Comprehensive characterization of the OCT1 phenylalanine-244-alanine substitution reveals highly substrate-dependent effects on transporter function.

Organic cation transporters (OCTs) can transport structurally highly diverse substrates. The molecular basis of this extensive polyspecificity has been further elucidated by cryo-EM. Apparently, in addition to negatively charged amino acids, aromatic residues may contribute to substrate binding and substrate selectivity. In this study, we provide a comprehensive characterization of phenylalanine 244 in OCT1 function. We analyzed the uptake of 144 OCT1 substrates for the phenylalanine 244 to alanine substitution compared to WTOCT1. This substitution had highly substrate-specific effects ranging from transport reduced to 10% of WT activity up to 8-fold increased transport rates. Four percent of substrates showed strongly increased uptake (>200% of WT) whereas 39% showed strongly reduced transport (<50% of WT). Particularly with larger, more hydrophobic, and more aromatic substrates, the Phe244Ala substitution resulted in higher transport rates and lower inhibition of the transporter. In contrast, substrates with a lower molecular weight and less aromatic rings showed generally decreased uptake rates. A comparison of our data to available transport kinetic data demonstrates that generally, high-affinity low-capacity substrates show increased uptake by the Phe244Ala substitution, whereas low-affinity high-capacity substrates are characterized by reduced transport rates. Altogether, our study provides the first comprehensive characterization of the functional role of an aromatic amino acid within the substrate translocation pathway of OCT1. The pleiotropic function further highlights that phenylalanine 244 interacts in a highly specific manner with OCT1 substrates and inhibitors.