Department of Pharmaceutics, University of Washington, Seattle, Washington
Submitted 19 April 2004 ; accepted in final form 17 September 2004
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ABSTRACT |
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localization; inhibition; polymorphism
While investigators at our laboratory (15, 20) and others (17, 18) have made considerable progress in elucidating the structural basis of hENT1 function, progress in dissecting the structural and functional relationships of the CNT has been relatively limited. Nevertheless, chimera studies of hCNT1 followed by site-directed mutagenesis have shown that converting Ser319/Gln320 of transmembrane domain (TM)7 and Ser353/Leu354 of TM8 to the corresponding residues in hCNT2 (Gly313/Met314 and Thr347/Val348) changes the substrate selectivity of hCNT1 to resemble that of an hCNT2-like transporter (7). On the other hand, mutation of only the two TM7 residues of hCNT1 produced a protein with an intermediate, CNT3-like activity. In addition, TM7 and TM8 have been identified as potential determinants of substrate selectivity in rat rCNT1 and rCNT2 (21), and mutation of rCNT1 Ser318 (corresponding to hCNT1 Ser319) yields a broad, CNT3-like phenotype similar to that resulting from the hCNT1 Ser319 mutation (22).
Such studies, however, did not indicate which other amino acid residues might be critical for hCNT1 function. We therefore performed a bioinformatic analysis of CNT1-like transporters from a range of animal species, with the aim of identifying conserved amino acid sequences that might highlight other domains of functional significance. This sequence alignment revealed two highly conserved amino acid positions, F316 and G476, in TM7 and TM11, respectively, that (along with 6 others) are invariant across 17 concentrative transporters drawn from 8 different species. These amino acid residues therefore became a focal point for our initial studies examining the impact of conservative mutations on the transport function and membrane localization of hCNT1. To this end, we engineered a series of single conservative amino acid changes at the F316 and G476 positions of hCNT1 and generated fusion proteins between such CNT1 mutants and green fluorescent protein (GFP). The fusion proteins were then expressed in MDCK cells and characterized for their kinetic properties and plasma membrane expression. In addition, a panel of DNA samples derived from an ethnically diverse group of individuals was genotyped for variants at the F316 position.
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EXPERIMENTAL PROCEDURES |
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Gene construction and site-directed mutagenesis.
As described previously, an 2.0-kb hCNT1 fragment cloned from human intestine was subcloned into GFP vector pEGFP-C1 (Clontech, Palo Alto, CA) (6). Mutants with single amino acid substitution (G476A, G476L, F316A, F316Y and F316H) fused to GFP were generated by means of the QuickChange site-directed mutagenesis kit (Stratagene) using the hCNT1-GFP construct as a template. The nucleotide sequences of the primers used to generate the above mutants were as follows: 1) G476A: 5'-TGTAGCCTTCTTGATGGCAGTGGCGTG, 2) G476L: 5'-TGTAGCCTTCTTGATGCTTGTGGCGTG, 3) F316A: 5'-GCTGGAAACATCGCAGTGAGCCAGA, 4) F316Y: 5'-GCTGGAAACATCTATGTGAGCCAGA, and 5) F316H: 5'-GCTG GAAACATCCATGTG AGCCAGA. All mutant constructs were sequenced to confirm that the correct mutations had been introduced.
Selection of MDCK cells stably expressing hCNT1 and the mutants. All cell lines were routinely maintained in MEM with L-glutamine containing 10% fetal bovine serum (FBS), 100 U of penicillin, and 100 µg/ml of streptomycin (GIBCO, Grand Island, NY) at 37°C in 95% air-5% CO2 at 95% humidity. hCNT1-GFP and the mutants were transfected into MDCK cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The transfected cells were subsequently selected by G418 as described previously (6). High-expressing transfectants were identified by the GFP fluorescence intensity using a fluorescence microscope (Carl Zeiss, Thornwood, NY), subsequently isolated by means of cloning cylinders, and then propagated.
Na+-dependent [3H]nucleoside transport. The Na+-dependent [3H]nucleoside transport experiments were conducted in Na+-containing buffer (in mM: 20 Tris·HCl, 3 K2HPO4, 1 MgCl2, 6 H2O, 2 CaCl2, 5 glucose, and 130 NaCl, pH 7.4) or Na+-free buffer in which NaCl was replaced by 130 mM N-methyl-D-glucamine (pH 7.4). Cells grown on 24-well plates were washed three times with Na+-free buffer and then preincubated with Na+-free buffer containing 10 µM NBMPR for 15 min at 37°C. When necessary, transport substrates and inhibitors were dissolved in DMSO at a maximum concentration of 0.1% DMSO because previous experiments have shown that DMSO concentrations of up to 0.1% have no effect on total [3H]nucleoside transport (6). [3H]nucleoside (1 µM, 2 µCi/ml) in either Na+ or Na+-free buffer containing 10 µM NBMPR was added to each well [to inhibit endogenous (canine) ENT1 equilibrative transporter activity]. After incubating at 37°C for different time intervals, the plates were rapidly washed three times with ice-cold Na+-free buffer containing 10 µM NBMPR. The cells were solubilized with 0.3 ml of 1 N NaOH by shaking for 15 min at room temperature and then neutralized with 0.3 ml of 1 N HCl. Next, 0.5 ml of the cell lysates was counted on a scintillation counter. The protein content of the cell lysates was determined spectrophotometrically using the bicinchoninic acid protein assay kit (Pierce Biochemicals, Rockford, IL) with bovine serum albumin (BSA) used as the standard.
Visualization of hCNT1-GFP and GFP-tagged mutant proteins. Stably expressing cells were grown in two-well Lab-Tek borosilicated coverglass chambers (Nalge Nunc International, Naperville, IL) for 23 days (undifferentiated) or for 68 days after reaching confluence (differentiated). Images were obtained using a Leica TCS NT laser scanning confocal microscope equipped with a krypton/argon laser as the light source. Images were captured by excitation at 488 nm and emission at 540 nm, a wavelength suitable for GFP imaging.
Inhibition profile and data analysis. [3H]nucleoside transport was performed in the presence of the unlabeled nucleoside at concentrations varying from 0 to 5 mM. IC50 values of [3H]nucleoside transport were estimated using nonlinear regression (WinNonlin; Pharsight, Mountain View, CA) and the model E = Emax (Emax E0)·[C/(C + IC50)], where E and Emax are the transport of [3H]nucleoside in the presence and absence of unlabeled nucleoside, respectively. C is the unlabeled nucleoside concentration, and E0 is the transport of [3H]nucleoside obtained in the presence of excess (10 mM) nucleosides. Data are expressed as means ± SD of transport values obtained in three wells. Data shown are representative of a minimum of two experiments performed on different days with different batches of cells.
Sequencing analysis for exons 11 and 12 of hCNT1. Exon 11 was amplified from genomic DNA using the forward primer 5'-ACTGTGTAAGGTTCTTCCACTCCAGC-3' and the reverse primer 5'-AGTATCCCTGGAGCAGCAGAATTTGATG-3'. Exon 12 was amplified using the forward primer 5'-TCTATCTCAGAAGGTTGTGAGTAAC-3' and the reverse primer 5'-AGAGAGAAGATCTGAGGGCTGCTCTG-3'. A 316- or 528-bp DNA fragment flanking exon 11 or 12 of hCNT1 was amplified by performing PCR. To increase the fidelity of PCR, Pfu DNA polymerase (Stratagene) was used. The PCR products were purified by 1.5% agarose gel filtration and then cycle sequenced using BigDye sequencing reagent, version 2 (ABI). To ascertain that the mutant did not arise as an artifact of PCR amplification, we repeated the PCR and reanalyzed the product by cycle sequencing.
TaqMan genotyping assay for human hCNT1 F316H mutant. Genotyping was performed using the ABI Prism Sequence Detection System 7000 instrument in a 96-well format. Dual-labeled TaqMan/TAMRA probes bearing either FAM (6-carboxyfluorescein) or VIC as reporter was custom synthesized by ABI, while PCR primers were made by Invitrogen. The sequences of the TaqMan probes were as follows: 1) hCNT1F316Htg-FAM (mutant allele on complementary strand): 5'-6FAM-CTGGCTCA CAtgGATGTTTCCAGC-TAMRA-3', and 2) hCNT1F316HAA-VIC (wild-type allele on complementary strand): 5'-VIC-CCTGGCTCACAAAGATGTTTCCAGC-TAMRA-3'. The underlined letters indicate the polymorphism. The sequences for the PCR primers were hCNT1F316H-F1, 5'-GGCACCACAGCCACTGAGA-3'; and hCNT1F316H-R1, 5'-GAGGGTAGGAGGCTTCCATACC-3'. These sequences generated a 79-bp amplicon. Sequence-verified, cloned genomic PCR products of the 316 bp containing the F316H polymorphism, derived from the HK114B kidney sample, were used as positive controls for the wild-type AA/AA and mutant CA/CA genotypes in the TaqMan experiments. The heterozygous TT/CA genotype was simulated using an equimolar mixture of the TT and CA genomic PCR products. Each experiment included three no-template controls.
Genotyping. A total of 260 DNA samples were genotyped for this study, of which 19 were isolated from human kidney tissue samples from anonymous organ donors (tissue samples kindly provided by Drs. Kenneth E. Thummel and Evan D. Kharasch, University of Washington, Seattle, WA). The remainder were purchased from the Coriell cell repositories (Camden, NJ) as follows: African-American (87 tissue samples), Caucasian (87), Indo-Pakistani (9), Middle Eastern (10), South American (Andean) (10), South American (Brazil) (10), Russian (Krasnodar) (9), Russian (Moscow) (10), and Ashkenazi Jew (9).
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RESULTS AND DISCUSSION |
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Characterization of F316H-GFP-expressing cells.
To determine whether the hCNT1 F316H variant demonstrates the same inhibition profile as F316Y-GFP, we used site-directed mutagenesis to create an F316H-GFP fusion construct and expressed it stably in MDCK cells. Tracking the variant by its green fluorescence, we found that F316H-GFP was distributed evenly on the plasma membrane in undifferentiated MDCK cells but was sorted to the apical membrane upon differentiation (data not shown). Like F316Y-GFP-expressing cells, transport of [3H]uridine by F316H-GFP-expressing MDCK cells was not inhibited by 1.0 mM inosine but was significantly inhibited by the purine nucleoside guanosine (Fig. 7A). The IC50 of guanosine inhibition of [3H]uridine transport by F316H-GFP-expressing MDCK cells was 148 ± 31 µM (mean ± SE). This value is comparable to the data of Gutierrez and Giacomini (5) showing 40% inhibition of [3H] thymidine uptake at a guanosine concentration of 100 µM. The guanosine sensitivity of the F316 mutants was reminiscent of that of the N4 nucleoside transport system, previously observed in brush-border membrane vesicles prepared from human kidneys. There is currently no evidence that the N4 transporter is encoded by an additional, distinct nucleoside transporter gene apart from the three known examples, hCNT13. In fact, with the completion of the Human Genome Project, it appears that all orthologs of the human concentrative nucleoside transporter gene family (SLC28) have been accounted for. It is thus more plausible that the reported N4 transporter activity corresponds to the phenotype of some naturally occurring variant of hCNT1, perhaps at the F316 position.
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The low allele frequency of the F316H in the general population suggests that its discovery was indeed a fortuitous occurrence. However, it is known that rare SNP in particular can frequently display marked ethnicity-dependent distribution or founder effects within specific population subgroups (16). It is possible that our study and that of Gutierrez and Giacomini (5), who examined a small (<8) but indeterminate number of kidneys, might have sampled such a subgroup in which the F316H mutant is present in relatively higher abundance.
In summary, the experiments conducted in the present study demonstrate that the highly conserved residues F316 (TM7) and G476 (TM11) have strong structural and functional significance with regard to the membrane localization and guanosine sensitivity, respectively, of the hCNT1 nucleoside transporter. Of particular novelty is the discovery of the natural variant F316H, hitherto unobserved in previous surveys (1). This variant occurred in TM7, within which no other mutations, coding or otherwise, have been reported. Thus the natural paucity of F316H, coupled with the functional impact of the variations at both of the conserved residues studied, suggests that the remaining conserved sites within TM7/TM11 are likely to play important functional roles as well. Indeed, the F316 residue may well be important in nucleoside transport by hCNT2 and hCNT3. Mutational analysis of conserved residues within TM7 and TM11 will likely yield additional insights into the workings of the CNT transporters.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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