cations as well [36–39]. Various homology‐based approaches have been used to identify additional Oat family members. For example, Oat3/Slc22a8 was originally identified in a differential display assay comparing gene expression in wild‐type and mice homozygous for the juvenile cystic kidney mutation [40]. Initially identified as Roct (reduced in osteosclerosis transporter), this transporter was then found to share a high degree of identity with Oat3/Slc22a8 genes cloned from both rat brain and human kidney [41, 42]. Meanwhile database searches for clones with homology to Oat family members led to the identification of Oat4/Slc22a11, expressed in the kidney and placenta [43]. In silico homology‐based analyses resulted in the identification of Oat5/Slc22a19 [44] and Oat6/Slc22a20 [45] (which was found, intriguingly, to be expressed mainly in the olfactory mucosa). Somewhat similar approaches led to the identification of other OATs and other transporters (Table 4.1) [9, 10,14–26].
4.1.3 Nomenclature
The nomenclature of the OATs can be challenging. This is largely due to the history of their discovery. For example, as described above, the original Oat1 cloned from mouse kidney in 1996 was initially identified as an organic ion transporter due to its high homology to Oct1 and Nlt and was termed Nkt (novel kidney transporter) [30–32]. The homology of these transporters raised the notion that these transporters might be representatives of a new transporter subfamily [32], now called SLC22 [8], and consisting of over 30 members in mammals depending on which species is being considered, about half of which appear to be OATs [5, 6, 46]. In addition, a number of so‐called unknown substrate transporters (Usts) with similarity to the SLC22 family have also been identified, many of which appear likely to be functionally more similar to the OAT group than other SLC22 groups [6, 47].
4.2 MOLECULAR CHARACTERIZATION
4.2.1 Genomics
A relatively unique feature of the SLC22 family of genes is that many members tend to appear as pairs or clusters in the genome [48–51]. For example, some of the known OATs are found in three tightly linked pairs (i.e., as adjoining neighbors without other genes interposed between them): SLC22A6—SLC22A8, SLC22A9—SLC22A10, and SLC22A11—SLC22A12. The SLC22A6—SLC22A8 pair is found as a tandem repeat on human chromosome 11q12.3. In addition, SLC22A6 and SLC22A8, together with other less well‐characterized transporters in the SLC22 family (e.g., SLC22A9, SLC22A10, SLC22A24, and SLC22A25), are found clustered together in a 0.6 megabase region on human chromosome 11 in order from centromere to telomere [6]. The SLC22A11—SLC22A12 pair is also found in a tandem repeat (separated by less than 20 kilobase pairs) and is also located on human chromosome 11 (11q13.1), but it is about a million base pairs away from the SLC22A6—SLC22A8 cluster. Nevertheless, these observations raise the possibility that the chromosomal arrangement of these OAT genes might exist to facilitate the coordinated transcription (co‐regulation) of pair‐members [5, 6, 50].
4.2.2 Protein Structure
OATs not only share high sequence homology, but they share many structural characteristics as well, including a sequence length of 500–600 amino acids, 12 transmembrane domains (TMDs) (composed of two 6 transmembrane domains connected by a long intracellular loop), and cytosolic N and C termini (Fig. 4.1) [13, 52]. Many structure–function analyses have been performed for various OATs, though no crystal structures are available. There are two large interconnecting loops, one between TMD 1/2 and another between TMD 6/7. The first large extracellular loop contains multiple consensus N‐glycosylation sites, as well as conserved cysteine residues [32]. The large intracellular loop, along with the intracellular carboxy terminus, contains several potential phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) [53], casein kinase II, and tyrosine kinase [13, 32]. The functional importance of kinases in the regulation of OAT function is described later in this chapter.
FIGURE 4.1 Schematic illustrating the transmembrane topology of organic anion transporters. Hydropathy analyses indicate that the OATs comprise twelve TMDs (numbered in the figure). The large loops between TMD 1 and 2 (extracellular) and TMD 6 and 7 (intracellular) contain several consensus glycosylation (G) and PKC phosphorylation (P) sites, respectively. Amino acid residues that are critical for OAT function are H (histidine), Y (tyrosine), F (phenylalanine), W (tryptophan), K (lysine), and R (arginine).
With permission from Ref. 49.
4.2.3 Mechanism of Substrate Translocation
OAT‐mediated counter‐transport is driven by the concentration gradient of intracellular dicarboxylates, which are exchanged for extracellular anions. This is ATP‐independent. Nevertheless, the high concentration gradient of dicarboxylates is maintained by the sodium‐dicarboxylate co‐transporter, which in turn is driven by the inwardly directed sodium gradient generated by Na+/K+‐ATPase [54, 55] Recent studies using proximal tubule cells overexpressing OAT1 and OAT3 have also revealed that the expression of these OATs results in an oxidative phenotype characterized by decreased extracellular lactate and unchanged intracellular ATP [56]. While there is little direct data on the molecular interactions of substrate ions with amino acid residues lining the channel of the OATs which might indicate the exact properties that determine substrate binding, mutagenesis studies have been conducted targeting highly conserved amino acid residues that appear to be important for transport function [57]. Certain conserved basic residues are believed to be a major component in the determination of the substrate charge specificity of the OATs. Interestingly, a rat Oat3 double mutant with the Lys370 and Arg454 residues substituted by one neutral and one acidic residue (K370A/R454D) has been reported to change its substrate orientation from anions to cations [58]. Mutagenesis studies provide some insight into how these highly similar transporters discriminate between structurally similar compounds [59].
Although the structure of the OATs has yet to be defined, the crystal structure has been determined for related MFS proteins found in bacteria, including the glycerol‐3‐phosphate transporter from the Escherichia coli inner membrane (a G3P/Pi antiporter, GlpT), the E. coli lactose permease (a lactose/H+ symporter, LacY), and the oxalate transporter from Oxalobacter formigenes (an oxalate/formate antiporter, OxlT) [1]. These transporters have similar topology, raising the possibility that other MFS proteins, including the OATs, might share similar structural designs. Therefore, the resolved structure of these bacterial transporters, with substrate‐binding sites located at the interface between the N‐ and C‐terminal halves of the protein, have been suggested as templates for structural modeling of the OATs and other SLC22 transporters. Along these lines, modeling of human OAT1—based on the tertiary structure of GlpT—revealed a large putative active site open to the cytoplasm, as well as identifying two amino acid residues important for substrate interaction [60]. Computational modeling has also been conducted using a model of OAT1 embedded within an artificial phospholipid bilayer [61].
4.3 EXPRESSION AND REGULATION OF OATS
4.3.1 Tissue Distribution
While Oats were initially shown to be predominantly expressed in the kidney [32], it is now clear that these transporters are found in most barrier epithelia of the body, including the choroid plexus, blood–brain barrier, biliary tract, intestine, retinal–blood barrier, the nose–brain barrier, blood–testis barrier, the maternal‐fetal barrier among others (
