The SLC22A family of genes encodes the OCT (Organic cation transporter) family of proteins, which include OCT1-3 (SLC22A1-3), and OCTN1 (SLC22A4) and OCTN2 (SLC22A5).
1.4.4.1 Tissue distribution
The OCT1 (SLC22A1) protein is predominately expressed at the basolateral surface of hepatocytes (Nies at al, 2008) and the apical surface of lung epithelia (Lips et al, 2005). In situ hybridisation techniques failed to identify expression in the kidney. Low levels of OCT1 mRNA expression have been detected in the heart, brain and placenta (Gorboulev et al, 1997). OCT2 protein expression is mainly localised to the convoluted tubules of the kidney (Gorboulev et al, 1997). The spleen and small intestine have been shown to express low levels of OCT2 (SLC22A2) expression. OCT3 is the most ubiquitously expressed OCT family drug transporter, and is expressed at protein level in hepatocytes (Nies et al, 2009), at the luminal surface of the lungs (Lips et al, 2005) and also in trophoblasts (Sata et al, 2005).
OCTN1 mRNA expression is found in the bone marrow, kidney and trachea. Protein expression has been confirmed on the apical surface of the enterocytes and epithelial cells lining the proximal tubule (Tamai et al, 1997). OCTN2 protein expression has been confirmed at the apical borders of the proximal tubules (Masuda et al, 2006) and syncytiotrophoblasts (Grube et al, 2005), while OCTN2
mRNA expression is strongly expressed in the heart, placenta, kidney and pancreas (Wu et al, 1998).
1.4.4.2 Substrate specificity
OCT family proteins translocate organic cations by facilitated diffusion along an electrochemical gradient. This transport is not mediated by sodium exchange but can be influenced by pH. The pH can affect the ionisation of a substrate and hence change its affinity for a specific OCT binding site. The OCT family of drug transporters transport monoamine neurotransmitters, catecholamines and steroid hormones as endogenous substrates. 1-methyl-4-phenylpyridinium (MPP) is a common substrate for OCT1-3, while Tetra ethyl ammonium (TEA) is transported by OCT1 and OCT2, but not by OCT3 (Grundemann et al, 1998). Studies have shown OCT1 binding to substrates is stereoselective, and a pharmacophore model based on 22 diverse substrates revealed the importance of a positive ionisable site, a hydrophobic site and two hydrogen acceptor sites for substrate interaction. However, this model does not fit all substrates, and dopamine and thiamine only have two interactive binding sites with the pharmacophore model (Moaddel R et al, 2007).
OCTN1 and OCTN2 transport both cations and carnitine. The endogenous substrate for OCTN1 is ergothioneine, although TEA and L-carnitine are also transported by OCTN1 but with less efficiency. Ergothioneine is an antioxidant though its role in the body is not clear (Gründemann D et al, 2005). Carnitine and TEA are also transported by OCTN2, but the substrate binding sites vary as certain mutations affecting OCTN2 inhibit carnitine transport but have no effect on TEA transport. Ergothioneine was poorly transported by OCTN2 (Seth et al, 1999).
1.4.4.3 Regulation of OCT expression
The regulation of OCT occurs both at a transcriptional and protein level, and varies with respect to tissue distribution and the specific drug transporter. In the promoter region of hOCT1, there are two response elements which interact with HNF4α. Both of these elements are vital for OCT1 transcription, as site mutagenesis reduced the expression of hOCT1. Small heterodimer partner (SHP), which is a bile acid inducible transcriptional repressor, can also inhibit hOCT1 transcription (Saborowski et al, 2006). OCT2 mRNA expression level was noted to increase with dexamethasone and hydrocortisone, but reduced with oestradiol. This would suggest the OCT2 promoter regions may contain androgen receptor elements (Shu et al, 2001). OCTN1 (SLC22A4) regulation is affected by RUNX1, a transcriptional repressor, which can bind to the intronic SNP of OCTN1 (SLC22A4) and reduce transcription of the SLC22A4 gene (Tokuhiro S et al, 2003). The PDZ proteins are known to interact with both OCTN1 and OCTN2, and help with protein stabilisation at the luminal surface of enterocytes. One such PDZ protein, PDZK1, has been shown to enhance substrate translocation when interacting with OCTN2 (Kato et al, 2007).
1.4.4.4 OCT transporter structure
The OCT family of drug transporter proteins vary in size from 543-557 amino acids in length. They have 12 transmembrane domains with the carboxyl and amine termini groups located intracellularly. The N-glycosylated sites are predominately located between the first and second transmembrane domains, while the phosphorylation sites are located in a large intracellular loop between the sixth and
seventh domains. The amino acid sequences of OCT1 and OCT2 are 70% homologous, while there is 50% homology between OCT3 and OCT1/OCT2 (Gorboulev et al., 1997). OCTN1 and OCTN2 also share similar amino acid sequences with 77% homology.
The predicted 3 dimensional structure for OCT1 has given rise to the theory substrates interact with OCT1 within a certain region as opposed to a single binding site. During substrate electrogenic facilitated transfer, the transporter undergoes 3 distinct conformational changes which include an outward facing conformation, followed by an occluded state and then finally an inward facing state (Koepsell et al., 2011).
1.4.4.5 Pathology and significance
OCT 1-3 polymorphisms have not been associated with any human diseases to date, and OCT knockout mice do not develop a distinct phenotype. Single nucleotide polymorphisms (SNPs) of hOCT1 alter functional characteristics of the protein as demonstrated by different binding affinities to known substrates when compared to the wild type (Kerb et al, 2002). However, OCTN1 and OCTN2 have been associated with familial inflammatory bowel disease (Lin Z et al., 2010) and systemic carnitine deficiency (Lahjouji et al, 2001) respectively. Mutations affecting OCTN2 impair the nephrons from reabsorbing carnitine. Carnitine is an amine which is vital for mitochondrial β-oxidation. Systemic carnitine deficiency manifests with skeletal myopathy, cardiomyopathy and hypoglycaemia. This disorder is inherited in an autosomal recessive pattern (Lahjouji et al, 2001).