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-beta-d-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-beta-d-ribofuranosyl)-2,4-difluoro-5-iodobenzene. Apparent K(m) values for 5-fluorouridine, 5-fluoro-2'-deoxyuridine and zidovudine were 18, 15 and 450 microm, 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 [(14)C]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 x 10(10) per cell). The hCNT1 turnover rate at -50 mV was 9.6 molecules of uridine transported per second.
Smith
Kyla M
KM
Membrane Protein Research Group, Department of Physiology, Faculty of Pharmacy, 7-55 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Ng
Amy M L
AM
Yao
Sylvia Y M
SY
Labedz
Kathy A
KA
Knaus
Edward E
EE
Wiebe
Leonard I
LI
Cass
Carol E
CE
Baldwin
Stephen A
SA
Chen
Xing-Zhen
XZ
Karpinski
Edward
E
Young
James D
JD
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005260:Female; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism / Q000502:physiology*; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism*; D014982:Xenopus laevis
D000970:Antineoplastic Agents; D026901:Membrane Transport Proteins; D009705:Nucleosides; D011994:Recombinant Proteins; C065171:cif nucleoside transporter