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

Abstract

To identify functionally relevant amino acids in the rat organic cation transporter 1 (rOCT1), 18 consecutive amino acids in the presumed fourth transmembrane ␣ helix (TMH) were mutated and functionally characterized after expression in Xenopus laevis oocytes. After mutation of three amino acids on successive turns of the ␣ 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 ␣ 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.

Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)

“…21 Because ASP + was used as a probe substrate, we first docked ASP + into two predicted binding sites and selected the site with the best score (−6.08) for all subsequent docking. Next, we validated the accuracy of the comparative model of the OCT1 transporter by confirming that (1) known OCT1 substrates docked favorably against the predicted binding site and (2) residues implicated in OCT1 transport 12,14,16,17 were localized in the predicted binding site, as follows. First, we docked 15 known OCT1 endogenous and drug substrates against the predicted binding site ( Figure 1A).…”

“…Additionally, in rat, mutations of Y221 and D474 resulted in reduced uptake of tetraethylammonium (TEA). 16 Finally, docked substrates formed non-covalent interactions with W217, T225, I449, and Q447 in TMHs 4, 10, and 11. The equivalent residues in rat have also been implicated in ligand−transporter interactions.…”

“…First, although several atomic structures have been resolved for bacterial members of the major facilitator superfamily of transporters, low sequence identity between bacterial homologues and human OCT1 casts doubt on the accuracy of comparative models built using these structures as templates. 16 Second, OCT1 is considered a polyspecific transporter that transports compounds of different sizes and Here we built a comparative model of human OCT1 in an inward-facing occluded conformation using a recently determined structure of its eukaryotic homologue, a phosphate transporter from Piriformospora indica (PiPT). 18 We confirmed that 80% of the known structurally diverse substrates can be docked favorably against the predicted binding site (Table S1).…”

Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3

“…2 The abbreviations used are: OCT, organic cation transporter; MPP ϩ , methyl-4-phenylpyridinium; DA-7, 1,7-diaminoheptane; DA-10, 1,10-diaminodecane; TMA, tetramethylammonium; TMA-6, 1,6 bis-trimethylaminohexane; TMA-10, decamethonium; TEA, tetraethylammonium; TEA-6, 1,6-bistriethylaminohexane; TEA-10, 1,10-bistriethylaminodecane; Quin-6, bisquinuclidinehexane; Quin-10, 1,10-bisquinuclidinedecane; TM, transmembrane. 26), suggest that seven of the predicted 12 transmembrane helices (TM) of OCTs, namely TMs 1, 2, 4, 5, 7, 10, and 11, fold in a large hydrophobic cleft capable of accommodating a wide variety of chemical species (27,28), within which certain amino acid residues underlie substrate-transporter interactions ( Fig. 1) (28,29).…”

“…26), suggest that seven of the predicted 12 transmembrane helices (TM) of OCTs, namely TMs 1, 2, 4, 5, 7, 10, and 11, fold in a large hydrophobic cleft capable of accommodating a wide variety of chemical species (27,28), within which certain amino acid residues underlie substrate-transporter interactions ( Fig. 1) (28,29). In particular, inward and outward oriented homology models of the rat OCT1 (rOCT1) tertiary structure highlight amino acids within a putative substrate binding pocket that have been implicated in substrate affinity as determined by site-directed mutagenesis experiments (28,30).…”