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Physiological nucleosides and nucleoside analogues have important biochemical, physiological and pharmacological activities in humans. Adenosine, for example, has cell-surface receptor-mediated functions in processes such as modulation of immune responses, platelet aggregation, renal function and coronary vasodilatation (Fredholm, 1997; Shryock & Belardinelli, 1997). Nucleoside analogues are commonly used in the therapy of cancer and viral infections (Handschumacher et al. 2000; Perigaud et al. 1994). Most nucleosides, including those with antineoplastic and/or antiviral activities are hydrophilic, and specialized plasma membrane nucleoside transporter (NT) proteins are often required for uptake into or release from cells (Baldwin et al. 1999; Mackey et al. 1999; Young et al. 2001). NT-mediated transport is therefore a critical determinant of metabolism and, for nucleoside drugs, their pharmacological actions. Multiple nucleoside transport systems that differ in their cation dependence, permeant selectivities and inhibitor sensitivities have been observed in human and other mammalian cells and tissues (Cass, 1995; Griffiths & Jarvis, 1996; Young et al. 2001). The major concentrative systems (cit, cif and cib) are inwardly directed Na+-dependent processes that have been described primarily in specialized cells, such as intestinal and renal epithelia, hepatocytes, choroid plexus, macrophages, splenocytes and leukaemic cells (Cass, 1995; Griffiths & Jarvis, 1996; Young et al. 2001). The equilibrative (bidirectional) transport processes (es and ei) mediate passive downhill transport of nucleosides, have generally lower permeant affinities than the concentrative systems and occur in most, possibly all, cell types (Cass, 1995; Griffiths & Jarvis, 1996; Young et al. 2001). Systems cit and cif are generally pyrimidine and purine nucleoside selective, respectively, whereas systems cib, es and ei transport both pyrimidine and purine nucleosides. The es process is inhibited by NBMPR (nitrobenzylthioinosine, 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine), while system ei also transports nucleobases (Yao et al. 2002b). Molecular cloning studies have resulted in the isolation and functional expression of cDNAs encoding the human and rodent proteins responsible for each of these nucleoside transport processes (Huang et al. 1994; Che et al. 1995; Yao et al. 1996b; Ritzel et al. 1997; Wang et al. 1997; Crawford et al. 1998; Ritzel et al. 1998, 2001). They belong to two unrelated and previously unrecognized protein families, the concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) proteins. Their relationship to the processes defined by functional studies is: CNT1 (cit), CNT2 (cif), CNT3 (cib), ENT1 (es) and ENT2 (ei). Three further ENTs (ENT3, ENT4 and CLN3) of undetermined function have recently been identified (Hyde et al. 2001; Acimovic & Coe, 2002; Baldwin et al. 2004). Mammalian CNTs have 13 predicted transmembrane helices (TMs), with an intracellular N-terminus and an extracellular glycosylated C-terminus (Hamilton et al. 2001). NupC, an H+-coupled CNT from Escherichia coli, has a similar predicted topology, but lacks TMs 1–3 (Craig et al. 1994; Hamilton et al. 2001). Other characterized CNTs include hfCNT from Eptatretus stouti (Loewen et al. 1999; Yao et al. 2002a), CeCNT3 from Caenorhabditis elegans (Xiao et al. 2001) and CaCNT from Candida albicans (Loewen et al. 2003). Human CNT1 (hCNT1, 650 amino acid residues) (Huang et al. 1994) and rat CNT1 (rCNT1, 648 amino acid residues) (Ritzel et al. 1997) are 83% identical in amino acid sequence and have been studied functionally as recombinant proteins produced in Xenopus oocytes, Saccharomyces cerevisiae and cultured mammalian cells. Radioisotope flux studies have demonstrated pyrimidine nucleoside-selective (cit-type) Na+-dependent fluxes of both 3H- and 14C-labelled physiological nucleosides and nucleoside drugs (Huang et al. 1994; Fang et al. 1996; Yao et al. 1996a,b; Ritzel et al. 1997; Mackey et al. 1998; Yao et al. 2001). In the present study, the two-microelectrode voltage-clamp technique was used to undertake an in-depth steady-state and presteady-state electrophysiological analysis of recombinant hCNT1 produced in Xenopus oocytes. |
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