Received for publication July 19, 2007.
Revised October 16, 2007.
Accepted for publication October 16, 2007.

High-affinity Cation Binding to Transporter OCT1 Induces Movement of Helix 11 and Blocks Transport after Mutations in a Modelled Interaction Domain between Two Helices

Abstract

Voltage clamp fluorometry was performed with a cysteine-deprived mutant of rat organic cation transporter 1 (rOCT1) in which phenylalanine 483 in the 11^th transmembrane -helix (TMH) close to the extracellular surface was replaced by cysteine and labeled with tetramethylrhodamine-6-maleimide (TMRM). Potential-dependent fluorescence changes were observed that were sensitive to presence of substrates choline, tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) and of the non-transported inhibitor tetrabutylammonium (TBuA). Using potential-dependent fluorescence changes as readout one high-affinity binding site per substrate and two high-affinity binding sites for TBuA were identified in addition to the previously described single interaction sites. In a structure model of rOCT1 with an inward open cleft that was derived from a known crystal structure of lacY permease, phenylalanine 483 is close to tryptophan 147 in the 2^nd TMH. In contrast, in a model with an outward open cleft these amino acids are far apart. After replacement of phenylalanine 483 or tryptophan 147 by cysteine or serine, high-affinity binding of TBuA leads to inhibition of MPP or TEA uptake whereas it has no effect on cation uptake by wildtype rOCT1. Coexisting high-affinity cation binding sites in organic cation transporters may collect low concentration xenobiotics and drugs, however, translocation including transitions between outward and inward oriented conformations may only be induced when a low-affinity cation binding site is loaded. We propose that cations bound to high-affinity sites may be translocated together with cations bound to low affinity sites or may block the translocation mechanism.

Key words: Biogenic Amine, Ion transporters (SERCA, Na/K ATPase, CFTR), Structure-activity relationships and modeling, Fluorescence techniques, Mutagenesis/Chimeric approaches, Liver transporters, Structure/function/mechanism