Identification and Mutational Analysis of Amino Acid Residues Involved in Dipyridamole Interactions with Human and Caenorhabditis elegans Equilibrative Nucleoside Transporters

Abstract

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile 429 in TM 11 of CeENT1 and Met 33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC 50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu 442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)

“…The V max of the F394L mutant was 2-fold lower than wild-type PfENT1. The (29) demonstrated that mutagenesis at a position in those proteins analogous to PfENT1 L393 (Fig. 1) altered both transport K m as well as sensitivity to the nucleoside transport inhibitor dipyridamole.…”

“…The ENT family members are predicted to have eleven transmembrane (TM) segments with a cytoplasmic N terminus and extracellular C terminus (21,22). Mutations in multiple transmembrane segments have been shown to affect substrate affinity and/or inhibitor efficacy including residues in TM1 (M33, hENT1), TM2 (M89 and L92, hENT1), TM4 (G154, hENT1 ϭ C140, rENT2; S160, hENT1; K155, CfNT2), TM8 (F334, N338, hENT1), and TM11 (L442, hENT1 ϭ I429, CeENT1; S469 & T478, LdNT1.1) (23)(24)(25)(26)(27)(28)(29)(30)(31). For many of these residues it is not known whether they line the permeation pathway or lie elsewhere in the protein where the mutations alter the protein structure leading to the observed functional effects of the mutations.…”

Characterization of mENT1Δ11, a Novel Alternative Splice Variant of the Mouse Equilibrative Nucleoside Transporter 1

“…Regions outside of TMs 3-6 have also been shown to affect ligand binding to ENTs, but almost all of the studies implicating specific regions/residues in ligand binding and transporter function have focused on amino acids upstream of TM9 (Visser et al, 2005b. The exceptions are a study showing that Arg404 of the Leishmania donovani nucleoside transporter LdNT1.1 (corresponding to Arg369 in TM9 of hENT1) is important for function and substrate specificity (Valdés et al, 2006) and a study showing that Leu442 in TM11 of hENT1 is important for substrate selectivity (Visser et al, 2005a). These results are difficult to reconcile with fact that mENT1⌬11, which is missing TMs 9 -11 transports both the purine nucleoside 2-chloroadenosine and the pyrimidine nucleoside uridine as effectively as the full-length mENT1.…”

Cysteine Residues in the Transmembrane (TM) 9 to TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function

“…Although this result suggested that the region was not essential for transporter function, it did imply that NEM sensitivity of NBMPR binding may be due to modification of cysteines encompassed by TM9 to TM11. In addition, Visser et al (2005Visser et al ( , 2007) have shown Fig. 1.…”