Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake.

The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium (MPP) and tetraethylammonium (TEA) or inhibition of MPP uptake by the nontransported inhibitors tetrabutylammonium (TBuA), tetrapentylammonium (TPeA), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant () values, different IC values, and varying effects of mutations were determined. In addition, varying IC values for the inhibition of MPP uptake and varying effects of mutations were obtained when different MPP concentrations far below the apparent value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 M MPP for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent values for TEA and/or MPP Mutation of Trp218 and Asp475 led to altered IC values for TBuA, TPeA, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.