Received for publication October 29, 2004.
Revised January 18, 2005.
Accepted for publication January 19, 2005.

Subtype-specific Affinity for Corticosterone of Rat Organic Cation Transporters rOCT1 and rOCT2 Depends on Three Amino Acids within the Substrate Binding Region

Abstract

The affinity of corticosterone to organic cation transporters (OCTs) is subtype and species-dependent. For example, the IC₅₀ values for corticosterone inhibition of cation uptake by transporters rOCT1 and OCT2 are ~150 and ~4 µM, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that exchange of three amino acids in the presumed 10^th transmembrane -helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (A443, L447, Q448 in rOCT1 and I443, Y447, E448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants the Michaelis-Menten constant (K_M) values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC₅₀ values for inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E) the IC₅₀ value for the inhibition of [³H]MPP (0.1 µM) uptake by corticosterone (24 ± 4 µM) was significantly higher compared to the IC₅₀ value for inhibition of [¹⁴C]TEA (10 µM) uptake (5.3 ± 1.7 µM). This finding suggests an allosteric interaction between transported cation and corticosterone. Since this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP vs. TEA, and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short range allosteric effect on the corticosterone binding site.

Key words: Biogenic Amine, Organic anion, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches