Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine

•Crithidia fasciculata has multiple adenosine transporters of the ENT family.•Like CfNT1, CfAT1 and its allele CfAT1.2 are adenosine transporters.•CfNT3 may encode a third adenosine-transporting ENT.•A Lys residue in TM4 plays a key role in ligand affinity in CfAT1.

Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.

Graphical abstractA lysine (K) residue in transmembrane domain 4 plays a key role in the ligand affinity of Crithidia fasciculata adenosine transporters CfAT1 and CfNT1.Figure optionsDownload full-size imageDownload high-quality image (74 K)Download as PowerPoint slide