Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes.
The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.
Loewen
Shaun K
SK
Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.
Yao
Sylvia Y M
SY
Slugoski
Melissa D
MD
Mohabir
Nadira N
NN
Turner
Raymond J
RJ
Mackey
John R
JR
Weiner
Joel H
JH
Gallagher
Maurice P
MP
Henderson
Peter J F
PJ
Baldwin
Stephen A
SA
Cass
Carol E
CE
Young
James D
JD
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics; D000998:Antiviral Agents / Q000378:metabolism / Q000493:pharmacokinetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D029968:Escherichia coli Proteins; D005260:Female; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009705:Nucleosides / Q000378:metabolism / Q000493:pharmacokinetics; D009865:Oocytes; D011994:Recombinant Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014982:Xenopus laevis
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009705:Nucleosides; C088081:NupC protein, Bacteria; C490231:NupC protein, E coli; D011994:Recombinant Proteins; C065171:cif nucleoside transporter