Five Amino Acids in the Innermost Cavity of the Substrate Binding Cleft of Organic Cation Transporter 1 Interact with Extracellular and Intracellular Corticosterone

Abstract

We have shown previously that Leu447 and Gln448 in the transmembrane helix (TMH) 10 of rat organic cation transporter rOCT1 are critical for inhibition of cation uptake by corticosterone. Here, we tested whether the affinity of corticosterone is different when applied from the extracellular or intracellular side. The affinity of corticosterone was determined by measuring the inhibition of currents induced by tetraethylammonium ϩ (TEA ϩ ) in Xenopus laevis oocytes expressing rOCT1. Either corticosterone and TEA ϩ were added to the bath simultaneously or the oocytes were preincubated with corticosterone, washed, and TEA ϩ -induced currents were determined subsequently. In mutant L447Y, K i values for extracellular and intracellular corticosterone were decreased, whereas in mutant Q448E, only the K i for intracellular corticosterone was changed. Modeling of the interaction of corticosterone with rOCT1 in the inward-or outward-facing conformation predicted direct binding to Leu447, Phe160 (TMH2), Trp218 (TMH4), Arg440 (TMH10), and Asp475 (TM11) from both sides. In mutant F160A, affinities for extracellular and intracellular corticosterone were increased, whereas maximal inhibition was reduced in W218F and R440K. In stably transfected epithelial cells, the affinities for inhibition of 1-methyl-4-phenyl-pyridinium ϩ (MPP ϩ ) uptake by extracellular and intracellular corticosterone were decreased when Asp475 was replaced by glutamate. In mutants F160A, W218Y, R440K, and L447F, the affinities for MPP ϩ uptake were changed, and in mutant D475E, the affinity for TEA ϩ uptake was changed. The data suggest that Phe160, Trp218, Arg440, Leu447, and Asp475 are located within an innermost cavity of the binding cleft that is alternatingly exposed to the extracellular or intracellular side during substrate transport.Polyspecific organic cation transporters (OCTs) of the SLC22 family play important roles in the elimination, distribution, and homeostasis of drugs. In addition, they are involved in the reuptake of neurotransmitters into neurons and release of acetylcholine during non-neuronal cholinergic reactions (Lips et al., 2005;Koepsell et al., 2007;Reitman and Schadt, 2007). Three OCT subtypes with overlapping substrate specificities and tissue distributions have been identified . OCT1, OCT2, and OCT3 are polyspecific facilitated diffusion systems that are able to translocate organic cations in both directions across the plasma membrane. However, it remains unclear how OCTs recognize substrates and inhibitors exhibiting different chemical structures and how substrate translocation is achieved. Extensive mutagenesis experiments performed in rat OCT1 (rOCT1) revealed that seven amino acids localized in the 4th, 10th, and 11th transmembrane helices (TMH) are critical for substrate and inhibitor specificity (Gorboulev et al., 1999Popp et al., 2005). Modeling of the rOCT1 structures using the inward-facing structure of lactose permease (LacY) from Escherichia coli (Abramson et al., 2003;Popp et al., 2005) suggested that the...

The Large Extracellular Loop of Organic Cation Transporter 1 Influences Substrate Affinity and Is Pivotal for Oligomerization

“…Assuming monomeric function of rOct1, well supported hypotheses concerning substrate binding and translocation have been raised. Neglecting allosteric effects of substrate binding to different monomers, differential effects of single point mutations on different substrates were interpreted to indicate that the substrate binding region of rOct1 contains overlapping binding domains for different substrates (10,37). Although our present data do not exclude subtle allosteric effects between monomers, they show that allosteric interaction between monomers does not have main functional importance.…”

Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

“…Moreover, X-ray crystal structures of P-gp (Aller et al, 2009) and the multidrug-binding transcription regulator QacR (Schumacher and Brennan, 2003) reveal the presence of multisite binding pockets. With respect to the OCTs, a number of studies have suggested that inhibitors of OCT transport display allosteric interactions at multiple sites (e.g., Volk et al, 2003Volk et al, , 2009Popp et al, 2005;Lee et al, 2009;Minuesa et al, 2009;Egenberger et al, 2012). Furthermore, the results of site-directed studies of OCT function consistently suggest that substrates must interact with spatially distinct sites within the large hydrophilic translocation pathway suspected to exist with OCT1 (Popp et al, 2005) and OCT2 (Zhang et al, 2005;Pelis et al, 2006).…”

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).…”

“…The equivalent residues in rat have also been implicated in ligand−transporter interactions. 17 Prediction of New Ligands by Virtual Screening and Validation by HTS. We predicted new ligands of OCT1 by docking each one of the 1780 compounds in the Pharmakon drug library (MicroSource Discovery Systems; http://www.…”