Organic anions are taken up from the blood into proximal tubule cells by organic anion transporters 1 and 3 (OAT1 and OAT3) in exchange for dicarboxylates. The released dicarboxylates are recycled by the sodium dicarboxylate cotransporter 3 (NaDC3). In this study, we tested the substrate specificities of human NaDC3, OAT1, and OAT3 to identify those dicarboxylates for which the three cooperating transporters have common high affinities. All transporters were stably expressed in HEK293 cells, and extracellularly added dicarboxylates were used as inhibitors of3 H]estrone-3-sulfate (OAT3) uptake. Human NaDC3 was stably expressed as proven by immunochemical methods and by sodiumdependent uptake of succinate (K 0.5 for sodium activation, 44.6 mM; Hill coefficient, 2.1; K m for succinate, 18 M). NaDC3 was best inhibited by succinate (IC 50 25.5 M) and less by ␣-ketoglutarate (IC 50 69.2 M) and fumarate (IC50 95.2 M). Dicarboxylates with longer carbon backbones (adipate, pimelate, suberate) had low or no affinity for NaDC3. OAT1 exhibited the highest affinity for glutarate, ␣-ketoglutarate, and adipate (IC50 between 3.3 and 6.2 M), followed by pimelate (18.6 M) and suberate (19.3 M). The affinity of OAT1 to succinate and fumarate was low. OAT3 showed the same dicarboxylate selectivity with ϳ13-fold higher IC50 values compared with OAT1. The data 1) reveal ␣-ketoglutarate as a common high-affinity substrate of NaDC3, OAT1, and OAT3 and 2) suggest potentially similar molecular structures of the binding sites in OAT1 and OAT3 for dicarboxylates.EFFICIENT EXCRETION OF ANIONIC endogenous waste products and exogenous compounds including drugs and toxins is an important task of the kidneys. Many organic anions (OA) undergo active trans-cellular secretion in renal proximal tubules, involving OA uptake across the basolateral membrane and OA release across the luminal membrane of proximal tubule cells. The uptake of OA from blood across the basolateral membrane into tubule cells is remarkable in several ways. First, OA uptake is considered the rate-limiting step in secretion; second, the uptake occurs against an opposing intracellular negative membrane potential difference; third, a multitude of chemically different OA is accepted as substrates for secretion (8,30,36). The mechanism by which OA overcome the opposing driving force during basolateral uptake was clarified in studies with basolateral membrane vesicles isolated from rat kidneys. Shimada et al. (29) and Pritchard (25) suggested the cooperation of two transporters: a sodium-driven dicarboxylate cotransporter and an OA/dicarboxylate exchanger.As regards the molecular identity of these cooperating transporters, the sodium-dicarboxylate cotransporter 3 (NaDC3, SLC13A3) takes up dicarboxylates into the cells and thus provides substrates for exchange against extracellular OA through the organic anion transporters 1 and 3 (OAT1, SLC22A6; and OAT3, SLC22A8). NaDC3 was cloned from human, rat, mouse, and winter flounder and found to be expressed in kidneys, liver, placenta, and...

= 252 (E = 25500) and 292 m p (9200), in dioxane]; its NMR spectrum has only an AABB' system, centered at T = 3.05, and an AB system at TA = 6.87 and TB = 8.43 (J = 13.5 cps) (8:4), so that structure (10) must be ascribed to it. Since this product is0 colorless, and, as far as established, has no pronounced oxidizing capacity, it cannot be regarded as a quinone, in contrast to the yellow 15,16-dihydro-trans-15.16-dimethyl-2,7-pyrenedione (11) [81. The main reason for this difference between (11) and (10) is doubtless to be sought in the steric hindrance to conjugation present in the latter. Received: M a y 16th, 1966 [Z 236 lE] German version: Angew. Chem. 78, 642 (1966) Received: April 18th. 1965 [ 2 213 IE] German version: Angeh. Chem. i d . 615 (196s)

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Modell des hOAT1 mit 12 TransmembrandomänenDas Aminoende ist links, das Carboxyende rechts, die extrazelluläre Seite ist oben, die intrazelluläre Seite unten. Die 1. Transmembrandomäne ist links, die 12. rechts.