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Functional and pharmacological characterization of equilibrative nucleoside transporter splice variants
Endogenous nucleosides, as well as nucleoside analogues used in chemotherapy, are accumulated by cells via a family of equilibrative nucleoside transporters (ENTs) (Baldwin et al, 2004). An alternative splice variant of the ENT1 transporter subtype has been identified in mice which is missing one of the protein kinase CK2 (casein kinase 2) consensus sites (S254) in the central intracellular loop of this eleven transmembrane domain protein (Kiss et al, 2000). Little is known about the regulation of ENT1, and we hypothesized that the relative expression of these splice variants would affect the ability of CK2 to regulate transport activity. Stable transfectants of each variant (mENT1a, one CK2 site; mENT1b, two CK2 sites) were made using the PK15-NTD (nucleoside transporter deficient) cell line (Ward et al, 2000). These cells were characterized for their ability to bind the ENT1-selective probe [3H]nitrobenzylthioinosine (NBMPR)(Baldwin et al, 2004) and to mediate the cellular uptake of [3H]2-chloroadenosine, with and without incubation of the cells for 48 h with an inhibitor of CK2, 4,5,6,7-tetrabromobenzotriazole (TBB, 10 µ:M) (Litchfield, 2003), or the vehicle (DMSO, 0.01%). All experiments were repeated at least four times and comparisons were made using the Students t-test (paired or unpaired as appropriate, P<0.05). Relative to PK15-mENT1b, the PK15-mENT1a cells (in the absence of TBB) had significantly lower [3H]NBMPR binding (BMAX 460000 ± 16000 and 70000 ± 13000 sites/cell, respectively) and [3H2]-chloroadenosine uptake (VMAX 3.5 ± 0.3 and 1.8 ± 0.2 pmol/µl/s respectively). While this could represent differences in transfection efficiency, the fact that it was a consistent difference in a number of clonal cell lines tested suggests that it may be due to the CK2 site difference. In contrast, the affinity of the two cell lines for [3H]NBMPR (KD ~ 0.08 nM) or [3H2]-chloroadenosine (KM ~ 45 μM) was not significantly different. Likewise, a series of ENT1 inhibitors and substrates also blocked both mENT1a and mENT1b-mediated [3H2]-chloroadenosine uptake with comparable KI values, and an order of potency compatible with the mouse ENT1 (NBMPR > dilazep > draflazine > dipyridamole > adenosine > uridine). Incubation of the cells with TBB for 48 h decreased significantly the binding of [3H]NBMPR (20 ± 5%) and the uptake of [3H2]-chloroadenosine (33 ± 6%) by PK15-mENT1b cells, but had no effect on PK15-mENT1a cells. These data suggest that the loss of the CK2 site (S254) in mENT1a does not affect its affinity for substrates/inhibitors, but does remove a potential regulatory site involving CK2-mediated phosphorylation. Further direct phosphorylation studies are required to confirm these results, however we reason that phosphorylation at S254 may decrease the rate of internalization of the transporter, or enhance recycling from intracellular compartments to the plasma membrane. This work was supported by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada.
Baldwin, SA et al. (2004) Pflugers Archives, 447, 735-743. |
