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• W2072863040 abstract "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. 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. Most nucleosides, including nucleoside analogs with antineoplastic and/or antiviral activity, are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT) 4The abbreviations used are: NT, nucleoside transporter; CNT, concentrative nucleoside transporter; ENT, equilibrative nucleoside transporter; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl) methanethiosulfonate; MTSET, [(triethylammonium)ethyl] methanethiosulfonate bromide; PCMBS, sodium p-chloromercuriphenyl sulfonate; TM, putative transmembrane helix; h, human.4The abbreviations used are: NT, nucleoside transporter; CNT, concentrative nucleoside transporter; ENT, equilibrative nucleoside transporter; MTS, methanethiosulfonate; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl) methanethiosulfonate; MTSET, [(triethylammonium)ethyl] methanethiosulfonate bromide; PCMBS, sodium p-chloromercuriphenyl sulfonate; TM, putative transmembrane helix; h, human. proteins for uptake into or release from cells (1Cass, C. E. (1995) in Drug Transport in Antimicrobial and Anticancer Chemotherapy (Georgopapadakou, N. H., ed) pp. 403-451, Marcel Dekker, Inc., New YorkGoogle Scholar, 2Griffith D.A. Jarvis S.M. Biochim. Biophys. Acta. 1996; 1286: 153-181Crossref PubMed Scopus (453) Google Scholar, 3Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Curr. Top. Membr. 2000; 50: 329-378Crossref Google Scholar). NT-mediated transport is a critical determinant of nucleoside and nucleotide metabolism and, for nucleoside drugs, their pharmacologic actions (3Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Curr. Top. Membr. 2000; 50: 329-378Crossref Google Scholar, 4Damaraju V.L. Damaraju S. Young J.D. Baldwin S.A. Mackey J. Sawyer M.B. Cass C.E. Oncogene. 2003; 22: 7524-7536Crossref PubMed Scopus (250) Google Scholar, 5Latini S. Pedata F. J. Neurochem. 2001; 79: 463-484Crossref PubMed Scopus (643) Google Scholar). By regulating adenosine availability to cell-surface purinoreceptors, NTs also profoundly affect neurotransmission, vascular tone, and other physiological processes (5Latini S. Pedata F. J. Neurochem. 2001; 79: 463-484Crossref PubMed Scopus (643) Google Scholar, 6King A.E. Ackley M.A. Cass C.E. Young J.D. Baldwin S.A. Trends Pharmacol. Sci. 2006; 27: 416-425Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Two structurally unrelated families of integral membrane proteins exist in human and other mammalian cells as follows: the SLC28 concentrative nucleoside transporter (CNT) family, and the SLC29 equilibrative nucleoside transporter (ENT) family (3Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Curr. Top. Membr. 2000; 50: 329-378Crossref Google Scholar, 6King A.E. Ackley M.A. Cass C.E. Young J.D. Baldwin S.A. Trends Pharmacol. Sci. 2006; 27: 416-425Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 7Gray J.H. Owen R.P. Giacomini K.M. Pfluegers Arch. 2004; 447: 728-734Crossref PubMed Scopus (346) Google Scholar, 8Baldwin S.A. Beal P.R. Yao S.Y.M. King A.E. Cass C.E. Young J.D. Pfluegers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar). ENTs are normally present in most, possibly all, cell types (8Baldwin S.A. Beal P.R. Yao S.Y.M. King A.E. Cass C.E. Young J.D. Pfluegers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cells, suggesting important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1Cass, C. E. (1995) in Drug Transport in Antimicrobial and Anticancer Chemotherapy (Georgopapadakou, N. H., ed) pp. 403-451, Marcel Dekker, Inc., New YorkGoogle Scholar, 2Griffith D.A. Jarvis S.M. Biochim. Biophys. Acta. 1996; 1286: 153-181Crossref PubMed Scopus (453) Google Scholar, 3Young J.D. Cheeseman C.I. Mackey J.R. Cass C.E. Baldwin S.A. Curr. Top. Membr. 2000; 50: 329-378Crossref Google Scholar, 4Damaraju V.L. Damaraju S. Young J.D. Baldwin S.A. Mackey J. Sawyer M.B. Cass C.E. Oncogene. 2003; 22: 7524-7536Crossref PubMed Scopus (250) Google Scholar, 6King A.E. Ackley M.A. Cass C.E. Young J.D. Baldwin S.A. Trends Pharmacol. Sci. 2006; 27: 416-425Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 7Gray J.H. Owen R.P. Giacomini K.M. Pfluegers Arch. 2004; 447: 728-734Crossref PubMed Scopus (346) Google Scholar). In humans (h), hCNT1 and hCNT2 are pyrimidine nucleoside-selective and purine nucleoside-selective, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides (9Ritzel M.W.L. Yao S.Y.M. Huang M.Y. Elliott J.F. Cass C.E. Young J.D. Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar, 10Ritzel M.W.L. Yao S.Y.M. Ng A.M.L. Mackey J.R. Cass C.E. Young J.D. Mol. Membr. Biol. 1998; 15: 203-211Crossref PubMed Scopus (178) Google Scholar, 11Ritzel M.W.L. Ng A.M.L. Yao S.Y.M. Graham K. Loewen S.K. Smith K.M. Ritzel R.G. Mowles D.A. Carpenter P. Chen X.Z. Karpinski E. Hyde R.J. Baldwin S.A. Cass C.E. Young J.D. J. Biol. Chem. 2001; 276: 2914-2927Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Together, these proteins and their orthologs account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized include hfCNT from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (12Yao S.Y.M. Ng A.M.L. Loewen S.K. Cass C.E. Baldwin S.A. Young J.D. Am. J. Physiol. 2002; 283: C155-C168Crossref PubMed Scopus (31) Google Scholar), CaCNT from the pathogenic yeast Candida albicans (13Loewen S.K. Ng A.M.L. Mohabir N.N. Baldwin S.A. Cass C.E. Young J.D. Yeast. 2003; 20: 661-675Crossref PubMed Scopus (25) Google Scholar), CeCNT3 from the nematode Caenorhabditis elegans (14Xiao G. Wang J. Tangen T. Giacomini K.M. Mol. Pharmacol. 2001; 59: 339-348Crossref PubMed Scopus (29) Google Scholar), and NupC from the bacterium Escherichia coli (15Loewen S.K. Yao S.Y.M. Slugoski M.D. Mohabir N.N. Turner R.J. Mackey J.R. Weiner J.H. Gallagher M.P. Henderson P.J. Baldwin S.A. Cass C.E. Young J.D. Mol. Membr. Biol. 2004; 21: 1-10Crossref PubMed Scopus (35) Google Scholar). hCNT1, hCNT2, and hfCNT are predominantly Na+-coupled nucleoside transporters, whereas hCNT3 can utilize electrochemical gradients of either Na+ or H+ to accumulate nucleosides within cells (12Yao S.Y.M. Ng A.M.L. Loewen S.K. Cass C.E. Baldwin S.A. Young J.D. Am. J. Physiol. 2002; 283: C155-C168Crossref PubMed Scopus (31) Google Scholar, 16Smith K.M. Ng A.M.L. Yao S.Y.M. Labedz K.A. Knaus E.E. Wiebe L.I. Cass C.E. Baldwin S.A. Chen X.Z. Karpinski E. Young J.D. J. Physiol. (Lond.). 2004; 558: 807-823Crossref Scopus (79) Google Scholar, 17Smith K.M. Slugoski M.D. Loewen S.K. Ng A.M.L. Yao S.Y.M. Chen X.-Z. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2005; 280: 25436-25449Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Ritzel M.W.L. Ng A.M.L. Yao S.Y.M. Graham K. Loewen S.K. Smith K.M. Hyde R.J. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Mol. Membr. Biol. 2001; 18: 65-72Crossref PubMed Scopus (120) Google Scholar). CaCNT, CeCNT3, and NupC function exclusively as H+/nucleoside cotransporters (13Loewen S.K. Ng A.M.L. Mohabir N.N. Baldwin S.A. Cass C.E. Young J.D. Yeast. 2003; 20: 661-675Crossref PubMed Scopus (25) Google Scholar, 14Xiao G. Wang J. Tangen T. Giacomini K.M. Mol. Pharmacol. 2001; 59: 339-348Crossref PubMed Scopus (29) Google Scholar, 15Loewen S.K. Yao S.Y.M. Slugoski M.D. Mohabir N.N. Turner R.J. Mackey J.R. Weiner J.H. Gallagher M.P. Henderson P.J. Baldwin S.A. Cass C.E. Young J.D. Mol. Membr. Biol. 2004; 21: 1-10Crossref PubMed Scopus (35) Google Scholar). Na+/nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT (12Yao S.Y.M. Ng A.M.L. Loewen S.K. Cass C.E. Baldwin S.A. Young J.D. Am. J. Physiol. 2002; 283: C155-C168Crossref PubMed Scopus (31) Google Scholar, 16Smith K.M. Ng A.M.L. Yao S.Y.M. Labedz K.A. Knaus E.E. Wiebe L.I. Cass C.E. Baldwin S.A. Chen X.Z. Karpinski E. Young J.D. J. Physiol. (Lond.). 2004; 558: 807-823Crossref Scopus (79) Google Scholar, 17Smith K.M. Slugoski M.D. Loewen S.K. Ng A.M.L. Yao S.Y.M. Chen X.-Z. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2005; 280: 25436-25449Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Ritzel M.W.L. Ng A.M.L. Yao S.Y.M. Graham K. Loewen S.K. Smith K.M. Hyde R.J. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Mol. Membr. Biol. 2001; 18: 65-72Crossref PubMed Scopus (120) Google Scholar, 19Smith K.M. Slugoski M.D. Cass C.E. Baldwin S.A. Karpinski E. Young J.D. Mol. Membr. Biol. 2007; 24: 53-64Crossref PubMed Scopus (27) Google Scholar). H+/nucleoside coupling ratios for hCNT3 and CaCNT are both 1:1 (13Loewen S.K. Ng A.M.L. Mohabir N.N. Baldwin S.A. Cass C.E. Young J.D. Yeast. 2003; 20: 661-675Crossref PubMed Scopus (25) Google Scholar, 17Smith K.M. Slugoski M.D. Loewen S.K. Ng A.M.L. Yao S.Y.M. Chen X.-Z. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2005; 280: 25436-25449Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Ritzel M.W.L. Ng A.M.L. Yao S.Y.M. Graham K. Loewen S.K. Smith K.M. Hyde R.J. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Mol. Membr. Biol. 2001; 18: 65-72Crossref PubMed Scopus (120) Google Scholar, 19Smith K.M. Slugoski M.D. Cass C.E. Baldwin S.A. Karpinski E. Young J.D. Mol. Membr. Biol. 2007; 24: 53-64Crossref PubMed Scopus (27) Google Scholar). Although considerable progress has been made in molecular studies of ENT proteins (6King A.E. Ackley M.A. Cass C.E. Young J.D. Baldwin S.A. Trends Pharmacol. Sci. 2006; 27: 416-425Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 8Baldwin S.A. Beal P.R. Yao S.Y.M. King A.E. Cass C.E. Young J.D. Pfluegers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar), studies of structurally and functionally important residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (20Hamilton S.R. Yao S.Y.M. Ingram J.C. Hadden D.A. Ritzel M.W.L. Gallagher M.P. Henderson P.J.F. Cass C.E. Young J.D. Baldwin S.A. J. Biol. Chem. 2001; 276: 27981-27988Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Prokaryote CNTs lack the first three TMs of their eukaryote counterparts, and functional expression of N-terminally truncated human/rat CNT1 in Xenopus oocytes has established that the first three TMs are not required for Na+-dependent uridine transport activity (20Hamilton S.R. Yao S.Y.M. Ingram J.C. Hadden D.A. Ritzel M.W.L. Gallagher M.P. Henderson P.J.F. Cass C.E. Young J.D. Baldwin S.A. J. Biol. Chem. 2001; 276: 27981-27988Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Consistent with these findings, chimeric studies between hCNT1 and hfCNT (12Yao S.Y.M. Ng A.M.L. Loewen S.K. Cass C.E. Baldwin S.A. Young J.D. Am. J. Physiol. 2002; 283: C155-C168Crossref PubMed Scopus (31) Google Scholar) and between hCNT1 and hCNT3 (17Smith K.M. Slugoski M.D. Loewen S.K. Ng A.M.L. Yao S.Y.M. Chen X.-Z. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2005; 280: 25436-25449Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) have demonstrated that residues involved in Na+- and H+-coupling reside in the C-terminal half of the protein. In hCNT1, two sets of adjacent residues in TM 7 and 8 have been identified (Ser-319/Gln-320 and Ser-353/Leu-354) that, when converted to the corresponding residues in hCNT2, change the nucleoside specificity of the transporter from CNT1-type to CNT2-type (21Loewen S.K. Ng A.M.L. Yao S.Y.M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1999; 274: 24475-24484Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Mutation of Ser-319 in TM 7 of hCNT1 to glycine was sufficient to enable transport of purine nucleosides, whereas mutation of the adjacent residue Gln-320 to methionine (which had no effect on its own) augmented this transport. The additional mutation of Ser-353 in TM 8 of hCNT1 to threonine converted S319G/Q320M from broadly selective (CNT3-type) to purine nucleoside-selective (CNT2-type) but with relatively low adenosine transport activity. Further mutation of Leu-354 to valine increased the adenosine transport capability of S319G/Q320M/S353T, producing a full CNT2-type phenotype. Residues in both TMs 7 and 8 therefore play key roles in determining hCNT1/2 nucleoside selectivities. Confirming this, the double TM 8 mutant (S353T/L354V) was recently shown to exhibit a unique uridine-preferring transport phenotype (22Slugoski M.D. Loewen S.K. Ng A.M.L. Smith K.M. Yao S.Y.M. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Biochemistry. 2007; 46: 1684-1693Crossref PubMed Scopus (15) Google Scholar). Mutation of Leu-354 alone markedly increased the affinity of the transporter for Na+ and Li+, demonstrating that TM 8 also has a role in cation coupling (22Slugoski M.D. Loewen S.K. Ng A.M.L. Smith K.M. Yao S.Y.M. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Biochemistry. 2007; 46: 1684-1693Crossref PubMed Scopus (15) Google Scholar). Although negatively charged amino acid residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (23Pourcher T. Zani M.L. Leblanc G. J. Biol. Chem. 1993; 268: 3209-3215Abstract Full Text PDF PubMed Google Scholar, 24Franco P.J. Brooker R.J. J. Biol. Chem. 1994; 269: 7379-7386Abstract Full Text PDF PubMed Google Scholar, 25Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5508-5543Crossref PubMed Scopus (74) Google Scholar, 26Quick M. Jung H. Biochemistry. 1997; 36: 4631-4636Crossref PubMed Scopus (42) Google Scholar, 27Quick M. Jung H. Biochemistry. 1998; 37: 13800-13806Crossref PubMed Scopus (41) Google Scholar, 28Griffith D.A. Pajor A.M. Biochemistry. 1999; 38: 7524-7531Crossref PubMed Scopus (27) Google Scholar, 29Pirch T. Quick M. Nietschke M. Langkamp M. Jung H. J. Biol. Chem. 2002; 277: 8790-8796Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 30Grewer C. Watzke N. Rauen T. Bicho A. J. Biol. Chem. 2003; 278: 2585-2592Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 31Noel J. Germain D. Vadnais J. Biochemistry. 2003; 42: 15361-15368Crossref PubMed Scopus (26) Google Scholar, 32Chen N. Rickey J. Berfield J.L. Reith M.E. J. Biol. Chem. 2004; 279: 5508-5519Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1221) Google Scholar), essentially nothing is known about their roles in CNTs. Using hCNT1 as the template, we report here the consequences of individually mutating 10 aspartate and glutamate residues that are highly conserved in mammalian CNTs. We identified three residues (Glu-308, Glu-322, and Glu-498) with roles in CNT Na+ and nucleoside binding and translocation. Site-directed Mutagenesis and DNA Sequencing—hCNT1 cDNA (GenBank™ accession number U62968) in the Xenopus expression vector pGEM-HE (34Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (979) Google Scholar) provided the template for construction of hCNT1 mutants by the oligonucleotide-directed technique (35Kirsch R.D. Joly E. Nucleic Acids Res. 1998; 26: 1848-1850Crossref PubMed Scopus (205) Google Scholar), using reagents from the QuikChange® site-directed mutagenesis kit (Stratagene) according to the manufacturer's directions. Constructs were sequenced in both directions by Taq dye deoxy terminator cycle sequencing to ensure that only the correct mutation had been introduced. Production of Wild-type and Mutant hCNT1 Proteins in Xenopus Oocytes—hCNT1 cDNAs were transcribed with T7 polymerase and expressed in oocytes of Xenopus laevis by standard procedures (36Yao, S. Y. M., Cass, C. E., and Young, J. D. (2000) in Membrane Transport: A Practical Approach (Baldwin, S. A., ed) pp. 47-78, Oxford University Press, OxfordGoogle Scholar). Healthy defolliculated stage VI oocytes were microinjected with 10 nl of water or 10 nl of water containing RNA transcript (1 ng/nl) and incubated in modified Barth's medium at 18 °C for 72 h prior to the assay of transport activity. Flux Assays—Transport was traced using 14C/3H-labeled nucleosides at 1 μCi/ml. Flux measurements were performed at room temperature (20 °C) as described previously (36Yao, S. Y. M., Cass, C. E., and Young, J. D. (2000) in Membrane Transport: A Practical Approach (Baldwin, S. A., ed) pp. 47-78, Oxford University Press, OxfordGoogle Scholar, 37Huang Q.Q. Yao S.Y.M. Ritzel M.W. Paterson A.R. Cass C.E. Young J.D. J. Biol. Chem. 1994; 269: 17757-17760Abstract Full Text PDF PubMed Google Scholar). Briefly, groups of 12 oocytes were incubated in 200 μl of transport medium containing either 100 mm NaCl or choline chloride and 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.5. Unless otherwise indicated, the uridine concentration was 10 μm. At the end of the incubation period, extracellular label was removed by six rapid washes in ice-cold Na+-free (choline chloride) transport medium, and individual oocytes were dissolved in 1% (w/v) SDS for quantitation of oocyte-associated radioactivity by liquid scintillation counting (LS 6000 IC; Beckman). Initial rates of transport (influx) were determined using an incubation period of 1 min, except for mutants E322Q and E322D, which had low transport activity and required a longer incubation time (5 min) to achieve cellular uptake comparable with that of wild-type hCNT1 and the other mutants. In PCMBS inhibition studies, oocytes were pretreated with PCMBS (0.1 mm) on ice for 30 min and then washed five times with ice-cold transport medium to remove excess organomercurial before the assay of transport activity. Corresponding pretreatment with the MTS reagents MTSEA, MTSES, and MTSET (2.5, 10, and 1 mm, respectively) was performed at room temperature for 5 min. To demonstrate substrate protection, unlabeled uridine (20 mm) was included along with inhibitor during the preincubation step (38Yao S.Y.M. Sundaram M. Chomey E.G. Cass C.E. Baldwin S.A. Young J.D. Biochem. J. 2001; 353: 387-393Crossref PubMed Google Scholar). The flux values shown are means ± S.E. of 10–12 oocytes, and each experiment was performed at least twice on different batches of cells. Kinetic (Km, K50, Vmax, and Hill coefficient) parameters (±S.E.) were determined using SigmaPlot software (Jandel Scientific). Statistical significance of the reported data sets was evaluated using t tests. Electrophysiology—Steady-state and presteady-state currents were measured using the two-microelectrode voltage clamp as described previously (16Smith K.M. Ng A.M.L. Yao S.Y.M. Labedz K.A. Knaus E.E. Wiebe L.I. Cass C.E. Baldwin S.A. Chen X.Z. Karpinski E. Young J.D. J. Physiol. (Lond.). 2004; 558: 807-823Crossref Scopus (79) Google Scholar). Na+/Nucleoside Stoichiometry—Coupling ratios were determined by direct charge/flux measurements (16Smith K.M. Ng A.M.L. Yao S.Y.M. Labedz K.A. Knaus E.E. Wiebe L.I. Cass C.E. Baldwin S.A. Chen X.Z. Karpinski E. Young J.D. J. Physiol. (Lond.). 2004; 558: 807-823Crossref Scopus (79) Google Scholar, 17Smith K.M. Slugoski M.D. Loewen S.K. Ng A.M.L. Yao S.Y.M. Chen X.-Z. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2005; 280: 25436-25449Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Smith K.M. Slugoski M.D. Cass C.E. Baldwin S.A. Karpinski E. Young J.D. Mol. Membr. Biol. 2007; 24: 53-64Crossref PubMed Scopus (27) Google Scholar). Isolation of Membranes and Immunoblotting—Total membranes (plasma + intracellular membranes) and purified plasma membranes were isolated by centrifugation from groups of 100 oocytes at 4 °C in the presence of protease inhibitors as described previously (39Sundaram M. Yao S.Y.M. Ingram J.C. Berry Z.A. Abidi F. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2001; 276: 45270-45272Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 40Kamsteeg E.-J. Deen P.M.T. Biochim. Biophys. Res. Commun. 2001; 282: 683-690Crossref PubMed Scopus (45) Google Scholar). Colloidal silica (Sigma) was used to increase the gravitational density of plasma membranes and enhance their yield and purity (40Kamsteeg E.-J. Deen P.M.T. Biochim. Biophys. Res. Commun. 2001; 282: 683-690Crossref PubMed Scopus (45) Google Scholar). Protein was determined by the bicinchoninic acid protein assay (Pierce) using bovine serum albumin as standard. For immunoblotting, oocyte membranes (1 μg of plasma membrane protein or total membrane protein) were resolved on 12% SDS-polyacrylamide gels (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar). The electrophoresed proteins were transferred to polyvinylidene difluoride membranes and probed with affinity-purified anti-hCNT1-(31–55) polyclonal antibodies (22Slugoski M.D. Loewen S.K. Ng A.M.L. Smith K.M. Yao S.Y.M. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Biochemistry. 2007; 46: 1684-1693Crossref PubMed Scopus (15) Google Scholar). Blots were then incubated with horseradish peroxidase-conjugated anti-rabbit antibodies (Amersham Biosciences) and developed with enhanced chemiluminescence reagents (Amersham Biosciences). Residues Identified for Mutagenesis—In this study, we employed site-directed mutagenesis and heterologous expression in Xenopus oocytes to analyze the roles of hCNT1 acidic amino acid residues. The locations of the residues selected for study are shown in Fig. 1. hCNT1 contains 51 aspartate and glutamate residues. Of these, 10 are conserved in other mammalian CNT family members and were included in the present analysis (hCNT1 residues Asp-172, Glu-308, Glu-322, Glu-338, Glu-389, Glu-413, Asp-482, Glu-498, Glu-532, and Asp-565). All but one (Asp-172) were located in the C-terminal half of the protein. In initial mutagenesis experiments, each of the 10 hCNT1 aspartate and glutamate residues were individually replaced by the corresponding neutral amino acids (asparagine or glutamine, respectively). All mutations were verified by sequencing the entire coding region of the double-stranded plasmid DNA in both directions. Except for the desired base changes, all sequences were identical to wild-type hCNT1. Uridine Uptake and Cell-surface Expression of Wild-type hCNT1 and Mutants—The hCNT1 mutant transporters were expressed in Xenopus oocytes and assayed for uridine transport activity (10 μm uridine influx, 1 min fluxes) in the presence and absence of Na+ as described under “Experimental Procedures.” Representative values for mediated uridine uptake, corrected for basal nonmediated uptake in control water-injected oocytes, are presented in Table 1. Five of the mutants (D172N, E338Q, E389Q, E413Q, and D565N) exhibited >75% of wild-type Na+-dependent transport activity and were not investigated further. In contrast, substitution of Glu-308 reduced transport activity by almost 90%, whereas mutation of E322Q, D482N, E498Q, and E532Q resulted in >99% loss of uridine transport activity. The time course of uridine uptake by wild-type hCNT1 shown in Fig. 2A demonstrates that the measured fluxes corresponded to initial rates of transport. Fig. 2A also demonstrates the absence of uridine uptake in water-injected oocytes.TABLE 1Uridine uptake by wild-type hCNT1 and mutants expressed in Xenopus oocytes Oocytes producing recombinant hCNT1 and hCNT1 mutants were incubated in transport medium with or without Na+ at 20 °C as described under “Experimental Procedures.” Each value is the mean ± S.E. from 10 to 12 oocytes. For influx in the presence of Na+, significant differences in mediated uridine uptake (p < 0.05) compared with wild-type hCNT1 are indicated by*.Mediated uridine uptakeProtein expressionaRelative expression in the plasma membrane is from Fig. 3Na+ mediumNa+-free mediumpmol/oocyte·min-1hCNT15.0 ± 0.70.06 ± 0.01+D172N4.9 ± 0.40.12 ± 0.02NDbND indicates not determinedE308Q0.7 ± 0.1*<0.01+E322Q0.2 ± 0.1*<0.01+E338Q4.7 ± 0.60.03 ± 0.01NDE389Q3.8 ± 0.30.05 ± 0.01NDE413Q5.4 ± 0.60.07 ± 0.02NDD482N0.1 ± 0.1*<0.01-E498Q<0.01*<0.01-E532Q<0.01*<0.01-D565N5.4 ± 0.50.08 ± 0.02NDa Relative expression in the plasma membrane is from Fig. 3b ND indicates not determined Open table in a new tab Cell-surface expression of mutants E308Q, E322Q, D482N, E498Q, and E532Q was investigated by immunoblotting of purified oocyte plasma membranes using polyclonal antibodies (22Slugoski M.D. Loewen S.K. Ng A.M.L. Smith K.M. Yao S.Y.M. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. Biochemistry. 2007; 46: 1684-1693Crossref PubMed Scopus (15) Google Scholar) directed against amino acid residues 31–55 at the N terminus of the protein (Fig. 3A). Wild-type hCNT1 and transporters with mutations at positions 308 and 322 were present in similar amounts, indicating that these single amino acid substitutions resulted in loss of intrinsic hCNT1 transport activity without altering surface quantities in the oocyte plasma membrane. Antibody specificity was confirmed by lack of immunoreactivity in membranes prepared from control water-injected oocytes. Unlike E308Q and E322Q, very little plasma membrane immunoreactivity was detected for the transporters having mutations at positions 482, 498, and 532, indicating that the lack of transport activity was associated with reduced cell-surface expression. In a second round of mutagenesis experiments, Glu-308, Glu-322, Asp-482, Glu-498, and Glu-532 of hCNT1 were replaced by the alternative negatively charged amino acid (i.e. glutamate to aspartate or aspartate to glutamate). Time courses of uridine accumulation in the presence of Na+ and the initial rate of uridine uptake in the presence and absence of Na+ by E308D, E322D, D482E," @default.
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• W2072863040 title "Conserved Glutamate Residues Are Critically Involved in Na+/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter 1 (hCNT1)" @default.
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