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.

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 (HEK) 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 non-transported inhibitors tetrabutylammonium + (TBuA + ), tetrapentylammonium + (TPeA + ) and corticosterone was measured. Uptake measurements were performed on confluent cell layers using 2 min-incubation or in dissociated cells using incubation times of 1, 5, or 10 s. With both methods different apparent Michaelis-Menten K m values, different half-maximal inhibitor concentrations ( IC 50 ), and varying effects of mutations were determined. In addition, varying IC 50 values for 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 IC 50 values for TBuA + , TPeA + and corticosterone, whereas mutation of Phe160 and Leu447 changed the IC 50 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.