Four main steps, known as ADME, determinate the pharmacokinetics of drugs in the human body: absorption, distribution, metabolism, and excretion. During this process drugs pass through a number of membrane barriers and cell monolayers including epithelial cells, intestinal mucosa, and alveolar epithelium. Due to their low membrane permeability, hydrophilic and charged substances require carrier-mediated transport to penetrate cell membranes.
Increasing evidence underlines the importance of membrane transporters like OCT1 in drug disposition and response (Gong and Kim, 2013; Tzvetkov et al., 2016). The route of orally administered drugs through the body is influenced by various factors upon administration: transporters located in the intestine determine the reabsorption of a drug and its uptake in the circulation system. At this point pre-systemic elimination via bile excretion can already negatively influence the bioavailability of a drug (Sparreboom et al., 1997). Furthermore, transporters localized in the liver and kidneys mediate drug clearance.
Transporters located in membranes of the blood-brain barrier or the blood-placental barrier, at so called immune-privileged sites, stringently restrict access of drugs to these special compartments (Kim et al., 1998; de Boer et al., 2003; Molsa et al., 2005; Vahakangas and Myllynen, 2009). Considering efflux pumps they decrease the intracellular concentration and may impair drug efficacy. Finally, co-administered drugs may affect pharmacokinetics as they can act as inhibitors of transporters (Shitara et al., 2003). Therefore, next to the expression profile of a transporter in various tissues and its substrate specificity, factors like genetic variability, cooperation with other transport systems as well as drug-drug interactions play a crucial role when analyzing the pharmacokinetics and pharmacodyna-mics of a drug.
The uptake and excretion of positively charged drugs and weak bases at physiological pH is mediated by members of the organic cation transporters of the SLC22 family (solute carrier family 22) as well as by members of the multidrug and toxin extrusion family SLC47 (recently reviewed in (Motohashi and Inui, 2016)).
The SLC22 family is part of the major facilitator superfamily (MFS), which is one of the largest families of membrane transporters, next to the ATP-binding cassette superfamily (ABC family). The MFS family comprises 74 families containing uniporters, symporters, and antiporters (Reddy et al., 2012). Members of the MFS are found in bacteria, archaea, and eukaryotes (Pao et al., 1998). In contrast to the ABC family, members of the MFS are single polypeptide secondary active transporters that only transport small molecules in response of a chemiosmotic ion gradient (Pao et al., 1998).
The SLC22 family of the MFS contains organic cation transporters (OCTs), organic zwitterionic and cationic transporters (OCTNs), and organic anion transporters (OATs) (for a recent overview see (Koepsell, 2013)). The organic cation transporters of the SLC22 family comprise three transporters: OCT1, OCT2, and OCT3. These three transporters are characterized by electrogenic, reversible, and Na+-independent transport (Gorboulev et al., 1997; Nagel et al., 1997; Koepsell and Endou, 2004). All three transporters OCT1, OCT2, and OCT3 are poly-specific. They are characterized by different but partially overlapping substrate specificities (Nies et al., 2011; Hendrickx et al., 2013; Sala-Rabanal et al., 2013;
Ciarimboli, 2016). OCT1, OCT2, and OCT3 are tissue-specifically expressed: Whereas OCT1 is predominantly expressed in the sinusoidal membrane of hepatocytes (Figure 1.4), OCT2 is specifically expressed in the basolateral membrane of tubular epithelial cells (Zhang et al., 1997; Motohashi et al., 2002; Nies et al., 2009; Tzvetkov et al., 2009). In contrast, OCT3 transcripts are detectable in several tissues (Zhang et al., 1997; Wu et al., 2000; Motohashi et al., 2002; Nies et al., 2009).
OCT1 is a facilitated diffusion system whose transport mechanism is described with the help of the alternating access model (Figure 1.1) (Volk et al., 2009): This model suggests that the binding site of OCT1 is accessible from both sides of the membrane. For translocation of a substrate from the extracellular to the intracellular side, the substrate needs to be bound to the extracellular facing substrate binding site of the transporter.
During translocation of the substrate across the plasma membrane the transporter passes through a state described as “occluded state”, in which the substrate is enclosed by the transporter. After the transporter achieved its inward-facing conformation the substrate is released to the intracellular side. Finally, the empty transporter flips back to its outward-facing conformation (Koepsell, 2011). While the transporter resides in the open outward or open inward conformation, the individual transmembrane domains are not bent. Instead,
the translocation process of a bound substrate, especially when concerning the occluded state, requires large structural changes of the transporter (Gorbunov et al., 2008;
Egenberger et al., 2012).
There were no restrictions in vitality and fertility in Oct1-/- knock out mice (Jonker et al., 2001), but differences in the pharmacokinetics of drugs, exogenous substances, and toxins were reported (Shu et al., 2007; Nies et al., 2008; Chen et al., 2014). Hence, although membrane transporters just seem to be a small part of the complex interplay of different factors that need to be considered when evaluating the profile of a drug, membrane transporters represent a key role as they mediate the first step of metabolism and excretion of a drug.
Figure 1.1 (previous page) Structure and function of OCT1. (A) The proposed secondary structure of OCT1. The OCT1 protein consists of 12 transmembrane helices with an intracellularly located N-terminal and C-terminal end. The big extracellular loop between the 1st and 2nd transmembrane domain contains putative glycosylation sites (ψ). The big intracellular loop between the 6th and 7th transmembrane domain contains putative phosphorylation sites (P). In rat Oct1 the amino acids F160, W218, Y222, T226, R440, L447, Q448, and D475 were reported to be located in the substrate binding region and to be involved in substrate binding and/or translocation (Gorboulev et al., 1999; Popp et al., 2005; Gorbunov et al., 2008; Volk et al., 2009). The corresponding amino acids in the human ortholog are indicated with green triangles. The corresponding amino acids in the human ortholog are: F159, W217, Y221, T225, R439, L446, I447, and D474. (B) Schematic representation of the alternating access model: During the outward open conformation a substrate binds at the binding cleft. This induces conformational changes leading to translocation of the substrate to the intracellular side and thereby the transporter passes through an occluded state. The substrate dissociates from the binding site and is released to the cytosol. The transporter flips back from its inward open conformation to the extracellular side. Based on (Koepsell and Keller, 2016). (C) Homology model of the outward and inward open conformation of rat Oct1. Outward open conformation of rat OCT1 from the side and from extracellular showing the substrate binding cleft. Inward open conformation of rat OCT1 from the side and from the intracellular showing the substrate binding cleft. The amino acids F160, W218, Y222, R440, L447, Q448, and D475, which are known to be involved in transport mechanism, are labeled in the substrate binding cleft shown from the extracellular and intracellular side. The transmembrane domains are colored as indicated. The 3D model of rat Oct1 is based on the crystal structure of LacY of E.coli (Popp et al., 2005; Gorbunov et al., 2008). The PDB file was kindly provided by Prof. Thomas Mueller from the University of Würzburg. Presentation and editing was made using Swiss Pdb Viewer v4.1
Rat Oct1 was the first gene of SLC22 transporters cloned and characterized in 1994 (Grundemann et al., 1994). The human OCT1 gene was cloned together with OCT2 in 1997 (Gorboulev et al., 1997; Zhang et al., 1997). The human OCT3 gene was cloned in 1998 (Grundemann et al., 1998). The human OCT1 protein shares 71 % and 50 % identical amino acids with the two other human paralogs OCT2 and OCT3, respectively (OCT1:
NP_003048.1, OCT2: NP_003049.2, OCT3: NP_068812, protein alignment using http://blast.ncbi.nlm.nih.gov). The secondary structure of the OCT1 protein is characterized by 12 transmembrane helices with an intracellularly located amino and carboxyl terminus (Figure 1.1).
In human, the genes encoding OCT1, OCT2, and OCT3 are clustered together on the long arm of chromosome six (6q26-q27). Each gene compromises 10 introns and 11 exons. The OCT1 protein is characterized by a pseudosymmetric structure, which is common for all members of the MFS. Both, the N-terminal and C-terminal part, comprise six
transmembrane domains (Koepsell and Keller, 2016). There is a big extracellular loop between the first and second transmembrane helix in OCT1 containing putative glycosylation sites according to the motive N-X-S/T at position N71, N96, and N112 (X means any amino acid) (Zhang et al., 1997). The largest intracellular loop between the 6th and 7th transmembrane domain contains putative phosphorylation sites at position S285, S291, T327, T340, and T524 (Gorboulev et al., 1997; Zhang et al., 1997).