the membrane electric field. Data were normalized to Q_T, plotted as a function of voltage and fitted to the Boltzmann equation to determine z_d and V_0.5. The dashed line indicates V_0.5. B, mean values for V_0.5 in mV (± s.e.m.) for groups of 5 individual oocytes are plotted versus log [Na⁺]. The fitted line corresponds to a voltage shift of 41 ± 1 mV for an e-fold change in Na⁺ concentration. The expression vector was pGEM-HE.

Figure 8. Turnover rate of recombinant hCNT1

The total charge (Q_T) displaced for the OFF response during voltage steps from V_h = −50 mV to V_t ranging from −170 to +150 mV (40 mV increments) was correlated with hCNT1 transport activity in the same cell determined as steady-state currents induced by 100 μm uridine superfusion at V_h = −50 mV. Linear regression analysis of results for 12 individual oocytes gave a slope of 17.2 ± 4.4 s⁻¹ (continuous line), corresponding to an hCNT1 uridine turnover rate of 9.6 ± 2.5 s⁻¹. The expression vector was pGEM-HE.