QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.107.040170v1 73/1/50    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Gorbunov, D. Articles by Koepsell, H. PubMed PubMed Citation Articles by Gorbunov, D. Articles by Koepsell, H.

High-Affinity Cation Binding to Organic Cation Transporter 1 Induces Movement of Helix 11 and Blocks Transport after Mutations in a Modeled Interaction Domain between Two Helices

Institutes of Anatomy and Cell Biology (D.G., V.G., N.S., H.K.) and Physiological Chemistry (T.M.), University Würzburg, Würzburg, Germany; Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt, Germany (E.B.); and Institute of Chemistry, Technical University of Berlin, Berlin, Germany (T.F.)

Voltage-clamp fluorometry was performed with a cysteine-deprived mutant of rat organic cation transporter 1 (rOCT1) in which Phe483 in transmembrane -helix (TMH) 11 close to the extracellular surface was replaced by cysteine and labeled with tetramethylrhodamine-6-maleimide. 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 nontransported 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, Phe483 is close to Trp147 in TMH 2. In contrast, in a model with an outward open cleft these amino acids are far apart. After replacement of Phe483 or Trp147 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 wild-type 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 that they may block the translocation mechanism.

Address correspondence to: Dr. Hermann Koepsell, Institute of Anatomy and Cell Biology, Koellikerstr. 6, 97070 Würzburg, Germany. E-mail: hermann{at}koepsell.de