Sodium and Lithium Interactions with the Na+/Dicarboxylate Cotransporter

Abstract

The two-electrode voltage clamp was used to study the currents associated with transport of succinate by the cloned Na ؉ /dicarboxylate cotransporter, NaDC-1, expressed in Xenopus oocytes. The presence of succinate induced inward currents which were dependent on the concentrations of succinate and sodium, and on the membrane potential. At ؊50 mV, the K 0.5 succinate was 180 M and the K 0.5 Na؉ was 19 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na ؉ :1 divalent anion substrate. Currents were induced in NaDC-1 by a range of di-and tricarboxylates, including citrate, methylsuccinate, fumarate, and tricarballylate. Although Na ؉ is the preferred cation, Li ؉ was also able to support transport. The K 0.5 succinate was approximately 10-fold higher in Li ؉ compared with Na ؉ . In the presence of Na ؉ , however, Li ؉ was a potent inhibitor of transport. Millimolar concentrations of Li ؉ resulted in decreases in apparent succinate affinity and in the I max succinate . Furthermore, lithium inhibition under saturating sodium concentrations showed hyperbolic kinetics, suggesting that one of the three cation binding sites in NaDC-1 has a higher affinity for Li ؉ than Na ؉ . We conclude that NaDC-1 is an electrogenic anion transporter that accepts either Na ؉ or Li ؉ as coupling cations. However, NaDC-1 contains a single high affinity binding site for Li ؉ that, when occupied, results in transport inhibition, which may account for its potent inhibitory effects on renal dicarboxylate transport.The active transport of Krebs cycle intermediates, such as succinate and citrate, is mediated by a specific sodium-coupled transporter found on the apical membrane in epithelial cells of the kidney proximal tubule (1, 2). The Na ϩ -dicarboxylate cotransporter reabsorbs a wide range of di-and tricarboxylic acids in the form of divalent anions. This transporter is sensitive to inhibition by lithium (3), and patients receiving therapeutic doses of lithium exhibit increased renal excretion of ␣-ketoglutarate and glutarate (4). The cDNA coding for the rabbit renal Na ϩ /dicarboxylate cotransporter, NaDC-1, 1 has been cloned and sequenced (5), and the protein has been identified in renal brush border membranes (6). NaDC-1 belongs to a distinct gene family of sodium-coupled anion transporters that includes the Na ϩ /dicarboxylate cotransporters, hNaDC-1, from human kidney (7), and NaDC-2, from Xenopus intestine (8), and the renal Na ϩ /sulfate cotransporter, NaSi-1 (9). The transport mechanism of NaDC-1 is thought to involve the ordered binding of four charged substrates: 3 Na ϩ ions and 1 divalent anion substrate (10 -12), resulting in one net inward positive charge across the membrane per cycle. Experiments with rabbit renal brush border membrane vesicles support this hypothesis: sodium-dependent transport of succinate was affected by changes in membrane potential, and transport of succinate also caused a depolarization of membrane potential (12-14). However, the dependence of transport kinetics o...

Primary Structure and Functional Characteristics of a Mammalian Sodium-coupled High Affinity Dicarboxylate Transporter

“…Furthermore, the present study shows that Li ϩ can substitute for Na ϩ , although to a small extent, in supporting succinate transport by NaDC3. Similar results have been obtained for rabbit NaDC1 (28). Thus, Li ϩ is a stimulator as well as an inhibitor for NaDC3 and NaDC1.…”

“…These differences in K t values among NaDC1s from various animal species may not be entirely due to the differences in the experimental approaches employed in the determination of the K t values. A recent study (28) has shown that rabbit NaDC1 has a K t of 180 M in X. laevis oocytes when analyzed electrophysiologically, in contrast to the K t value of 25-30 M for rat NaDC1 obtained with similar experimental approaches (15,16). Therefore, the possibility of actual differences in the affinity of NaDC1 among various animal species cannot be ruled out.…”

Expression Cloning and Characterization of a Novel Sodium-Dicarboxylate Cotransporter from Winter Flounder Kidney

“…In contrast, neither the cloned rabbit or human NaDC-1, nor X. laevis NaDC-2 were inhibited by dimethylsuccinate (13,14,18). Additionally, dimethylsuccinate did not evoke a significant current in oocytes injected with cRNA from rabbit NaDC-1 (35) or rat SDCT1 (16). The effect of cis-aconitate on the cloned luminal transporters has not been tested so far.…”

“…Using the Michaelis-Menten equation, the best fit to the data could be obtained assuming a cotransport of 3 sodium ions, resulting in an apparent affinity constant of 39.6 Ϯ 3.3 mM Na brane depolarization and the inward current induced by succinate (not shown). The inhibition by lithium, which is probably due to the replacement of one of the sodium ions by lithium, has been shown previously for dicarboxylate transport in the renal luminal (33,34) and basolateral membranes (8,10), as well as for the cloned NaDC-1 of rabbit (13,35) and rat kidney (16). In contrast, lithium binds probably to all sodium binding sites of the intestinal transporter NaDC-2, albeit the V max of succinate uptake in the presence of Li ϩ was approximately one-third of that in the presence of Na ϩ (18).…”

The Renal Na+-Dependent Dicarboxylate Transporter, NaDC-3, Translocates Dimethyl- and Disulfhydryl-Compounds and Contributes to Renal Heavy Metal Detoxification

“…In contrast, 2,3-DMS was a very weak inhibitor of dicarboxylate transport at the luminal membrane (K i , 3.76 Ϯ 0.73 mM) (19). Within the cloned NaDC-1 orthologs, DMS analogs left either radiolabeled succinate uptake unaffected (41,45,46) or induced currents comparable in magnitude to succinate under two-electrode voltage clamp conditions (44,47,48). In contrast, NaDC-3 orthologs were generally sensitive to DMS analogs (12)(13)(14)(15)(16).…”