adrenal gland, skin, adipose, kidney
2.2.2 Structure–Function Relationships
In human, the genes SLC22A1, SLC22A2, and SLC22A3, which encode OCT1, OCT2, and OCT3, respectively, are localized within a cluster on chromosome 6q26‐27 [1]. Each of these genes comprise 11 exons and 10 introns. OCT1–3 contain 554, 555, and 556 amino acids, respectively. hOCT1 and hOCT2 are approximately 70% identical in amino acid sequence, whereas hOCT3 shares 50% sequence identity with hOCT1 and hOCT2 [1]. The precise mechanism of binding and transport is not fully uncovered due to the lack of a high‐resolution crystal structure [10]. The predicted 2 and 3D structures of hOCTs consist of a typical major facilitator superfamily (MFS) fold of 12 α‐transmembrane helix domains arranged in a barrel‐shaped structure with a large cleft that opens in cytoplasm. The modeled 3D structures of all hOCTs are based on the crystal structure of the human GLUT3 (SLC2A3) transporter and display an outward‐open conformation and putative cyclic C1 protein symmetry [10]. The NH2‐ and COOH‐terminal ends of the OCTs are intracellular [1]. All three transporters contain a large (100+ amino acids) extracellular loop between transmembrane domain (TMD) 1 and TMD2 and a relatively large intracellular loop between TMD6 and TMD7 [1, 10]. The large extracellular loop contains N‐glycosylation sites (Asn‐Xaa‐Ser/Thr) and cysteine residues, features indicative of putative roles in drug binding and uptake. The large intracellular loop contains several predicted sites for protein kinase C (PKC)‐dependent phosphorylation. Phosphorylation of these sites changes substrate selectivity [1] [10]. In addition, homology models of inward‐facing and outward‐facing tertiary structures of OCTs have been generated based on E. coli transporters, lactose permease LacY and the glycerol‐3‐phosphate transporter GlpT [11]. The transmembrane domains and, in particular, the 4th and 10th transmembrane domains are thought to be critically involved in substrate recognition by the OCTs, and differences between the three isoforms in terms of substrate specificity may be related to differences in these regions. Extensive site‐directed mutagenesis followed by functional characterization of mutants has indicated that transported organic cations bind to amino acids in the innermost cavity of the outward open binding cleft. The binding sites for different transported organic cations are overlapping but nonidentical so that exchange of one amino acid in this innermost cleft may change affinity for one substrate but not another [11]. These results suggest that OCT1, and likely all of the OCTs, contains multiple overlapping but nonidentical recognition sites for the various structurally diverse substrates. Further mutational analyses in OCT1 and OCT2 support the occurrence of a complex binding pocket in these transporters. The binding pocket might appear in inward‐ or outward‐oriented conformation and these conformations can differ in substrate affinity [12]. On the basis of the uptake studies for hOCT2, a model has been suggested where two substrates can bind simultaneously to the transporter. Upon binding, the resulting transporter/substrate1/substrate2 complex cannot be translocated [13], suggesting an inhibition mechanism. However, it is important to note that given the broad substrate selectivity of the OCTs, the key domains or residues involved in substrate recognition may differ by substrate, even within the same protein.
2.2.3 Transport Mechanism
OCT1–3 function as uniporters (Fig. 2.2b), facilitating diffusion of substrates across the plasma membrane. Transport can be bidirectional, depending on substrate, and is driven by electrochemical gradients. The OCTs share several common features related to transport mechanism. First, modeling and mutational analysis suggest that OCTs follow an alternating‐access transport model. The substrate binds to the outward‐open conformation of the transporter, which induces a conformational change. Then the substrate–transporter complex passes a transient occluded state to the inward‐open conformation. Lastly, the substrate is released to the cytoplasm and the transporter returns to the outward open conformation [12] (Fig. 2.2a). The structural changes of OCTs during the transport cycle require a rigid body movement of the six N‐terminal TMDs against the six C‐terminal TMDs, and a hinge domain in TMD 11 is crucial for this movement [11, 14]. Second, the translocation of organic cations by OCTs is electrogenic and independent of sodium and chloride ions [11]. The net transport of organic cations is driven by the intracellular negative membrane potential and the concentration gradient. Positively charged cations are taken up into cells according to the electrochemical gradient, and this process is membrane sensitive. Artificially modulating the membrane potential by replacement of extracellular Na+ with K+ changes the rate of transport by OCTs [15]. Third, the transport direction of OCTs is bidirectional, and as noted, net transmembrane flux is dependent on the electrochemical gradient. In addition to cation influx, OCTs acting as efflux transporters have been demonstrated in multiple studies [1]. Fourth, OCT1‐3, defined as “poly‐specific” OCTs, can transport a variety of substrates with diverse molecular structures. As such, their substrates tend to have higher K m values than those of the substrates for the more specific transporters, such as the neurotransmitter transporters (SLC6). In addition, OCT1–3 can be inhibited by a large number of compounds that are not transported. Common substrates of all OCTs are relatively low molecular mass (below 500 g/mol) and hydrophilic organic cations such as the prototypical cation tetraethylammonium (TEA), the neurotoxin MPP+, and the endogenous compound N‐methylnicotinamide (NMN). Several clinically important drugs have been shown to interact with all of the OCTs, including the antidiabetic drug metformin. Besides this, endogenous compounds such as the biogenic amine neurotransmitters (i.e., dopamine, epinephrine, norepinephrine, histamine, and serotonin) have been shown to interact with one or more OCT transporters [1]. Although the OCT family shows broad overlap in substrate
