Abstract

Organic cation transporters OCT1 (SLC22A1) and OCT2 (SLC22A2) are critically involved in absorption and excretion of diverse cationic drugs. Because drug-drug interactions at these transporters may induce adverse drug effects in patients, in vitro testing for interaction with the human transporters during drug development is mandatory. Recent data performed with rat OCT1 (rOCT1) suggest that currently performed in vitro tests assuming one polyspecific binding site are insufficient. Here we measured binding and transport of model substrate 1-methyl-4-phenylpyridinium⁺ (MPP⁺) by cell-free-expressed fusion proteins of rOCT1 and various rOCT1 mutants with green fluorescent protein that were reconstituted into nanodiscs or proteoliposomes. The nanodiscs were formed with major scaffold protein MSP and different phospholipids whereas the proteoliposomes were formed with a mixture of cholesterol, phosphatidylserine and phosphatidylcholine. In nanodiscs formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or cholesterol, phosphatidylserine and phosphatidylcholine two low-affinity MPP⁺ binding sites and one high-affinity MPP⁺ binding site per transporter monomer were determined. Mutagenesis revealed that tryptophan 218 and aspartate 475 in neighboring positions in the modeled outward-open cleft contribute to one low-affinity binding site whereas arginine 440 located distantly in the cleft is critical for MPP⁺ binding to another low-affinity site. Comparing MPP⁺ binding with MPP⁺ transport suggests that the low-affinity sites are involved in MPP⁺ transport whereas high-affinity MPP⁺ binding influences transport allosterically. The data provide a rationale for future reliable, more sophisticated in vitro testing and for the generation of pharmacophore models with high predictive power. They will be helpful to interprete future crystal structures.