Intestinal cells have a poor capacity for de novo nucleoside biosynthesis and therefore have to rely heavily on salvage pathways for their nucleoside needs. This necessitates the intestinal epithelial cells to synthesis and express nucleoside transporters on their brush border membranes to allow for efficient salvage of nucleosides from the lumen. Since many antiviral and anti-cancer nucleoside drugs are substrates of nucleoside transporters, understanding their mechanism of uptake by the nucleoside transporters in the human intestine is important for prediction of potential beneficial or deleterious pharmacokinetic and pharmacodynamic drug-drug interactions and the design of nucleoside drugs with better oral absorption profiles. Therefore, the overall goal of this proposal is to elucidate the mechanisms of nucleoside transport in the human intestinal epithelium. In the current funding period, we have found that equilibrative (es and ei) and two concentrative (N1 and N2) nucleoside transporters are expressed in Xenopus-oocytes microinjected with intestinal mRNA. Because only the concentrative (N1 and N2) nucleoside transporters are present on the brush border membrane on the human intestinal epithelium, we have hypothesized that the equilibrative transporters are expressed on the basolateral membrane and perhaps on the intracellular membranes. Therefore, the specific aims of the present proposal are to: 1. Identify and map the critical structural determinants of the human es, N1 and the N2 transporters and of the nucleosides necessary for high affinity nucleoside-transporter interaction. We will accomplish this aim: a) by selecting PCR-generated mutant clones for null or reduced transporter activity in a high throughput expression system, Saccharomyces cerevisiae, and b) by binding a three dimensional model of the nucleoside binding sites of the transporters using Comparative Molecular Field Analysis (CoMFA). 2. Test the hypothesis that cellular differentiation in the crypt-to-villus axis results in a switch in the types and cellular localization of the expressed nucleoside transporters. We will accomplish this aim by characterizing the cellular localization and expression of the es and N2 nucleoside transporters and their mRNAs in the crypt-to-villus axis in both the adult and the fetal human intestine.
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