Functional characterization of CitM, the Mg2+-citrate transporter.

The CitM transporter from Bacillus subtilis transports citrate as a complex with Mg2+. In this study, CitM was functionally expressed and characterized in E. coli DH5a cells. In the presence of saturating Mg2+ concentrations, the Km for citrate in CitM was 274 mM, similar to previous studies using whole cells of B. subtilis. CitM has a high substrate specificity for citrate. Other di- and tricarboxylic acids including succinate, isocitrate, cis-aconitate and tricarballylic acid did not significantly inhibit the uptake of citrate in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The highest rate of citrate transport was seen in the presence of Mg2+, followed in order of preference by Mn2+, Ba2+, Ni2+, Co2+ and Ca2+. Citrate transport by CitM appears to be proton coupled. The transport was inhibited in transport buffers more alkaline than pH 7.5 and not affected by pH at acidic values. Transport was also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin. Valinomycin did not affect the uptake by CitM, suggesting that transport is electroneutral. In conclusion, the cloned CitM transporter from B. subtilis expressed in E. coli has properties similar to the transporter in intact B. subtilis cells. The results support a transport model with a coupling stoichiometry of one proton coupled to the uptake of one complex of (Mg2+-citrate)1-.

Conformationally sensitive residues in transmembrane domain 9 of the Na+/dicarboxylate co-transporter.

The Na(+)/dicarboxylate co-transporter, NaDC-1, couples the transport of sodium and Krebs cycle intermediates, such as succinate and citrate. Previous studies identified two functionally important amino acids, Glu-475 and Cys-476, located in transmembrane domain (TMD) 9 of NaDC-1. In the present study, each amino acid in TMD-9 was mutated to cysteine, one at a time, and the accessibility of the membrane-impermeant reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) to the replacement cysteines was determined. Cysteine substitution was tolerated at all but five of the sites: the A461C mutant was not present at the plasma membrane, whereas the F473C, T474C, E475C, and N479C mutants were inactive proteins located on the plasma membrane. Cysteine substitution of four residues found near the extracellular surface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in proteins that were sensitive to inhibition by MTSET. The accessibility of MTSET to the four substituted cysteines was highest in the presence of the transported cations, sodium or lithium, and low in choline. The four mutants also exhibited substrate protection of MTSET accessibility. The MTSET accessibility to S478C, A481C, and A480C was independent of voltage. In contrast, T482C was more accessible to MTSET in choline buffer at negative holding potentials, but there was no effect of voltage in sodium buffer. In conclusion, TMD-9 may be involved in transducing conformational changes between the cation-binding sites and the substrate-binding site in NaDC-1, and it may also form part of the translocation pathway through the transporter.

Cloning and functional characterization of a high-affinity Na(+)/dicarboxylate cotransporter from mouse brain.

Neurons contain a high-affinity Na(+)/dicarboxylate cotransporter for absorption of neurotransmitter precursor substrates, such as alpha-ketoglutarate and malate, which are subsequently metabolized to replenish pools of neurotransmitters, including glutamate. We have isolated the cDNA coding for a high-affinity Na(+)/dicarboxylate cotransporter from mouse brain, called mNaDC-3. The mRNA coding for mNaDC-3 is found in brain and choroid plexus as well as in kidney and liver. The mNaDC-3 transporter has a broad substrate specificity for dicarboxylates, including succinate, alpha-ketoglutarate, fumarate, malate, and dimethylsuccinate. The transport of citrate is relatively insensitive to pH, but the transport of succinate is inhibited by acidic pH. The Michaelis-Menten constant for succinate in mNaDC-3 is 140 microM in transport assays and 16 microM at -50 mV in two-electrode voltage clamp assays. Transport is dependent on sodium, although lithium can partially substitute for sodium. In conclusion, mNaDC-3 likely codes for the high-affinity Na(+)/dicarboxylate cotransporter in brain, and it has some unusual electrical properties compared with the other members of the family.

Topology of the Na(+)/dicarboxylate cotransporter: the N-terminus and hydrophilic loop 4 are located intracellularly.

The current secondary structure model of the Na(+)/dicarboxylate cotransporter, NaDC-1, contains 11 transmembrane domains. The model is based on hydropathy analysis and the extracellular location of the carboxy terminus, which contains an N-glycosylation site. In this study, the model was further tested using indirect immunofluorescence of COS-7 cells. The Flag epitope tag (DYKDDDDK) was fused to the amino terminus of NaDC-1 (Flag-NaDC-1), and a monoclonal antibody against the Flag epitope was used to determine the location of the N-terminus. Hydrophilic loop 4 of NaDC-1 was identified using polyclonal antibodies raised against a fusion protein containing amino acids 164--233 of NaDC-1. The expression of NaDC-1 and Flag-NaDC-1 in COS-7 cells was confirmed by functional assays of succinate transport and by Western blots of cell surface biotinylated proteins. Immunofluorescent labeling of cells expressing both NaDC-1 and Flag-NaDC-1 required permeabilization of the plasma membranes with digitonin whereas no immunofluorescence was visible in intact cells. The results of this study show that both the N-terminus and hydrophilic loop 4 of NaDC-1 are located intracellularly, which supports the current model of NaDC-1 structure.

Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1.

The role of cationic amino acids in the Na(+)/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased K(m) for succinate of 1.8 mM, compared with 0.3 mM in the wild-type NaDC-1. The R349A mutant had Na(+) and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney.

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations. (1) Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na(+)-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is approximately 24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant (K(m)) for succinate was 0.35 mM, and the K(m) for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at -50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.

The transport properties of the human renal Na(+)- dicarboxylate cotransporter under voltage-clamp conditions.

The transport properties of the human Na(+)-dicarboxylate cotransporter, (hNaDC-1), expressed in Xenopus laevis oocytes were characterized using the two-electrode voltage clamp technique. Steady-state succinate-evoked inward currents in hNaDC-1 were dependent on the concentrations of succinate and sodium, and on the membrane potential. At -50 mV, the half-saturation constant for succinate (K(0.5)(succinate)) was 1.1 mM and the half-saturation constant for sodium (K(0.5)(sodium)) was 65 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na(+):1 divalent anion substrate. The hNaDC-1 exhibits a high-cation selectivity. Sodium is the preferred cation and other cations, such as lithium, were not able to support transport of succinate. The preferred substrates of hNaDC-1 are fumarate (K(0.5) 1.8 mM) and succinate, followed by methylsuccinate (K(0.5) 2.8 mM), citrate (K(0. 5) 6.8 mM) and alpha-ketoglutarate (K(0.5) 16 mM). The hNaDC-1 may also transport sodium ions through an uncoupled leak pathway, which is sensitive to phloretin inhibition. We propose a transport model for hNaDC-1 in which the binding of three sodium ions is followed by substrate binding.

Chronic metabolic acidosis increases NaDC-1 mRNA and protein abundance in rat kidney.

BACKGROUND: Chronic metabolic acidosis increases, while alkali feeding inhibits, proximal tubule citrate absorption. The activity of the apical membrane Na+/citrate cotransporter is increased in metabolic acidosis, but is not altered by alkali feeding. METHODS: Renal cortical mRNA and brush border membrane protein abundances of sodium/dicarboxylate-1 (NaDC-1), the apical membrane Na+/citrate transporter, were measured. RESULTS: By immunohistochemistry, NaDC-1 was localized to the apical membrane of the proximal tubule. Chronic metabolic acidosis caused an increase in NaDC-1 protein abundance that was maximal in the S2 segment and that increased with time. Metabolic acidosis also increased NaDC-1 mRNA abundance, but this was first seen at three hours and correlated with the severity of the metabolic acidosis. Alkali feeding had no effect on NaDC-1 protein or mRNA abundance. CONCLUSIONS: Chronic metabolic acidosis increases renal cortical NaDC-1 mRNA abundance and apical membrane NaDC-1 protein abundance, while alkali feeding is without effect on NaDC-1.

Water transport by the renal Na(+)-dicarboxylate cotransporter.

This study investigated the ability of the renal Na(+)-dicarboxylate cotransporter, NaDC-1, to transport water. Rabbit NaDC-1 was expressed in Xenopus laevis oocytes, cotransporter activity was measured as the inward current generated by substrate (citrate or succinate), and water transport was monitored by the changes in oocyte volume. In the absence of substrates, oocytes expressing NaDC-1 showed an increase in osmotic water permeability, which was directly correlated with the expression level of NaDC-1. When NaDC-1 was transporting substrates, there was a concomitant increase in oocyte volume. This solute-coupled influx of water took place in the absence of, and even against, osmotic gradients. There was a strict stoichiometric relationship between Na(+), substrate, and water transport of 3 Na(+), 1 dicarboxylate, and 176 water molecules/transport cycle. These results indicate that the renal Na(+)-dicarboxylate cotransporter mediates water transport and, under physiological conditions, may contribute to fluid reabsorption across the proximal tubule.

Role of the efferent medial olivocochlear system in contralateral masking and binaural interactions: an electrophysiological study in guinea pigs.

Contralateral broadband noise (BBN) elevates ipsilateral auditory thresholds (central masking) and reduces the amplitude of ipsilateral brainstem auditory evoked potentials (BAEPs). Binaural interactions are complex psychophysical phenomena, but binaural interaction components are easily extracted from BAEPs to monaural versus binaural click stimulation. However, contralateral, or binaural, acoustical stimulation is known to activate simultaneously the crossed and uncrossed medial olivocochlear (MOC) efferent systems and decrease activity in both cochleas. Particularly, contralateral BBN stimulation suppresses in part ipsilateral peripheral activity. What is the role of such contralaterally induced peripheral suppression in the overall changes in central BAEPs observed during contralateral masking or binaural stimulation? Compound action potentials (CAPs) of the auditory nerve and BAEPs were recorded simultaneously in awake guinea pigs from electrodes chronically implanted on the round window of the cochlea and the surface of the brain. Peripheral and central measures of contralateral masking and binaural interactions were obtained from responses to monaural or binaural clicks, with or without contralateral BBN, recorded before, during, and after the reversible blockade of the MOC function following a single intramuscular injection of gentamicin. Contralateral BBN effectively reduced the amplitudes of CAP and of all BAEP peaks. CAP to ipsilateral click did not, however, change significantly from monaural to binaural click stimulation; still, normal binaural interaction components developed in the BAEPs. When the medial efferent function was blocked by gentamicin, the normal contralateral BBN suppression of CAP and of the earliest BAEP peak was lost; however, the later BAEP peaks were suppressed by contralateral BBN as before gentamicin, and the central binaural interaction components were unchanged. In these experimental conditions, the MOC efferent system seems to play little role in centrally recorded contralateral masking and binaural interactions.

Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1.

The role of cysteine residues in the Na(+)/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

Protein kinase C-mediated regulation of the renal Na(+)/dicarboxylate cotransporter, NaDC-1.

The Na(+)/dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the tubular filtrate. Although long-term regulation of this transporter by chronic metabolic acidosis and K(+) deficiency is well documented, there is no information on acute regulation of NaDC-1. In the present study, the transport of succinate in Xenopus oocytes expressing NaDC-1 was inhibited up to 95% by two activators of protein kinase C, phorbol 12-myristate, 13-acetate (PMA) and sn-1, 2-dioctanoylglycerol (DOG). Activation of protein kinase A had no effect on NaDC-1 activity. The inhibition of NaDC-1 transport by PMA was dose-dependent, and could be prevented by incubation of the oocytes with staurosporine. Mutations of the two consensus protein kinase C phosphorylation sites in NaDC-1 did not affect inhibition by PMA. The inhibitory effects of PMA were partially prevented by cytochalasin D, which disrupts microfilaments and endocytosis. PMA treatment was also associated with a decrease of approximately 30% in the amount of NaDC-1 protein found on the plasma membrane. We conclude that the inhibition of NaDC-1 transport activity by PMA occurs by a combination of endocytosis and inhibition of transport activity.

Acidic residues involved in cation and substrate interactions in the Na+/dicarboxylate cotransporter, NaDC-1.

The role of acidic amino acid residues in cation recognition and selectivity by the Na+/dicarboxylate cotransporter, NaDC-1, was investigated by site-directed mutagenesis and expression in Xenopus oocytes. Four of the residues tested, Asp-52, Glu-74, Glu-101, and Glu-332, were found to be unimportant for transport activity. However, substitutions of Asp-373 and Glu-475, conserved residues found in transmembrane domains M8 and M9, respectively, altered transport kinetics. Replacements of Asp-373 with Ala, Glu, Asn, and Gln resulted in changes in sodium affinity and cation selectivity in NaDC-1, indicating that the carbonyl oxygen at this position may play a role in the topological organization of the cation-binding site. In contrast, substitutions of Glu-475 led to dramatic reductions in transport activity and changes in transport kinetics. Substitution with Gln led to a transporter with increased substrate and sodium affinity, while the E475D mutant was inactive. The E475A mutant appeared to have poor sodium binding. Substrate-induced currents in the E475A mutant exhibited a strong voltage dependence, and a reversal of the current was seen at -30 mV. The results suggest that Glu-475 may play a role in cation binding and possibly also in mediating anion channel activity. Remarkably, mutations of both Asp-373 and Glu-475 affected the Km for succinate in NaDC-1, suggesting dual roles for these residues in determining the affinity for substrate and cations. We propose that at least one of the cation-binding sites and the substrate-binding site are close together in the carboxy-terminal portion of NaDC-1, and thus transmembrane domains M8 and M9 are candidate structures for the formation of the translocation pathway.

Determinants of substrate and cation affinities in the Na+/dicarboxylate cotransporter.

The Na+/dicarboxylate cotransporter (NaDC-1) couples the transport of sodium and tricarboxylic acid cycle intermediates, such as succinate and citrate. The rabbit and human homologues (rbNaDC-1 and hNaDC-1, respectively) are 78% identical in amino acid sequence but exhibit several differences in their functional properties. rbNaDC-1 has a greater apparent affinity for citrate and sodium than hNaDC-1. Furthermore, unlike hNaDC-1, rbNaDC-1 is inhibited by low concentrations of lithium. In this study, chimeric transporters were constructed to identify the protein domains responsible for the functional differences between rbNaDC-1 and hNaDC-1. Individual substitutions of transmembrane domain (TMD) 7, 10 or 11 produced transporters with intermediate properties. However, substitution of TMD 7, 10, and 11 together resulted in a transporter with the citrate Km of the donor, suggesting that interactions between these domains determine the differences in apparent citrate affinities. TMDs 10 and 11 are most important in determining the differences in apparent sodium affinities, and TMD 11 determines the sensitivity to lithium inhibition. We conclude that transmembrane domains 7, 10, and 11 in NaDC-1 may contain at least one of the cation binding sites in close proximity to the substrate binding domain.

Sodium-coupled transporters for Krebs cycle intermediates.

Krebs cycle intermediates such as succinate, citrate, and alpha-ketoglutarate are transferred across plasma membranes of cells by secondary active transporters that couple the downhill movement of sodium to the concentrative uptake of substrate. Several transporters have been identified in isolated membrane vesicles and cells based on their functional properties, suggesting the existence of at least three or more Na+/dicarboxylate cotransporter proteins in a given species. Recently, several cDNAs, called NaDC-1, coding for the low-affinity Na+/dicarboxylate cotransporters have been isolated from rabbit, human, and rat kidney. The Na+/dicarboxylate cotransporters are part of a distinct gene family that includes the renal and intestinal Na+/sulfate cotransporters. Other members of this family include a Na(+)- and Li(+)-dependent dicarboxylate transporter from Xenopus intestine and a putative Na+/dicarboxylate cotransporter from rat intestine. The current model of secondary structure in NaDC-1 contains 11 transmembrane domains and an extracellular N-glycosylated carboxy terminus.

Citrate transport by the kidney and intestine.

Citrate is an important metabolite that is transported in the kidney and intestine. Low urinary citrate concentrations, which may be determined in part by transport processes in the kidney, are associated with the development of kidney stones. Citrate is reabsorbed from the tubular filtrate in the renal proximal tubule on a sodium-coupled transporter, the Na+/dicarboxylate cotransporter, with a broad substrate specificity for Krebs cycle intermediates. The same transporter is found on the brush-border membrane of enterocytes. In contrast to the well-characterized apical pathway for citrate, there is relatively little information about citrate transport across the basolateral membrane. Recently, the complementary DNAs coding for the Na+/citrate transporters from the apical membranes of rabbit and human kidney, NaDC-1 and hNaDC-1, have been cloned and sequenced. These transporters belong to a separate gene family that includes the renal Na+/sulfate cotransporter, NaSi-1.

Primary structure and functional characteristics of a mammalian sodium-coupled high affinity dicarboxylate transporter.

We have cloned a Na+-dependent, high affinity dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na+-dependent, low affinity dicarboxylate transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na+-dependent transport of succinate with a Kt of 2 microM. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na+ concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li+ inhibits NaDC3 in the presence of Na+. Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8. 0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6. 5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na+-dependent, high affinity dicarboxylate transporter.

Sequence of a pyrimidine-selective Na+/nucleoside cotransporter from pig kidney, pkCNT1.

A 3 kb cDNA called pkCNT1, a new member of the Na+/nucleoside cotransporter family, was cloned from pig kidney and sequenced. The sequence of pkCNT1 encodes a 647 amino acid protein that is 84% identical to the sequence of the rat pyrimidine-selective Na+/nucleoside cotransporter, rCNT1. pkCNT1 transports pyrimidines, such as thymidine and uridine, and has a Km for uridine of 9 microM.

Sodium and lithium interactions with the Na+/Dicarboxylate cotransporter.

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 K0.5succinate was 180 microM and the K0.5Na+ 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 K0.5succinate 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 Imaxsuccinate. 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.