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Figure 1. hCNT3 topology. Shown is the sequence alignment of hCNT1 (U62968), hCNT2 (NP_004203.2), hCNT3 (AF305210), and vcCNT (NP_231982.1). Bars representing helices have the same color scheme as in Fig. 2. Residues studied by SCAM analysis in the present study and Refs. 28 and 29 are highlighted in gray.
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Figure 2. Predicted topology of human nucleoside transporter hCNT3 based upon that of its counterpart from V. cholerae (21). The N-terminal transmembrane helices designated TM1, TM2, and TM3 and the C-terminal extramembranous tail with N-glycosylation sites are not present in the bacterial protein.
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Figure 3. hCNT3 residues in the IH 2-TM9 region with altered Na+:H+ uridine uptake ratios. hCNT3(C−) mutants exhibiting Na+:H+ uridine uptake ratios of >2.5 are shown in yellow, and those with uptake ratios < 0.5 are shown in red. Low-activity mutants with uridine transport rates of <0.1 pmol/oocyte·min−1 in both Na+-containing, H+-reduced and Na+-free, acidified media (100 mm NaCl, pH 8.5, and ChCl, pH 5.5, respectively) are indicated by black triangles. Corresponding numerical values are given in supplemental Table S1.
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Figure 4. PCMBS inhibition of residues in the IH2-TM9 region of hCNT3(C−). Mediated influx of 10 μm radiolabeled uridine in Na+-containing, H+-reduced (A) or Na+-free, acidified (B) medium (100 mm NaCl, pH 8.5, or 100 mm ChCl, pH 5.5, respectively) was measured following 10 min of incubation on ice in the same medium (A or B, respectively) in the presence of 200 μm PCMBS. Black columns indicate residue positions inhibited by PCMBS; the asterisk identifies those residues that exhibited differential inhibition by PCMBS in the two media. Low-activity mutants for which inhibition was not determined are indicated by black triangles. The data are presented as mediated transport, calculated as uptake in RNA-injected oocytes minus uptake in water-injected oocytes, and normalized to influx of uridine in the absence of inhibitor. Each value is the mean ± S.E. of 10–12 oocytes. hCNT3 and hCNT3(C−) were included as controls in all experiments.
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Figure 5. hCNT3 IH2-TM9 region depicting PCMBS-inhibited and uridine-protected residues. hCNT3(C−) mutants exhibiting inhibition of uridine uptake following incubation with PCMBS in both Na+-containing, H+-reduced and Na+-free, acidified media are indicated in blue, and those that were inhibited only in Na+-free, acidified medium are indicated in red. Residues protected from PCMBS inhibition by excess unlabeled uridine are outlined in black. The three residues, Gly340 in the connecting region between HP1a and HP1b, Ile371 in the connecting region between TM7a and TM7b, and Ala392 in TM8a, which were inhibited by PCMBS in both media but protected from that inhibition only in the presence of Na+-containing, H+-reduced medium, are indicated by black arrows. Additional residues of interest with Na+:H+ uridine uptake ratios of >2.5 or <0.5 but not inhibited by PCMBS are highlighted in yellow and green, respectively. Low-activity mutants are indicated by black triangles, and those with altered permeant selectivity are indicated by asterisks. The helical wheel projection for TM9 in the inset shows clustering of the two PCMBS-sensitive residues to one face of the helix. The corresponding numerical values are given in Table 1.
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Figure 6. A, hCNT1 HP1a, HP1b, TM7a, and TM7b depicting PCMBS-inhibited and uridine-protected residues. hCNT1 TMs HP1a, HP1b, TM7a, and TM7b PCMBS-sensitive residues are highlighted in blue, and the single residue exhibiting greater inhibition in Na+-containing versus Na+-free medium is indicated in red. Residues protected from PCMBS inhibition by excess unlabeled uridine are outlined in black. Corresponding numerical values are given in supplemental Tables S2 and S3. B, NupC(C−) HP1a, HP1b, TM4a, and TM4b region depicting PCMBS-inhibited and uridine-protected residues. NupC(C−) mutants exhibiting inhibition of uridine uptake following incubation with PCMBS in both acidified and H+-reduced media are indicated in blue. The single residue exhibiting greater inhibition in acidified versus H+-reduced medium is indicated in pink. The residue protected from PCMBS inhibition by excess unlabeled uridine is outlined in black. Low-activity mutants are indicated by black triangles. Corresponding numerical values are given in supplemental Tables S5 and S6.
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Figure 7. Comparison of PCMBS inhibition and substrate protection of residues in the HP1a, HP1b, TM4a, and TM4b region of NupC with the corresponding HP1a, HP1b, TM7a, and TM7b regions of hCNT3(C−) and hCNT1. Residues sensitive to PCMBS inhibition are highlighted in gray, and those protected with excess 20 mm uridine are underlined. Low-activity mutants are indicated by black triangles. Some constructs were unavailable for testing and are denoted with a hyphen.
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Figure 8. Permeant selectivity of wild-type hCNT3, hCNT3(C−) and hCNT3(C−) single-cysteine mutants G340C(C−), Q341C(C−), T342C(C−), S374C(C−), and V375C(C−). Oocytes producing wild-type hCNT3 (A), hCNT3(C−) (B), or hCNT3(C−) single-cysteine mutants (C–G) were incubated with 10 μm nucleosides: U, uridine; C, cytidine; T, thymidine; A, adenosine; I, inosine; G, guanosine. The initial rates of uptake were measured in either Na+-containing, H+-reduced or Na+-free, acidified media, at 20 °C. The data are presented as mediated transport, calculated as uptake in RNA-injected oocytes minus uptake in water-injected oocytes. Each value is the mean ± S.E. of 10–12 oocytes.
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Figure 9. Proposed C-terminal topology of hCNT3. hCNT3(C−) mutants exhibiting inhibition of uridine uptake following incubation with PCMBS in both Na+-containing, H+-reduced and Na+-free, acidified media are indicated in blue, those that were inhibited only in Na+-containing, H+-reduced medium are indicated in yellow, and those that were inhibited only in Na+-free, acidified medium are indicated in red. Residues protected from PCMBS inhibition by excess unlabeled uridine are outlined in black. The asterisks represents residues that form the conserved CNT family (G/A)XKX3NEFVA(Y/M/F) motif. Low-activity mutants are indicated by a solid triangle. Residues of hCNT3(C−) mutants in IH3, HP2a, HP2b, TM10a, TM10b, and TM11 exhibiting inhibition of uridine uptake following incubation with PCMBS and residues protected from PCMBS inhibition by excess unlabeled uridine have been previously published (Fig. 5 in Ref. 28).
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Figure 10. Homology model of hCNT3. Shown are cartoon representations of hCNT3 3D models based upon the crystal structure of the bacterial nucleoside transporter vcCNT (Protein Data Bank accession code 3TIJ) using the program SWISS-MODEL (56). Molecular graphics and analyses were performed with the UCSF Chimera package (57). A and C, models of hCNT3 viewed parallel to the membrane. The extracellular boundaries of the hydrophobic core of the bilayer predicted using the PPM server are shown as gray lines (58). B and D, models of hCNT3 viewed from the extracellular surface of the membrane. The outer scaffold domain of hCNT3 (TM4, TM5, IH1, TM6, and TM9) is shown in yellow. For clarity the loop linking HP2 and TM10 is not shown. Side chains of PCMBS-sensitive residues (A and B) are shown in red. Side chains of PCMBS-sensitive and uridine-protected residues (C and D) are shown in purple. The bound uridine molecule is shown in space filling representation (green).
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