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  • Title: 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.
    Author: Gorbunov D, Gorboulev V, Shatskaya N, Mueller T, Bamberg E, Friedrich T, Koepsell H.
    Journal: Mol Pharmacol; 2008 Jan; 73(1):50-61. PubMed ID: 17940192.
    Abstract:
    Voltage-clamp fluorometry was performed with a cysteine-deprived mutant of rat organic cation transporter 1 (rOCT1) in which Phe483 in transmembrane alpha-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.
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