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

Abstract

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 (K_m) values, different IC₅₀ values, and varying effects of mutations were determined. In addition, varying IC₅₀ values for the inhibition of MPP⁺ uptake and varying effects of mutations were obtained when different MPP⁺ concentrations far below the apparent K_m value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 µM 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 K_m values for TEA⁺ and/or MPP⁺ Mutation of Trp218 and Asp475 led to altered IC₅₀ values for TBuA⁺, TPeA⁺, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC₅₀ 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.