QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.107.041871v1 74/1/264    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Robillard, K. R. Articles by Hammond, J. R. Search for Related Content PubMed PubMed Citation Articles by Robillard, K. R. Articles by Hammond, J. R.

Characterization of mENT111, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

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 (mENT111) missing the last three transmembrane domains of the full-length mENT1. The mENT111 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 mENT111 and assessed for nucleoside transport function. No significant differences were observed between the mENT1 and mENT111 in terms of transport function or inhibitor binding affinity. PK15-mENT111 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 mENT111 could not be photolabeled with [³H]NBMPR and that mENT111 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.

Received for publication September 14, 2007.

Accepted for publication April 14, 2008.

Address correspondence to: Dr. James R Hammond, Dept. of Physiology and Pharmacology, M266 Medical Sciences Building, University of Western Ontario, London, Ontario, N6A 5C1, Canada. E-mail: jhammo{at}uwo.ca