Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase

Abstract: Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium ([3H]-MPP+) as the marker ligand and MPP+, verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The Kd values calculated from the chromatographic studies correlated with previously reported Ki values (r2=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP+ and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil. [Copyright &y& Elsevier]