Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1)

Abstract

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.

FIGURE 1.

Uptake of radiolabeled uridine and a panel of purine and pyrimidine nucleobases by recombinant wild-type hENT1 and hENT2 produced in Xenopus oocytes. Uptake of uridine and nucleobases (20 μm) by oocytes microinjected with recombinant RNA transcripts for hENT1 or hENT2 (hatched and solid columns, respectively) or water alone (open columns) was measured at room temperature (20 °C) using 2-min uptake intervals. The inset shows uptake of uridine, cytosine, uracil, or guanine by hENT1-injected oocytes (solid columns) or water-injected oocytes (open columns) using extended uptake intervals of 30-min. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the thickness of lines.

FIGURE 2.

Time courses of uptake of radiolabeled adenine (A), hypoxanthine (B), or thymine (C) by recombinant wild-type hENT1 produced in Xenopus oocytes. Uptake of nucleobases (20 μm) by oocytes microinjected with recombinant RNA transcripts for hENT1 (solid circles) or water alone (open circles) was measured at room temperature (20 °C) using uptake intervals of 1–30 min. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the size of data points. In subsequent kinetic experiments, an uptake interval intermediate between the 1- and 3-min time points (2 min) was used to measure initial rates of transport.

FIGURE 3.

Uptake of radiolabeled uridine and a panel of purine and pyrimidine nucleobases by recombinant wild-type hCNT1, hCNT2, or hCNT3 produced in Xenopus oocytes. Uptake of uridine or various nucleobases (20 μm) by oocytes microinjected with recombinant RNA transcripts for hCNT1, hCNT2, or hCNT3 (solid, gray, and hatched columns, respectively) or water alone (open columns) was measured at room temperature (20 °C) using 30-min uptake intervals. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the thickness of lines.

FIGURE 4.

Concentration dependence of uptake of radiolabeled uridine (A), adenine (B), thymine (C), or hypoxanthine (D) by recombinant wild-type hENT1 produced in Xenopus oocytes. Uptake of uridine or nucleobases by oocytes microinjected with recombinant RNA transcripts for hENT1 (solid circles) or water alone (open circles) was measured at room temperature (20 °C). Calculated kinetic constants (K_m and V_max) for the mediated components of transport (uptake in RNA transcript-injected oocytes minus uptake in water-injected oocytes) are presented in Table 1. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the size of data points.

FIGURE 5.

Effects of NBMPR on nucleobase transport by recombinant wild-type hENT1 and hENT2 produced in Xenopus oocytes. A, mediated uptake of uridine, thymine, adenine, or hypoxanthine (20 μm, 20 °C) in oocytes producing recombinant hENT1 (solid and stippled columns) or hENT2 (hatched and open columns) was measured in the presence or absence of NBMPR (1 μm) (stippled/open and solid/hatched columns, respectively). B, concentration dependence of NBMPR inhibition of adenine uptake (20 μm, 20 °C) in oocytes producing recombinant hENT1. The values in A and B are corrected for endogenous uptake by water-injected oocytes. The calculated IC₅₀ and K_i values for NBMPR inhibition of adenine transport in B are presented in the text. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the size of data points.

FIGURE 6.

Effect of uridine on nucleobase transport by recombinant wild-type hENT1 produced in Xenopus oocytes. Mediated uptake of hypoxanthine (20 μm, 20 °C) in oocytes producing recombinant hENT1 was measured in the presence of graded concentrations of uridine. The inset is a corresponding Dixon plot of uridine inhibition (0.5–2.0 mm) of hENT1-mediated hypoxanthine transport (pmol/oocyte·min⁻¹) measured at two different substrate concentrations (1.0 and 5.0 mm). The values are corrected for endogenous uptake by water-injected oocytes. The calculated IC₅₀ value for uridine inhibition of 20 μm hypoxanthine uptake and the uridine K_i value determined by linear regression analysis of the Dixon plot are presented in the text.

FIGURE 7.

Time courses of uptake of radiolabeled 5-fluorouracil (A) and 6-mercaptopurine (B) by recombinant wild-type hENT1 and hENT2 produced in Xenopus oocytes. Uptake of 5-fluorouracil and 6-mercaptopurine (20 μm) by oocytes microinjected with recombinant RNA transcripts of hENT1 (solid circles) or hENT2 (open circles) or water alone (solid inverted triangles) was measured at room temperature (20 °C) using uptake intervals of 1–30 min. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the size of data points. In subsequent kinetic experiments, an uptake interval intermediate between the 1- and 3-min time points (2 min) was used to measure initial rates of transport.

FIGURE 8.

Concentration dependence of uptake of radiolabeled 5-fluorouracil (A and B) and 6-mercaptopurine (C and D) by recombinant wild-type hENT1 and hENT2 produced in Xenopus oocytes. Uptake of 5-fluorouracil or 6-mercaptopurine by oocytes microinjected with recombinant RNA transcripts for hENT1 or hENT2 (solid circles) or water alone (open circles) was measured at room temperature (20 °C). Calculated kinetic constants (K_m and V_max) for the mediated components of transport (uptake in RNA transcript-injected oocytes minus uptake in water-injected oocytes) are presented in Table 1. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the size of data points.

FIGURE 9.

Membrane topology of hENT1 showing locations of endogenous cysteine residues. The positions of putative TM helices are indicated as open rectangles and are numbered. Cysteine residues are indicated by the letter C. The single N-glycosylation site of hENT1 is highlighted by a solid star. *C414 is the single cysteine residue implicated in hENT1 nucleobase transportability.

FIGURE 10.

Uptake of radiolabeled uridine (A), adenine (B), hypoxanthine (C), or thymine (D) by recombinant wild-type hENT1, cysteine-less hENT1C−, or S-series hENT1C− mutants produced in Xenopus oocytes. Mediated uptake of uridine, adenine, hypoxanthine, or thymine (20 μm) in oocytes producing recombinant wild-type hENT1, hENT1C−, or S-series hENT1C− mutants was measured at room temperature (20 °C). The values are corrected for endogenous uridine and nucleobase uptake by water-injected oocytes.

FIGURE 11.

Concentration dependence of uptake of radiolabeled uridine (A) or adenine (B) by recombinant S-series mutant S414C/hENT1C− (S8) produced in Xenopus oocytes. Uptake of uridine or adenine by oocytes microinjected with recombinant RNA transcripts for S414C/hENT1C− (solid circles) or water alone (open circles) was measured at room temperature (20 °C). Calculated kinetic constants (K_m and V_max) for the mediated components of transport (uptake in RNA transcript-injected oocytes minus uptake in water-injected oocytes) are presented in the text. Error bars (S.E. of 10–12 oocytes) are not shown where values were smaller than the size of data points.