Читать книгу Drug Transporters - Группа авторов - Страница 32

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.