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  • Title: Amino acids critical for substrate affinity of rat organic cation transporter 1 line the substrate binding region in a model derived from the tertiary structure of lactose permease.
    Author: Popp C, Gorboulev V, Müller TD, Gorbunov D, Shatskaya N, Koepsell H.
    Journal: Mol Pharmacol; 2005 May; 67(5):1600-11. PubMed ID: 15662044.
    Abstract:
    To identify functionally relevant amino acids in the rat organic cation transporter 1 (rOCT1), 18 consecutive amino acids in the presumed fourth transmembrane alpha helix (TMH) were mutated and functionally characterized after expression in Xenopus laevis oocytes. After mutation of three amino acids on successive turns of the alpha helix, K(m) values for tetraethylammonium (TEA) and/or 1-methyl-4-phenylpyridinium (MPP) were decreased. After replacement of Trp218 by tyrosine (W218Y) and Tyr222 by leucine (Y222L), the K(m) values for both TEA and MPP were decreased. In mutants Y222F and T226A, only the K(m) values for TEA and MPP were decreased, respectively. The data suggest that amino acids Trp218 and Tyr222 participate in the binding of both TEA and MPP, whereas Thr226 is only involved in the binding of MPP. Using the crystal structure of the lactose permease LacY from Escherichia coli that belongs to the same major facilitator superfamily as rOCT1, we modeled the tertiary structure of the presumed 12 transmembrane alpha helices. The validity of the model was suggested because seven amino acids that have been shown to participate in the binding of cations by mutagenesis experiments [fourth TMH Trp218, Tyr222, and Thr226 (this work); 10th TMH Ala443, Leu447, and Gln448 (companion work in this issue of Molecular Pharmacology); 11th TMH Asp475 (previous report)] are located in one region surrounding a large cleft that opens to the intracellular side. The dimensions of TEA in comparison with the interacting amino acids in the modeled cleft suggest that more than one TEA molecule can bind in parallel to the modeled conformation of the transporter.
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