The intricate processes within the human body rely heavily on the precise movement of molecules across cellular membranes. Among these molecules, cationic and anionic species play pivotal roles in numerous physiological functions, ranging from waste elimination to neurotransmission. Understanding how these charged species are transported is crucial, and the SLC22 family of transporters, encompassing Organic Anion Transporters (OATs) and Organic Cation Transporters (OCTs), emerges as a key player in this domain. This article delves into the fascinating world of OATs and OCTs, exploring their mechanisms in facilitating the Transport Of Cationic And Anionic Species, their structural features, and the critical amino acid residues that govern their function.
Organic Anion Transporters (OATs): Mediating Anion Transport
Organic Anion Transporters (OATs), known as Oats in rodents, are encoded by the SLC22 gene family and are recognized for their broad substrate specificity. These transporters are responsible for the movement of a diverse array of relatively small and water-soluble anionic compounds. This includes vital molecules such as steroid hormone conjugates and biogenic amines, as well as a variety of drugs and environmental toxins [86]. To date, nine OAT family members have been identified in humans, designated OAT1 through OAT7 and OAT10 (encoded by genes SLC22A6, 7, 8, 11, 10, 16, 9, and 13 respectively) and URAT1 (encoded by SLC22A12) [87].
Among the OAT family, OAT1 and OAT3 are the most extensively studied. These transporters are classified as ‘tertiary active’ transporters. This designation stems from their unique transport mechanism: they move organic anions against their concentration gradient, even against a negative membrane potential. This is achieved through an exchange mechanism, where the influx of an organic anion is coupled with the efflux of α-ketoglutarate. The crucial gradient of α-ketoglutarate, driving this exchange, is maintained by a secondary active sodium-dicarboxylate co-transporter. This co-transporter, in turn, harnesses the sodium gradient established by the primary active Na+/K+ ATPase [86]. This intricate interplay highlights the energy-dependent nature of OAT-mediated transport of anionic species.
Organic Cation Transporters (OCTs) and Zwitterion/Cation Transporters (OCTNs): Facilitating Cation Transport
In addition to OATs, the SLC22 family also includes Organic Cation Transporters (OCTs), specifically OCT1, OCT2, and OCT3, and the organic zwitterion/cation transporters (OCTNs) like OCT6, OCTN1, and OCTN2. These transporters are essential for the transport of cationic species across epithelial membranes throughout the body. OCTs exhibit remarkable versatility in their substrate recognition, accommodating a wide range of structurally diverse small organic cations. Substrates include steroids, hormones, neurotransmitters, and a multitude of pharmaceuticals and xenobiotics.
OCTs function through facilitated diffusion, a passive transport mechanism. They mediate the movement of organic cations down their electrochemical gradients, meaning transport can occur in either direction depending on the prevailing electrochemical forces [86]. Notably, the uptake function of OCTs is independent of both sodium and pH gradients. However, the affinity of OCTs for certain substrates can be influenced by the degree of ionization of the substrate, leading to enhanced uptake at reduced pH [88]. In contrast to OCTs, OCTNs operate as Na+/zwitterion cotransporters [87], indicating a distinct mechanism for transport of cationic species in this subgroup.
Structural Insights into OAT/OCT Transporters: The Role of Transmembrane Domains
Members of the SLC22 family, including OATs and OCTs, share a predicted structural topology characterized by 12 transmembrane domains (TMs). In this model, both the amino and carboxyl termini of the protein are located within the intracellular space [89]. A prominent feature is a large extracellular loop situated between TM1 and TM2, and another significant intracellular loop positioned between TM6 and TM7.
To gain a deeper understanding of the structural basis of OAT function, a theoretical three-dimensional model was developed for human OAT1 (hOAT1). This model was built based on the crystal structure of the glycerol 3-phosphate transporter (GlpT) from Escherichia coli. The model revealed that helices 5, 7, 8, 10, and 11 are clustered around a potentially electronegative active site of the transporter. This active site is oriented towards the cytoplasm and is surrounded by specific amino acid residues: Tyr230 (TM5), Lys431, and Phe438 (TM10).
Studies involving alanine substitutions of these residues demonstrated their critical importance. Replacing Tyr230, Lys431, or Phe438 with alanine significantly reduced membrane expression levels of OAT1. Further investigation using conservative substitutions revealed substrate-specific effects. For instance, K431R and F438Y mutants showed a substantial decrease (approximately 70%) in the uptake of p-aminohippurate (PAH), while PAH transport by the Y230F mutant was comparable to the wild-type OAT1. However, all mutants exhibited significantly reduced transport of cidofovir, another hOAT1 substrate, which has a different affinity (Km value) compared to PAH.
Kinetic analysis of the Y230A and F438Y mutants provided further insights. Y230A showed a decreased Vmax for PAH uptake, while F438Y exhibited reduced Km and Vmax for cidofovir transport. These findings collectively suggest that these specific amino acid residues – Tyr230, Lys431, and Phe438 – are crucial for the interaction of hOAT1 with its substrates [90].
Further investigation into TM1 of OAT1 identified additional critical amino acid residues. Substitution of Leu30 with glycine, alanine, valine, or isoleucine showed that progressively increasing the size of the side chain at this position gradually restored both cell surface expression and uptake function of the transporter. This suggests that Leu30 is important for directing OAT1 to the plasma membrane. In contrast, substitutions at Thr36, whether conservative or non-conservative, did not affect cell surface expression. However, all Thr36 mutants showed negligible PAH transport function, indicating that both the methyl and hydroxyl groups of Thr36 are essential for OAT1 activity [91].
The role of GXXXG motifs within TMs, known to be important for protein processing and oligomerization, has also been studied in OAT1. Mutation of Gly144 and Gly148 (GXXXG motif in TM2) in human OAT1 resulted in a complete loss of both surface and total cellular expression of the transporter, leading to a loss of PAH uptake function. Similarly, alanine replacement of Gly227 (GXXXG motif in TM5) significantly reduced PAH uptake due to decreased surface and total protein expression. Kinetic analysis of the G227A mutant revealed a decrease in the maximum transport velocity (Vmax) [92].
Alanine-scanning mutagenesis of TM7 in OAT1 identified four critical residues: Trp346, Thr349, Tyr353, and Tyr354. Y353A and Y354A mutants showed a loss of PAH uptake function, while W346A and T349A exhibited dramatically reduced total and cell surface expression. Further investigation using the proteasome inhibitor MG132 indicated that Trp346 and Thr349 may be crucial for maintaining the stability of OAT1 [93].
TM12 has also been implicated in the stability and maturation efficiency of human OAT1. Replacing Tyr490 with alanine significantly reduced protein expression and uptake function. Conservative substitutions with tryptophan or phenylalanine partially or fully restored protein expression and transport activity, respectively. Pulse-chase labeling and treatment with protease inhibitors revealed that degradation of the Y490A mutant was accelerated, although its maturation efficiency was not affected. In addition to Tyr490, simultaneous mutation of Leu503 and Leu504 to alanine also dramatically affected transporter protein expression. Further studies demonstrated that the double mutant Leu503/504A was retained in the endoplasmic reticulum (ER) without maturing, suggesting that these residues are important for promoting the maturation efficiency of OAT1 [94].
Studies on conserved glycine residues in OAT4 showed that replacing Gly241 (TM5) and Gly400 (near TM8) with serine almost completely abolished estrone sulfate (ES) transport function. Substituting Gly241 and Gly400 with amino acids with progressively larger side chains (alanine, valine, leucine) resulted in increasingly impaired ES uptake and reduced cell surface expression. Biotinylation-labeling and immunocytochemical analysis suggested that mutations at these positions affected the trafficking of the transporter to the plasma membrane. Furthermore, G241A and G400A mutants exhibited decreased Vmax and increased Km for ES, indicating that both Gly241 and Gly400 are important for transporter targeting to the cell surface and substrate binding [95]. A mutation study focusing on histidine residues in OAT4 revealed that His469 in TM11 is responsible for the inhibitory effect of the histidine-modifying agent diethyl pyrocarbonate (DEPC). While the H469A mutant still transported ES, its ES uptake was no longer affected by DEPC treatment, suggesting that His469 is a binding site for DEPC within the hOAT4 structure [96].
Amino acid residues within the TMs of OCTs are equally critical for their proper function. However, research in this area has been more focused on rodent OCTs, with fewer studies directly examining human OCTs. Homology modeling, based on the crystal structure of the lactose permease LacY from Escherichia coli, suggests that substrates may interact with rat OCT1 within a region rather than at a single binding site [87]. This model proposes that Trp218, Tyr222, and Thr226 in TM4 [97], Ala443, Leu447, and Gln448 in TM10 [98], and Asp475 (TM11) [99] surround a large cleft that opens to the intracellular domain [97].
Replacement of these residues in rat OCT1 led to a decreased Km value for tetraethylammonium (TEA), and altered Km values for 1-methyl-4-phenylpyridinium (MPP+) were observed in most mutants. Interestingly, by replacing Ala443, Leu447, and Gln448 in rOCT1 with the corresponding residues from rOCT2, the affinity of rOCT1 for corticosterone (approximately 150 μM) increased to a level comparable to rOCT2 (approximately 4 μM). This suggests an allosteric interaction between cation substrates and corticosterone at these positions [98]. Furthermore, replacing Gly478 in TM11 of rat OCT1 with cysteine affected substrate interactions and voltage-dependent movements of TM5, 8, and 11. This indicates that Gly478 resides within a mechanistically important hinge domain of TM 11, which is essential for structural changes induced by substrate binding [100].
Homology modeling of rabbit OCT2 has also been used to guide site-directed mutagenesis studies of OCT/substrate interactions [101]. A conserved glutamate residue in TM10 was found to be located in a putative docking region within a hydrophilic cleft of the transporter protein. Biochemical analysis of the E447Q mutant revealed a shift in substrate specificity towards an OCT1-like phenotype. Replacing Glu447 with oppositely charged amino acids resulted in a loss of transport function. However, the E447L mutant showed reduced uptake of TEA and cimetidine, but not MPP+, indicating substrate-specific effects [101].
A secondary structure model of human OCT2, based on the rabbit OCT2 homology model, was used to examine thirteen cysteine residues, six in the extracellular loop and seven in TMs. Accessibility studies using hydrophilic thiol-reactive reagents revealed that Cys474 in TM11 may be adjacent to or involved in forming a transport pathway for substrates like TEA [102]. The importance of Cys474 in human OCT function was further confirmed by mutating this residue to alanine or phenylalanine. Both C474A and C474F mutants exhibited significantly reduced Kt and Jmax values for TEA uptake, and C474F completely failed to transport TEA. In contrast, only Jmax was decreased, while Kt remained unchanged for MPP+ transport in both C474A and C474F mutants, suggesting that Cys474 is specifically involved in the binding surface for TEA [103].
Studies on human OCTNs have identified functionally important TM regions. OCTN2, for example, transports carnitine in a sodium-dependent manner and organic cations in a sodium-independent manner. A single amino acid mutant (S467C in TM11) with impaired carnitine transport but retained organic cation transport activity was identified in a Japanese population. Further study showed that the Km value for carnitine was increased approximately 15-fold in this mutant compared to the wild-type. This selective reduction in carnitine transport in S467C was attributed to an altered affinity of a specific site that recognizes an anionic moiety for carnitine, but not for organic cations. These results suggest that OCTN2 interacts with carnitine at a site that overlaps but is distinct from the binding site for organic cations, and that an anion recognition site around TM11 is important for carnitine recognition [104].
P478L (TM11) is another missense mutant of OCTN2 identified in patients with primary carnitine deficiency [105]. Cells expressing this mutant lost carnitine transport function but showed significantly higher TEA transport. Conversely, the M352R (TM7) mutant, corresponding to the L352R mutation found in juvenile visceral steatosis mice [106], exhibited significantly reduced transport function for both carnitine and TEA.
Cross-species chimeric OCTN2 proteins have been generated to further dissect functional domains. These studies suggest that the sites responsible for carnitine transport and TEA transport are spatially separated. The N-terminal 122 amino acids and the region containing amino acids 240–449 are implicated in TEA transport, while the region containing amino acids 123–239 and the C-terminus are involved in carnitine transport [107]. By creating chimeric proteins of human OCTN2 (hOCTN2) and mouse OCTN3, it was found that TM1–7 of hOCTN2 is important for both organic cation uptake and sodium-dependent carnitine transport. Further studies pinpointed Gln180 in TM3 and Gln207 in TM4 as playing crucial roles in sodium dependency, but these residues did not appear to be involved in the recognition and transport of the OCTN2 substrate TEA [108]. These findings reinforce the idea that in OCTN2, the sites for organic cation transport and carnitine transport are overlapping but not identical [106].
Another study involving substitution of the C-terminus of OCTN2 (residues 342–557) with the corresponding region from OCTN1 resulted in a loss of carnitine transport function. Single replacements of Arg341 (TM7), Leu409 (TM9), Leu424, and Thr429 (TM10) with the corresponding amino acids from OCTN1 led to reduced carnitine transport (approximately 40% of wild-type activity). Further analysis showed that the triple mutant R341W/L409W/T429I exhibited dramatically decreased carnitine transport and an increased Km for the substrate. The involvement of these amino acids in carnitine transport highlights the crucial role of transmembrane domains in the recognition and transport of carnitine by OCTN2 [109].
Conclusion
The transport of cationic and anionic species is indispensable for maintaining cellular homeostasis and supporting a vast array of physiological processes. The SLC22 family transporters, particularly OATs and OCTs, are central to this transport. OATs facilitate the movement of anionic compounds through tertiary active transport, while OCTs and OCTNs mediate the facilitated diffusion of cationic species. Detailed investigations into the structural features of these transporters, especially the roles of specific amino acid residues within their transmembrane domains, have revealed critical insights into their mechanisms of action, substrate specificity, and cellular regulation. Further research in this area will continue to refine our understanding of these vital transporters and their implications in health and disease, potentially paving the way for targeted therapeutic interventions.
