Organic anion transporters

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Revision as of 13:23, 26 November 2023

Organic Anion Transporters are a group of proteins that belong to the solute carrier (SLC) superfamily, specifically categorized under the SLC22 family. These transporters are integral membrane proteins primarily found in various tissues, especially in the kidneys and the liver. OATs play a crucial role in transporting a wide range of organic anions across cell membranes, influencing the uptake, distribution, and excretion of diverse substances, including drugs, toxins, metabolites, and endogenous compounds.

History

Function

Structure

Figure 2: Molecular dynamics simulation of OAT1

The OAT family are transmembrane proteins of about 550 amino acids with intracellular and (TMDs). The transporter has 2 large loops: an one between TMD1/2 and an one between TMD6/7^[1]. Three highly conserved regions allow for substrate specificity and the activity of the transporter in this protein: the first is the loop between TMD1/2, which mediates homo-oligomerization^[2] and contains several ; the second is the intracellular loop between TMD6/7, which has phosphorylation sites and is involved in transcriptional regulation^[2]; and the third is domains 9 and 10^[1].

Among the different subtypes of OATs, the main structural differences are the number of phosphorylation sites (where, depending on the subtype, protein kinases A and C, casein kinase II, and tyrosine kinase can bind) and glycosylation sites, while maintaining the general structure among all members. The subtype that differs from the others most is OAT4, which possesses three C-terminal amino acids (threonine, serine, and leucine) that together form a PDZ binding motif. This motif is essential for the correct targeting and maintenance of the transporter in the renal cell's apical cell membrane ^[3].

Since the crystal structure of these transporters is yet unavailable, the studies carried out to observe the opening and closing mechanism have been performed on transporters homologous to these ^[4]. A transporter tilt that may be involved in the opening and closing of the transporter has been found in a molecular dynamics simulation for OAT1 based on the glycerol-3-phosphate transporter (GlpT). The opening of the carrier from the intracellular side of the membrane is depicted in this model, which displays the transporter's activity during a 100 ns period. Due to the brief duration of observation, it is not fully opened, but this tilting process may explain the initial stages of organic anion transport^[5].

Figure 2A shows the change in the conformation of the zone closest to the extracellular face, showing it at 40ns in yellow and at 90ns in black (Fiugre 2Aa). This simulation illustrates how the distance between the residues of SER139 and MET358 increases with time, as seen in Figures 2Ab and 2Ac^[5].

Figure 2B shows the change in the conformation of the zone closest to the intracellular face, with, as before, the structure at 40ns in yellow and at 94ns in black. In contrast to the extracellular zone, the distance between the residues (in this case VAL211 and GLY446) decreases with time (Figures 2Bb and 2Bc)^[5].

Thus, in a simplified scheme showing only the 4 helices that participate in the spin, the model would be shown as in Figure 2C, observing, once again, in yellow the conformation at 40ns and in black at 94ns^[5].

According to the results obtained in this model, the activation of the transporter would occur through the closure of the intracellular zone and the opening of the extracellular zone as seen in the simulations. Still, in order to examine this action more precisely, more models need to be created and the crystal structure of the transporter needs to be determined.

Mechanism of action

OATs are secondary active transporters, utilizing the energy stored in the electrochemical gradient of specific ions (like sodium or potassium) established by primary active transporters such as ATPases. The detailed breakdown of their mechanism is:

1. Substrate Binding. OATs recognize and bind to a wide array of organic anions, including endogenous substances like hormones (e.g., prostaglandins) and exogenous compounds such as drugs (e.g., antibiotics, NSAIDs). OATs bind substrates through interactions involving electrostatic forces, hydrogen bonds, and hydrophobic interactions between the transporter and the substrate molecule.

2. Conformational Changes. Upon substrate binding, the OAT undergoes conformational changes that allow it to transport the substrate across the cell membrane. These changes facilitate the movement of the substrate from the extracellular space into the cell or vice versa. The typical domains or regions within OATs that commonly undergo conformational changes are:

• Transmembrane Domains (TMDs): these domains span the lipid bilayer multiple times and contain the substrate-binding sites. Conformational changes in these regions occur upon substrate binding, altering their configuration to enable the transport of the substrate across the membrane.
• Extracellular and Intracellular Loops: these regions connect the transmembrane segments and play a role in substrate recognition, binding, and transport. Conformational changes in these loops occur as part of the overall structural rearrangement necessary for substrate translocation.
• Gate Domains or Gates: some transporters have specific gate domains or gating mechanisms that control access to the substrate-binding sites. Conformational changes in these gate domains upon substrate binding allow the transporter to open or close, regulating the entry or exit of substrates.
• Cytosolic Domains: regions facing the cell's interior may also undergo conformational changes. These cytosolic domains often interact with cellular components or signaling molecules, modulating the transporter's activity or facilitating intracellular trafficking.

3. Transport Process: OATs primarily work through a process called facilitated diffusion or exchange transport. They utilize the pre-existing electrochemical gradient of ions, typically sodium ions, established by ion pumps or cotransporters, as the driving force for substrate movement. By harnessing this gradient, OATs can transport substrates against their concentration gradient, moving them from areas of low concentration to high concentration (or vice versa), depending on the direction required.

Mechanism regulation

The regulation of Organic Anion Transporters (OATs) involves complex mechanisms that control their activity, expression levels, and localization within cells. These regulatory processes impact the transport of organic anions across cell membranes, influencing drug disposition, metabolic functions, and physiological homeostasis. The main regulation mechanisms regulating OATs are:

• Transcriptional Regulation. The expression of OAT genes is tightly controlled at the transcriptional level. Various transcription factors, such as nuclear receptors (e.g., PPARs - Peroxisome Proliferator-Activated Receptors), hormone receptors, and cytokine-induced signaling pathways, can modulate the expression of OAT genes in response to different stimuli. For instance, nuclear receptors play a role in regulating OAT expression in the liver and kidneys in response to changes in metabolic status.
• Post-Transcriptional and Post-Translational Modifications. After transcription, various mechanisms regulate OAT activity post-transcriptionally and post-translationally. This includes processes like alternative splicing, mRNA stability, and protein modifications (e.g., phosphorylation, glycosylation, ubiquitination) that can influence transporter activity, stability, and localization within the cell.
• Cellular Trafficking and Membrane Insertion. Regulation involves controlling the trafficking of OAT proteins within the cell and their insertion into the plasma membrane. Intracellular compartments, such as endosomes, lysosomes, and the endoplasmic reticulum, play roles in the sorting and trafficking of OAT proteins to their functional locations in the cell membrane. Regulation of these trafficking pathways affects the number of transporters available for substrate transport.
• Modulation by Signaling Pathways. Various signaling pathways, including those involving protein kinases, phosphatases, and G-protein-coupled receptors (GPCRs), can modulate OAT activity. Activation or inhibition of these signaling cascades can lead to changes in transporter function through phosphorylation, dephosphorylation, or alterations in transporter affinity for substrates.
• Drug-Induced Regulation. Some drugs can regulate OAT activity either by acting as substrates, inhibitors, or inducers of transporter expression. For example, certain medications may upregulate or downregulate OAT expression or function, thereby influencing their own pharmacokinetics or altering the disposition of co-administered drugs.

Inhibitors and activators

Alterations of the function

The pharmacokinetics and disposal of drugs may be significantly impacted by modifications in the activity of OATs, which can arise from a variety of causes.

Genetic polymorphisms^[6]

Examining the pharmacological effects of naturally occurring mutations in OATs is essential because they may explain different responses to drug treatment. As of right now, investigation has been conducted only into the genetic polymorphisms of OAT1-4.

• OAT1: numerous SNPs, both intronic and exonic, have been found in individuals representing the main ethno-geographical divisions. Among the results, we can observe cases, such as the R50H variant, where there is an increased uptake of acyclic nucleoside phosphonates, but no changes in the transport of para-aminohippurate (PAH) (used as a control in the study of these transporters) are identified; other variants, like K525I, P104L, or R293W, exhibit no change in function; lastly, certain variants, like R454Q, do not exhibit any uptake of PAH, but show normal renal clearance of acyclic nucleoside phosphonates.

• OAT2: compared to the other three transporters, OAT2 has far fewer SNPs, these being common among individuals of different ethnic groups. However, no major change in function has been identified in any of them.

• OAT3: the polymorphisms in this transporter that have been investigated primarily indicate changes in the transport of cimetidine and estrone-3-sulfate (ES), with some variants (like R149S or G239X) showing no uptake at all, and others (like R277W) solely showing reduced ES uptake. Nonetheless, the majority of the detected SNPs do not exhibit a change in function.

• OAT4: this transporter's polymorphisms are the most researched. Important variants include the E278K, which has decreased uptake of ES, Dehydroepiandrosterone sulfate, and ochratoxin A (OTA) due to a decrease in maximum transporting rate and transporter-substrate affinity; the L29P, R48X, and H469R variants, which have decreased uptake of ES, OTA, and uric acid; and the T392I variant, whose reduced function is caused by impaired expression of the transporter membrane.

Numerous questions, including what causes this change in function and why the alterations occur, remain unanswered in the study of genetic polymorphisms and their impacts.

Drug-drug interactions

OATs have a wide spectrum of substrate recognition, therefore when administered together, different drugs may interact—either in a competitive or non-competitive way—with the same transporters, which can lead to mutually affecting each other’s pharmacokinetic profiles^[1]. These interactions involve inhibitors and activators of OATs, discussed in the previous section.

Disease states^[1]

Kidney disorders and other diseases can significantly change the expression and activity of OATs, changing their function and ultimately impacting how the body handles different substances.

Acute kidney injury is caused by drug/toxin-induced renal toxicity and renal ischemia, and usually produces a decrease in the glomerular filtration rate and affects secretion and absorption in the renal tubules. Decreased levels of mRNA and protein expression of OAT1 and OAT3 have been observed in patients with this disease, being one of the possible explanations for this that the toxicity is caused by gentamicin, and this antibiotic down-regulates the expression of the transporters, which also contributes to the development of the disease by the reduction in renal function.

Chronic kidney failure produces a gradual decrease in glomerular filtration rate and renal clearance, resulting in the accumulation of various toxins and endogenous substances, leading to renal failure. It is believed that the buildup of toxins and metabolites is what causes the mRNA and protein expression of OAT1 and OAT3 to decline in people with this illness, inhibiting, in addition, the transport produced by the OATs.

In addition to renal diseases, alterations in OATs have been observed in patients with diabetes and cholestasis, observing a reduction in mRNA and protein expression levels of OAT1, OAT2 and OAT3 in the first condition. Cholestasis is a liver disease in which the flow of bile from the liver is reduced or obstructed, and reduced levels of protein expression of OAT1 and OAT3 have been observed.

These findings show that, in order to effectively treat patients with any of these diseases, drug dosages must be modified while accounting for the glomerular filtration rate and OAT expression.

Sex

Differences in mRNA and protein expression of OAT1, OAT2 and OAT3 have been identified between female and male mice, suggesting the possible regulatory role of sex hormones^[7]. Research has revealed that male mice's kidneys express more OAT1 and OAT3 than those of female mice, which may be a sign of androgen stimulation and estrogen inhibition. On the other hand, OAT2 expression has been found to be higher in females than in males, suggesting that androgen inhibition and estrogen and progesterone stimulation may be occurring^[8].

Despite these results, research is still ongoing to determine the reasons for the reported variations in OAT expression between the sexes as well as why these inhibitions and stimulations occur.

OAT family

References

1. ↑ ^{1.0 1.1 1.2 1.3} Zhang, J., Wang, H., Fan, Y., Yu, Z., & You, G. (2021). Regulation of organic anion transporters: Role in physiology, pathophysiology, and drug elimination. Pharmacology & therapeutics, 217, 107647. https://doi.org/10.1016/j.pharmthera.2020.107647
2. ↑ ^{2.0 2.1} Koepsell, H. (2013). The SLC22 family with transporters of organic cations, anions and zwitterions. Molecular aspects of medicine, 34(2-3), 413-435. https://doi.org/10.1016/j.mam.2012.10.010
3. ↑ Burckhardt, G., Burckhardt, B.C. (2011). In Vitro and In Vivo Evidence of the Importance of Organic Anion Transporters (OATs) in Drug Therapy. In: Fromm, M., Kim, R. (eds) Drug Transporters. Handbook of Experimental Pharmacology, vol 201. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-14541-4_2
4. ↑ Roth, M., Obaidat, A. and Hagenbuch, B. (2012), OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. British Journal of Pharmacology, 165: 1260-1287. https://doi.org/10.1111/j.1476-5381.2011.01724.x
5. ↑ ^{5.0 5.1 5.2 5.3} Tsigelny, I.F., Kovalskyy, D., Kouznetsova, V.L. et al. Conformational Changes of the Multispecific Transporter Organic Anion Transporter 1 (OAT1/SLC22A6) Suggests a Molecular Mechanism for Initial Stages of Drug and Metabolite Transport. Cell Biochem Biophys 61, 251–259 (2011). https://doi.org/10.1007/s12013-011-9191-7
6. ↑ Li, Z., Lam, P., Zhu, L., Wang, K., & Zhou, F. (2012). Current Updates in the Genetic Polymorphisms of Human Organic Anion Transporters (OATs). Journal of Pharmacogenomics and Pharmacoproteomics, 3, 1-8. https://doi.org/10.4172/2153-0645.1000E127
7. ↑ Anzai, N., Kanai, Y., & Endou, H. (2006). Organic anion transporter family: current knowledge. Journal of pharmacological sciences, 100(5), 411-426. https://doi.org/10.1254/jphs.CRJ06006X
8. ↑ Ljubojevic, M., Balen, D., Breljak, D., Kusan, M., Anzai, N., Bahn, A., ... & Sabolic, I. (2007). Renal expression of organic anion transporter OAT2 in rats and mice is regulated by sex hormones. American Journal of Physiology-Renal Physiology, 292(1), F361-F372. https://doi.org/10.1152/ajprenal.00207.2006