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.
BACKGROUND
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.


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 muM 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.


CONCLUSIONS
These results suggested a high degree of conservation of function for hCNT3 in the Caucasian population.
Damaraju
Sambasivarao
S
Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.
Zhang
Jing
J
Visser
Frank
F
Tackaberry
Tracey
T
Dufour
Jennifer
J
Smith
Kyla M
KM
Slugoski
Melissa
M
Ritzel
Mabel W L
MW
Baldwin
Stephen A
SA
Young
James D
JD
Cass
Carol E
CE
D000241:Adenosine / Q000737:chemistry; D000818:Animals; D001331:Automation; D001692:Biological Transport; D003562:Cytidine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D019992:Databases as Topic; D004566:Electrodes; D005091:Exons; D014644:Genetic Variation; D006801:Humans; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009705:Nucleosides / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D017434:Protein Structure, Tertiary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D017422:Sequence Analysis, DNA; D012964:Sodium / Q000378:metabolism; D014529:Uridine / Q000737:chemistry; D014982:Xenopus laevis
D017931:DNA Primers; D026901:Membrane Transport Proteins; D009705:Nucleosides; D011522:Protons; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; D007288:Inosine; D003562:Cytidine; D004247:DNA; D012964:Sodium; D000241:Adenosine; D014529:Uridine