GFP-Human high-affinity carnitine transporter OCTN2 protein: subcellular localization and functional restoration of carnitine uptake in mutant cell lines with the carnitine transporter defect.

Individuals with the plasmalemmal high-affinity carnitine transporter defect present with progressive infantile-onset carnitine-responsive cardiomyopathy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopathy, and failure to thrive. The carnitine uptake defect (CUD) has been documented in their cultured skin fibroblasts, lymphoblasts, and/or myoblasts. The cDNA encoding the high-affinity sodium-dependent human carnitine transporter OCTN2 has recently been cloned. We used the green fluorescent protein (GFP) as a living marker for positively transfected cells in our expression studies of the high-affinity carnitine transporter OCTN2 cDNA in cell lines with the CUD. Transfection of cell lines from 12 unrelated patients (nine fibroblast and three lymphoblastoid) with a GFP construct harboring the wild-type full-length OCTN2 cDNA was done using LipoTAXI. Transient and stable expression of the recombinant GFP-human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the fibroblast and stable transfection of the lymphoblastoid cell lines were achieved. There was functional restoration of carnitine uptake in the transfected mutant cell lines, thereby confirming the identity of the transfected cDNA. In addition, we report the first demonstration of the subcellular localization of an in-frame fusion GFP-human high-affinity carnitine transporter OCTN2 protein in the plasma membrane by confocal laser-scanning fluorescence microscopy.     

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Na(+)-coupled alternative to H(+)-coupled primary transport systems in bacteria.

Protons are the most common coupling ions in bacterial energy conversions. However, while many organisms, such as the alkaliphilic Bacilli, employ H(+)-bioenergetics for electron transport phosphorylation, they use Na+ as the coupling ion for transport and flagellar movement. The Na+ gradient required for these bioenergetic functions is established by the secondary Na+/H+ antiporter. In contrast, Vibrio alginolyticus and methanogenic bacteria have primary pumps for both H+ and Na+. They use the proton gradient for ATP synthesis while other, less energy-consuming membrane reactions are powered by the Na+ gradient. In a third mode, some anaerobic bacteria possess decarboxylases acting as primary Na+ pumps. For instance, in Klebsiella pneumoniae, the Na+ gradient established by oxaloacetate decarboxylase is used for the uptake of the growth substrate citrate, and Propionigenium modestum consumes the energy of the Na+ gradient formed by methylmalonyl-CoA decarboxylase directly for ATP synthesis.     

Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

l-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of l-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of l-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of l-[(3)H]carnitine uptake were investigated with or without specific inhibitors. l-Carnitine uptake in mature cells was sodium dependent and linear with time. K(m) and V(max) values for saturable uptake were 14.07 +/- 1.70 micro M and 26.3 +/- 0.80 pmol. mg protein(-1). 6 min(-1), respectively. l-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that l-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.     

Tyrosine residues affecting sodium stimulation of carnitine transport in the OCTN2 carnitine/organic cation transporter

Primary carnitine deficiency is a disorder of fatty acid oxidation caused by mutations in the Na+-dependent carnitine/organic cation transporter OCTN2. Studies with tyrosyl group-modifying reagents support the involvement of tyrosine residues in Na+ binding by sodium-coupled transporters. Here we report two new patients with carnitine deficiency caused by mutations affecting tyrosyl residues (Y447C and Y449D) close to a residue (Glu-452) previously shown to affect sodium stimulation of carnitine transport. Kinetic analysis indicated that the Y449D substitution, when expressed in Chinese hamster ovary cells, increased the concentration of sodium required to half-maximally stimulate carnitine transport from 14.8 +/- 1.8 to 34.9 +/- 5.8 mM (p<0.05), whereas Y447C completely abolished carnitine transport. Substitution of these tyrosine residues with phenylalanine restored normal carnitine transport in Y449F but resulted in markedly impaired carnitine transport by Y447F. This was associated with an increase in the concentration of sodium required to half-maximally stimulate carnitine transport to 57.8 +/- 7.4 mM (p<0.01 versus normal OCTN2). The Y447F and Y449D mutant transporters retained their ability to transport the organic cation tetraethylammonium indicating that their effect on carnitine transport was specific and likely associated with the impaired sodium stimulation of carnitine transport. By contrast, the Y447C natural mutation abolished the transport of organic cations in addition to carnitine. Confocal microscopy of OCTN2 transporters tagged with green fluorescent protein indicated that the Y447C mutant transporters failed to reach the plasma membrane, whereas Y447F, Y449D, and Y449F had normal membrane localization. These natural mutations identify tyrosine residues possibly involved in coupling the sodium electrochemical gradient to transmembrane solute transfer in the sodium-dependent co-transporter OCTN2.     

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane

L-carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+-free conditions chicken enterocytes take up L-carnitine until the cell to medium L-carnitine ratio is 1. This uptake was inhibited by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L-3H-carnitine uptake into BLMV showed no overshoot, and it was (i) Na+-independent, (ii) trans-stimulated by intravesicular L-carnitine, and (iii) cis-inhibited by TEA and cold L-carnitine. L-3H-carnitine efflux from L-3H-carnitine preloaded enterocytes was also Na+-independent, and trans-stimulated by L-carnitine, D-carnitine, gamma-butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L-carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl-L-carnitine. RT-PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH-independent L-carnitine transport activity measured in the three experimental conditions.     

Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342-557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 +/- 0.5 to 141 +/- 19 microM. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341-454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341-454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 +/- 5 microM). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 +/- 4.5 microm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition.     

Na(+)-dependent fructose transport via rNaGLT1 in rat kidney

We found a system of Na(+)-dependent uptake of fructose by rat renal brush-border membrane vesicles. It consisted of two saturable components, and was thought to involve at least two transporters. rNaGLT1, a novel glucose transporter in rat kidney, showed fructose uptake as well as alpha-methyl-D-glucopyranoside uptake by transfected HEK293 cells. The features of the lower affinity type of fructose transporter in the brush-border membranes, such as affinity and substrate recognition, were very comparable with those of rNaGLT1-transfected HEK293 cells. These results indicated that rNaGLT1 is a primary fructose transporter in rat renal brush-border membranes.     

Muscle contraction increases carnitine uptake via translocation of OCTN2

Since carnitine plays an important role in fat oxidation, influx of carnitine could be crucial for muscle metabolism. OCTN2 (SLC22A5), a sodium-dependent solute carrier, is assumed to transport carnitine into skeletal muscle cells. Acute regulation of OCTN2 activity in rat hindlimb muscles was investigated in response to electrically induced contractile activity. The tissue uptake clearance (CL(uptake)) of l-[(3)H]carnitine during muscle contraction was examined in vivo using integration plot analysis. The CL(uptake) of [(14)C]iodoantipyrine (IAP) was also determined as an index of tissue blood flow. To test the hypothesis that increased carnitine uptake involves the translocation of OCTN2, contraction-induced alteration in the subcellular localization of OCTN2 was examined. The CL(uptake) of l-[(3)H]carnitine in the contracting muscles increased 1.4-1.7-fold as compared to that in the contralateral resting muscles (p<0.05). The CL(uptake) of [(14)C]IAP was much higher than that of l-[(3)H]carnitine, but no association between the increase in carnitine uptake and blood flow was obtained. Co-immunostaining of OCTN2 and dystrophin (a muscle plasma membrane marker) showed an increase in OCTN2 signal in the plasma membrane after muscle contraction. Western blotting showed that the level of sarcolemmal OCTN2 was greater in contracting muscles than in resting muscles (p<0.05). The present study showed that muscle contraction facilitated carnitine uptake in skeletal muscles, possibly via the contraction-induced translocation of its specific transporter OCTN2 to the plasma membrane.     

Mutations in novel organic cation transporter (OCTN2), an organic cation/carnitine transporter, with differential effects on the organic cation transport function and the carnitine transport function

Novel organic cation transporter (OCTN2) is an organic cation/carnitine transporter, and two missense mutations, L352R and P478L, in OCTN2 have been identified as the cause for primary carnitine deficiency. In the present study, we assessed the influence of these two mutations on the carnitine transport function and the organic cation transport function of OCTN2. The L352R mutation resulted in a complete loss of both transport functions. In contrast, the P478L mutation resulted in a complete loss of only the carnitine transport function but significantly stimulated the organic cation transport function. Studies with human OCTN2/rat OCTN2 chimeric transporters indicated that the carnitine transport site and the organic cation transport site were not identical. Because carnitine transport is Na(+)-dependent whereas organic cation transport is Na(+)-independent, we investigated the possibility that the P478L mutation affected Na(+) binding. The Na(+) activation kinetics were found to be similar for the P478L mutant and wild type OCTN2. We then mutated nine different tyrosine residues located in or near transmembrane domains and assessed the transport function of these mutants. One of these mutations, Y211F, was found to have differential influence on the two transport activities of OCTN2 as did the P478L mutation. However, the Na(+) activation kinetics were not affected. These findings are of clinical relevance to patients with primary carnitine deficiency because whereas each and every mutation in these patients is expected to result in the loss of the carnitine transport function, all of these mutations may not interfere with the organic cation transport function.     

Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. We report novel localization of the organic cation/carnitine transporter, OCTN1, to mitochondria. We made GFP- and RFP-human OCTN1 cDNA constructs and showed expression of hOCTN1 in several transfected mammalian cell lines. Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L-[(3)H]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K(m) of 422 microM and Western blot with an anti-GFP antibody identified the expected GFP-hOCTN1 fusion protein (90 kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[(3)H]carnitine uptake (K(m) 1324 microM), solely attributable to OCTN1, in isolated mitochondria of mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase activity (alternate mitochondrial carnitine transporter). This mitochondrial localization was confirmed by TEM of murine heart incubated with highly specific rabbit anti-GST-hOCTN1 antibody and immunogold labeled goat anti-rabbit antibody. This suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis.     

Transport of butyryl-L-carnitine, a potential prodrug, via the carnitine transporter OCTN2 and the amino acid transporter ATB(0,+)

L-carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB(0,+). Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB(0,+) is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-L-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB(0,+) to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB(0,+) and OCTN2 in heterologous expression systems. BC inhibited ATB(0,+)-mediated glycine transport in mammalian cells (IC(50), 4.6 +/- 0.7 mM). In Xenopus laevis oocytes expressing human ATB(0,+), BC induced Na(+) -dependent inward currents under voltage-clamp conditions. The currents were saturable with a K(0.5) of 1.4 +/- 0.1 mM. Na(+) activation kinetics of BC-induced currents suggested involvement of two Na(+) per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC(50), 1.5 +/- 0.3 microM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na(+) dependent and saturable (K(0.5), 0.40 +/- 0.02 microM). We conclude that ATB(0,+) is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB(0,+) may mediate intestinal absorption of BC when OCTN2 is defective.     

Glycosylation of the OCTN2 carnitine transporter: Study of natural mutations identified in patients with primary carnitine deficiency

Primary carnitine deficiency is caused by impaired activity of the Na⁺-dependent OCTN2 carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary carnitine deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased carnitine transport. Substitution of two sites at a time caused a further decline in carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of carnitine and sodium-stimulated carnitine transport indicated that all substitutions decreased the Vmax for carnitine transport, but N64Q/N91Q also significantly increased the Km toward carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.     

Differential regulation of Na+,K+-ATPase and the Na+-coupled glucose transporter in hypertensive rat kidney

Several Na⁺ transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na⁺,K⁺-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na⁺,K⁺-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na⁺,K⁺-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K_m) for ATP, but not with a change of maximum velocity (V_max). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V_max for α-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na⁺,K⁺-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na⁺,K⁺-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter

We have clonedand functionally characterized the human Na⁺-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa⁺ [concentration of substrate necessary for half-maximaltransport (K_t) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa⁺-to-succinate stoichiometry is 3:1 and concentration ofNa⁺ necessary for half-maximal transport(K^Na+_0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na⁺-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,K^Suc_0.5 is 102 ± 20 µM andK^Na+_0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na⁺-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li⁺inhibits succinate-induced currents in the presence of Na⁺.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

     

Expression of novel organic cation/carnitine transporter (OCTN2) in the mouse pancreas

Among the organic cation transporters, OCTN2 is identified as the most important carnitine transporter owing to the ability to transport carnitine. Although the OCTN2 is previously found in various tissues, there have been no reports showing the OCTN2 in the pancreas. In this study, we examined the expression and localization of OCTN2 in the mouse pancreas by the aid of an in situ hybridization technique and immunohistochemistry with anti-OCTN2 antibody. As a result, the OCTN2 expression was found in the A-cells for the first time. OCTN2 was not expressed in B-cells, notwithstanding that the metabolism of long-chain fatty acids, which are transported into the mitochondria with the help of carnitine, was expected for fatty acid-stimulated insulin secretion. Thus, this study suggests the possibility of carnitine uptake in the pancreatic A-cells through OCTN2 and implies the presence of carnitine transporter(s) other than OCTN2 in the B-cell.     

Human Na+ -coupled citrate transporter: primary structure,genomic organization,and transport function

This paper describes the cloning and functional characterization of the human Na(+)-coupled citrate transporter (NaCT). The cloned human NaCT shows 77% sequence identity with rat NaCT. The nact gene is located on human chromosome 17 at p12-13. NaCT mRNA is expressed most predominantly in the liver, with moderate expression detectable in the brain and testis. When functionally expressed in mammalian cells, human NaCT mediates the Na(+)-coupled transport of citrate. Studies with several monocarboxylates, dicarboxylates, and tricarboxylates show that the transporter is selective for citrate with comparatively several-fold lower affinity for other intermediates of citric acid cycle. The Michelis-Menten constant for citrate is approximately 650 microM. The activation of citrate transport by Na(+) is sigmoidal, suggesting involvement of multiple Na(+) ions in the activation process. The transport process is electrogenic. This represents the first plasma membrane transporter in humans that mediates the preferential entry of citrate into cells. Citrate occupies a pivotal position in many important biochemical pathways. Among various citric acid cycle intermediates, citrate is present at the highest concentrations in human blood. The selectivity of NaCT towards citrate and its predominant expression in the liver suggest that this transporter may facilitate the utilization of circulating citrate for the generation of metabolic energy and for the synthesis of fatty acids and cholesterol.