Kyla M. Smith

Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib)

The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2, found primarily in specialized epithelia, are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have orthologs in other mammalian species and belong to a gene family (CNT) that also includes members in lower vertebrates, insects, nematodes, pathogenic yeast and bacteria. The CNT transporter family also includes a newly identified human and mouse CNT3 transporter isoform. This paper reviews the studies of CNT transport proteins that led to the identification of hCNT3 and mCNT3, and gives an overview of the structural and functional properties of these latest CNT family members. hCNT3 and mCNT3 have primary structures that place them in a CNT subfamily separate from CNT1/2, transport a wide range of physiological pyrimidine and purine nucleosides and antineoplastic and antiviral nucleoside drugs (system cib), and exhibit a Na+:uridine coupling ratio of at least 2:1 (cf 1:1 for hCNT1/2). Cells and tissues containing hCNT3 transcripts include mammary gland, differentiated HL-60 cells, pancreas, bone marrow, trachea, liver, prostrate and regions of intestine, brain and heart. In HL-60 cells, hCNT3 is transcriptionally regulated by phorbol myristate (PMA). The hCNT3 gene, which contains an upstream PMA response element, mapped to 9q22.2 (cf chromosome 15 for hCNT1 and hCNT2).The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1 and hCNT2, found primarily in specialized epithelia, are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have orthologs in other mammalian species and belong to a gene family (CNT) that also includes members in lower vertebrates, insects, nematodes, pathogenic yeast and bacteria. The CNT transporter family also includes a newly identified human and mouse CNT3 transporter isoform. This paper reviews the studies of CNT transport proteins that led to the identification of hCNT3 and mCNT3, and gives an overview of the structural and functional properties of these latest CNT family members. hCNT3 and mCNT3 have primary structures that place them in a CNT subfamily separate from CNT1/2, transport a wide range of physiological pyrimidine and purine nucleosides and antineoplastic and antiviral nucleoside drugs (system cib), and exhibit a Na+:uridine coupling ratio of at least 2:1 (cf 1:1 for hCNT1/2). Cells and tissues containing hCNT3 transcripts include mammary gland, differentiated HL-60 cells, pancreas, bone marrow, trachea, liver, prostrate and regions of intestine, brain and heart. In HL-60 cells, hCNT3 is transcriptionally regulated by phorbol myristate (PMA). The hCNT3 gene, which contains an upstream PMA response element, mapped to 9q22.2 (cf chromosome 15 for hCNT1 and hCNT2).

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

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 Km 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 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 [14C]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 × 1010 per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.

Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses

Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidneys proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.

Cation coupling properties of human concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3.

The SLC28 family of concentrative nucleoside transporter (CNT) proteins in mammalian cells contains members of two distinct phylogenic subfamilies. In humans, hCNT1 and hCNT2 belong to one subfamily, and hCNT3 to the other. All three CNTs mediate inwardly-directed Na+/nucleoside cotransport, and are either pyrimidine nucleoside-selective (hCNT1), purine nucleoside-selective (hCNT2), or broadly selective for both pyrimidine and purine nucleosides (hCNT3). While previous studies have characterized cation interactions with both hCNT1 and hCNT3, little is known about the corresponding properties of hCNT2. In the present study, heterologous expression in Xenopus oocytes in combination with radioisotope flux and electrophysiological techniques has allowed us to undertake a side-by-side comparison of hCNT2 with other hCNT family members. Apparent K50 values for Na+ activation were voltage-dependent, and similar in magnitude for all three transporters. Only hCNT3 was also able to couple transport of uridine to uptake of H+. The Na+/nucleoside stoichiometry of hCNT2, as determined from both Hill coefficients and direct charge/flux measurements, was 1:1. This result was the same as for hCNT1, but different from that of hCNT3 (2:1). The charge-to-22Na+ uptake stoichiometry was 1:1 for all three hCNTs. In parallel with their division into two separate CNT subfamilies, hCNT2 shares common cation specificity and coupling characteristics with hCNT1, which differ markedly from those of hCNT3.

Identification and functional characterization of variants in human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), arising from single nucleotide polymorphisms in coding regions of the hCNT3 gene.

Introduction Human concentrative nucleoside transporter 3, hCNT3 (SLC28A3), which mediates transport of purine and pyrimidine nucleosides and a variety of antiviral and anticancer nucleoside drugs, was investigated to determine if there are single nucleotide polymorphisms in the coding regions of the hCNT3 gene. Methods and results Ninety-six DNA samples from Caucasians (Coriell Panel) were sequenced and sixteen variants in exons and flanking intronic regions were identified, of which five were coding variants; three of these were non-synonymous (S5N, L131F, Y513F) and were further investigated for functional alterations of the resulting recombinant proteins in Saccharomyces cerevisiae and Xenopus laevis oocytes. In yeast, immunostaining and fluorescence quantitation of the reference (wild-type) and variant CNT3 proteins showed similar levels of expression. Kinetic studies were undertaken in yeast with a high through-put semi-automated assay process; reference hCNT3 exhibited Km values of 1.7±0.3, 3.6±1.3, 2.2±0.7, and 2.1±0.6 μM and Vmax values of 1402±286, 1310±113, 1020±44, and 1740±114 pmol/mg/min, respectively, for uridine, cytidine, adenosine and inosine. Similar Km and Vmax values were obtained for the three variant proteins assayed in yeast under identical conditions. All of the characterized hCNT3 variants produced in oocytes retained sodium and proton dependence of uridine transport based on measurements of radioisotope flux and two-electrode voltage-clamp studies. Conclusion These results suggested a high degree of conservation of function for hCNT3 in the Caucasian population.

The Role of Human Nucleoside Transporters in Cellular Uptake of 4′-Thio-β-d-arabinofuranosylcytosine and β-d-Arabinosylcytosine

4′-Thio-β-d-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 μM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-β-d-arabinofuranosyl cytosine (araC) were low (0.007 ± 003 and 0.034 ± 0.003, respectively) compared with that of [3H]uridine (0.317 ± 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 μM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 ± 0.003, 0.42 ± 0.03, and 0.51 ± 0.11, respectively) and mediated primarily by hENT1 (∼74, ∼65, and ∼61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 μM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

A Proton-mediated Conformational Shift Identifies a Mobile Pore-lining Cysteine Residue (Cys-561) in Human Concentrative Nucleoside Transporter 3

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 mediates transport of a broad range of purine and pyrimidine nucleosides and nucleoside drugs, whereas hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively. All three hCNTs are Na+-coupled. Unlike hCNT1/2, however, hCNT3 is also capable of H+-mediated nucleoside cotransport. Using site-directed mutagenesis in combination with heterologous expression in Xenopus oocytes, we have identified a C-terminal intramembranous cysteine residue of hCNT3 (Cys-561) that reversibly binds the hydrophilic thiol-reactive reagent p-chloromercuribenzene sulfonate (PCMBS). Access of this membrane-impermeant probe to Cys-561, as determined by inhibition of hCNT3 transport activity, required H+, but not Na+, and was blocked by extracellular uridine. Although this cysteine residue is also present in hCNT1 and hCNT2, neither transporter was affected by PCMBS. We conclude that Cys-561 is located in the translocation pore in a mobile region within or closely adjacent to the nucleoside binding pocket and that access of PCMBS to this residue reports a specific H+-induced conformational state of the protein.

A Conformationally Mobile Cysteine Residue (Cys-561) Modulates Na+ and H+ Activation of Human CNT3

In humans, the SLC28 concentrative nucleoside transporter (CNT) protein family is represented by three Na+-coupled members; human CNT1 (hCNT1) and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, whereas hCNT3 transports both purine and pyrimidine nucleosides and nucleoside drugs. Belonging to a phylogenetic CNT subfamily distinct from hCNT1/2, hCNT3 also mediates H+/nucleoside cotransport. Using heterologous expression in Xenopus oocytes, we have characterized a cysteineless version of hCNT3 (hCNT3C-). Processed normally to the cell surface, hCNT3C-exhibited hCNT3-like transport properties, but displayed a decrease in apparent affinity specific for Na+ and not H+. Site-directed mutagenesis experiments in wild-type and hCNT3C-backgrounds identified intramembranous Cys-561 as the residue responsible for this altered Na+-binding phenotype. Alanine at this position restored Na+ binding affinity, whereas substitution with larger neutral amino acids (threonine, valine, and isoleucine) abolished hCNT3 H+-dependent nucleoside transport activity. Independent of these findings, we have established that Cys-561 is located in a mobile region of the hCNT3 translocation pore adjacent to the nucleoside binding pocket and that access of p-chloromercuribenzene sulfonate to this residue reports a specific H+-induced conformational state of the protein ( Slugoski, M. D., Ng, A. M. L., Yao, S. Y. M., Smith, K. M., Lin, C. C., Zhang, J., Karpinski, E., Cass, C. E., Baldwin, S. A., and Young, J. D. (2008) J. Biol. Chem. 283, 8496-8507 ). The present investigation validates hCNT3C- as a template for substituted cysteine accessibility method studies of CNTs and reveals a pivotal functional role for Cys-561 in Na+- as well as H+-coupled modes of hCNT3 nucleoside transport.

Conserved Glutamate Residues Are Critically Involved in Na+/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50Na+ and/or Kmuridine values and diminished VmaxNa+ and Vmaxuridine values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

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

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na+ and H+ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na+/nucleoside and H+/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H+ dependence (all mutants), shift in Na+:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na+-specific hCNT1, uncoupled Na+ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.