OAT2 catalyses efflux of glutamate and uptake of orotic acid

Abstract

OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 μl·min⁻¹·mg of protein⁻¹) and human (46 μl·min⁻¹·mg of protein⁻¹). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [³H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.

Expression of Organic Anion Transporter 2 in the Human Kidney and Its Potential Role in the Tubular Secretion of Guanine-Containing Antiviral Drugs

“…In cloning OAT2 from a human kidney cDNA library, we obtained variant 1 (OAT2-546aa). This variant was stably expressed in HEK cells and supported robust cGMP transport, as had been reported previously (Cropp et al, 2008;Fork et al, 2011). In addition to cGMP, OAT2-546aa was previously found to transport a number of other guanine-containing compounds, including guanine itself, 2Ј-deoxyguanosine, guanosine monophosphate, guanosine diphosphate, and guanosine triphosphate (Cropp et al, 2008).…”

“…Although members of the OAT family are most known for their ability to interact with relatively small organic anions (Ͻ500 g/mol), acyclovir, ganciclovir, and penciclovir (225-250 g/mol) are neutral at physiological pH. Most recently, Fork et al (2011) showed that OAT2-546aa transports trigonelline, which is a small (137 g/mol) zwitterion. Together, these data indicate that the substrate specificity of OAT2-546aa is not restricted to compounds containing an anionic moiety but instead is relatively broad, as is the case with other drug transporters in the solute carrier family.…”

Organic Anion Transporters and Their Implications in Pharmacotherapy

“…Because orotic acid transport proteins have only been identified in bacteria (Defoor et al, 2007), OAT2 is one of few organic anion transporters that has been shown to carry out this function in mammals. As discussed by the authors, orotic acid is not retained in the cells, but rather, efficiently eliminated by the kidney; thus, it may seem that hepatic uptake of orotic acid by OAT2 is not imperative (Fork et al, 2011). However, OAT2 may play an important role in orotic aciduria, a disease state associated with excessive urinary excretion of orotic acid, by facilitating the transport of large stores of orotic acid from hepatocytes.…”

“…Furthermore, Kobayashi et al (2005) proposed that the 4-carbon dicarboxylates, succinate and fumarate, are transported by OAT2 and are also capable of trans-stimulating the uptake of estronesulfate, which was recently, however, questioned by Rizwan and . Fork et al (2011) studied the substrate specificity of this transporter again to clarify the confusion over its precise mechanism of transport. Their studies involved the use of human embryonic kidney (HEK) 293 cells, which may be a more reliable expression system than X. laevis oocytes.…”

“…1). Furthermore, in the article by Fork et al (2011), OAT2 was shown to transport orotic acid with high specificity (Fork et al, 2011). Because orotic acid transport proteins have only been identified in bacteria (Defoor et al, 2007), OAT2 is one of few organic anion transporters that has been shown to carry out this function in mammals.…”