CHIMERIC CONSTRUCTS BETWEEN HUMAN AND RAT EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS (HENT1 AND RENT1) REVEAL HENT1 STRUCTURAL DOMAINS INTERACTING WITH CORONARY VASOACTIVE DRUGS

We have recently isolated cDNAs from human placenta and rat jejunum en coding the prototypic human and rat equilibrative nitrobenzylthioinosi ne (NBMPR)-sensitive nucleoside transporters hENT1 and rENT1. The two proteins (456 and 457 residues, M-r 50,000) are 78% identical in amino acid sequence and contain 11 potential transmembrane segments (TMs) w ith a large putative extracellular loop between TMs 1 and 2 and a larg e cytoplasmic loop between TMs 6 and 7, When expressed in Xenopus oocy tes, recombinant hENT1 and rENT1 transport both purine and pyrimidine nucleosides, including adenosine, and are inhibited by nanomolar conce ntrations of NBMPR. hENT1 is also potently inhibited by coronary vasod ilator drugs (dipyridamole, dilazep, and draflazine), whereas rENT1 is insensitive to inhibition by these compounds (dipyridamole IC,, value s 190 nM (hENT1) and greater than or equal to 10 mu M (rENT1) at 10 mu M uridine). In the present study, we have generated reciprocal chimer as between hENT1 and rENT1, using splice sites at residues 99 (end of TM. 2) and 231 (end of TM 6), to identify structural domains of hENT1 responsible for transport inhibition by vasoactive compounds. Transpla nting the amino-terminal half of hENT1 into rENT1 converted rENT1 into a dipyridamole/dilazep-sensitive transporter, whereas the amino-termi nal half of rENT1 rendered hENT1 dipyridamole/dilazep-insensitive, Dom ain swaps within the amino-terminal halves of hENT1 and rENT1 identifi ed residues 100-231 (incorporating TMs 3-6) of hENT1 as the major site of vasodilator interaction. Since these drugs function as competitive inhibitors of nucleoside transport and NBMPR binding, TMs 3-6 are lik ely to form part of the substrate-binding site.