PT  - JOURNAL ARTICLE
AU  - Kevin R. Robillard
AU  - Derek B.J. Bone
AU  - Jamie S. Park
AU  - James R. Hammond
TI  - Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1
AID  - 10.1124/mol.107.041871
DP  - 2008 Jul 01
TA  - Molecular Pharmacology
PG  - 264--273
VI  - 74
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/74/1/264.short
4100  - http://molpharm.aspetjournals.org/content/74/1/264.full
SO  - Mol Pharmacol2008 Jul 01; 74
AB  - Mammalian cells require specific transport mechanisms for the cellular uptake and release of endogenous nucleosides such as adenosine, and nucleoside analogs used in chemotherapy. We have identified a novel splice variant of the mouse equilibrative nucleoside transporter, mENT1, that results from the exclusion of exon 11 during pre-RNA processing. This variant encodes a truncated protein (mENT1Δ11) missing the last three transmembrane domains of the full-length mENT1. The mENT1Δ11 transcript and protein were found to be differentially distributed among tissues relative to full-length mENT1. PK15-NTD (nucleoside transport deficient) cells were transfected with mENT1 or mENT1Δ11 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT1Δ11 in terms of transport function or inhibitor binding affinity. PK15-mENT1Δ11 transfected cells bound the ENT1 probe [3H]nitrobenzylthioinosine (NBMPR) with high affinity and mediated the cellular accumulation of both [3H]2-chloroadenosine and [3H]uridine. The only significant differences between the mENT1 variants were that mENT1Δ11 could not be photolabeled with [3H]NBMPR and that mENT1Δ11 was insensitive to the transporter-modifying effects of N-ethylmaleimide. These data suggest that the last three transmembrane domains of mENT1 are not necessary for transport activity, but this region does contain the cysteines responsible for the sensitivity of mENT1 to sulfhydryl reagents, and the residues important for covalent modification of the protein with NBMPR. These results provide important guidelines for future mutagenesis studies aimed at elucidating the tertiary structure of the ENT1 protein and the domains involved in inhibitor binding and substrate translocation. The American Society for Pharmacology and Experimental Therapeutics