Ala-504 is a determinant of substrate binding affinity in the mouse Na/dicarboxylate cotransporter

The Na⁺/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K_0.5 for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K_0.5 for both adipate and succinate.     

Transmembrane helices 3 and 4 are involved in substrate recognition by the Na+/dicarboxylate cotransporter, NaDC1

The Na(+)/dicarboxylate cotransporters (NaDC1) from mouse (m) and rabbit (rb) differ in their ability to handle glutarate. Substrate-dependent inward currents, measured using two-electrode voltage clamp, were similar for glutarate and succinate in Xenopus oocytes expressing mNaDC1. In contrast, currents evoked by glutarate in rbNaDC1 were only about 5% of the succinate-dependent currents. To identify domains involved in glutarate transport, we constructed a series of chimeric transporters between mouse and rabbit NaDC1. Although residues found in multiple transmembrane helices (TM) participate in glutarate transport, the most important contribution is made by TM 3 and 4 and the associated loops. The R(M3-4) chimera, consisting of rbNaDC1 with substitution of TM 3-4 from mNaDC1, had a decreased K(0.5)(glutarate) of 4 mM compared with 15 mM in wild-type rbNaDC1 without any effect on K(0.5)(succinate). The chimeras were also characterized using dual-label competitive uptakes with (14)C-glutarate and (3)H-succinate to calculate the transport specificity ratio (TSR), a measure of relative catalytic efficiency with the two substrates. The TSR analysis provides evidence for functional coupling in the transition state between TM 3 and 4. We conclude that TM 3 and 4 contain amino acid residues that are important determinants of substrate specificity and catalytic efficiency in NaDC1.     

Threonine-509 is a determinant of apparent affinity for both substrate and cations in the human Na+/dicarboxylate cotransporter

The Na+/dicarboxylate cotransporter (NaDC1) is involved in the absorption of citric acid cycle intermediates from the lumen of the renal proximal tubule and small intestine. The NaDC1 orthologues from human (h) and rabbit (rb) exhibit differences in citrate and cation transport properties. The citrate Km and sodium KNa values are much larger in human than rabbit NaDC1. Our previous study showed that transmembrane helices (TM) 7, 10, and 11 and associated loop regions contain the amino acids that are important in determining the differences in apparent citrate affinity, whereas TM10 and 11 determine differences in apparent sodium affinity. Chimera R10 (hNaDC1 with a substitution of TM10 and associated loop from rbNaDC1) contains only four amino acid differences between rb and hNaDC1. This chimera has similar apparent affinity for succinate and sodium as the wild-type rbNaDC1, and an intermediate Km for citrate. To identify individual residues in the TM10 region that determine functional differences between rb and hNaDC1, four mutants were made in which the rabbit sequence was substituted for that of the hNaDC1. Mutants with a serine or threonine at position 509 (or 512 in rbNaDC1) in TM10 have partial changes in Km for citrate and succinate but larger changes in apparent affinity for cations and substrate specificity for four-carbon dicarboxylates. The results show that the serine or threonine at position 509 (h) or 512 (rb) is the most important determinant of functional differences in apparent affinity for substrate and cations. Furthermore, the results suggest that the cation and substrate binding sites are located in close proximity to one another in NaDC1.     

Differential interaction of dicarboxylates with human sodium-dicarboxylate cotransporter 3 and organic anion transporters 1 and 3

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 of [(14)C]succinate (NaDC3), p-[(3)H]aminohippurate (OAT1), or [(3)H]estrone-3-sulfate (OAT3) uptake. Human NaDC3 was stably expressed as proven by immunochemical methods and by sodium-dependent uptake of succinate (K(0.5) for sodium activation, 44.6 mM; Hill coefficient, 2.1; K(m) for succinate, 18 μM). NaDC3 was best inhibited by succinate (IC(50) 25.5 μM) and less by α-ketoglutarate (IC(50) 69.2 μM) and fumarate (IC(50) 95.2 μM). Dicarboxylates with longer carbon backbones (adipate, pimelate, suberate) had low or no affinity for NaDC3. OAT1 exhibited the highest affinity for glutarate, α-ketoglutarate, and adipate (IC(50) between 3.3 and 6.2 μM), followed by pimelate (18.6 μM) and suberate (19.3 μM). The affinity of OAT1 to succinate and fumarate was low. OAT3 showed the same dicarboxylate selectivity with ～13-fold higher IC(50) values compared with OAT1. The data 1) reveal α-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.     

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.     

Role of conserved prolines in the structure and function of the Na+/dicarboxylate cotransporter 1, NaDC1

The Na+/dicarboxylate cotransporter 1 (NaDC1) is a low-affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TMs) 7 and 10. These transmembrane domains are of particular importance because they may be involved in determining the substrate or cation-binding affinity in NaDC1. Four conserved proline residues in TMs 7 and 10 of rabbit NaDC1 were replaced with alanine to promote ideal alpha helix or glycine to promote free conformation, and the mutant transporters were expressed in the HRPE cell line. Mutations of prolines in TM 10 produced decreased protein expression and activity, whereas mutations of prolines in TM 7 completely abolished protein expression and activity. The chemical chaperone glycerol was found to improve the expression of the Pro-351 mutants in TM 7, suggesting that these mutants had defects in trafficking. The inactive mutant transporters at position 351 could also be rescued by the addition of a proline at a second site. For example, the P351A-F347P mutant had restored activity, although its substrate specificity was altered. We conclude that, in TM 7, Pro-327 may be of particular importance in the function of the transporter, whereas Pro-351 may affect protein targeting. The prolines in TM 10, at positions 523 and 524, may not be directly involved in the transporter function but may be necessary for maintaining structure.     

Three aromatic amino acid residues critical for galactose transport in yeast Gal2 transporter

Tyr(446) in putative transmembrane segment 10 (TM10) of the yeast galactose transporter Gal2 has previously been identified as essential for galactose recognition. In the present study, alignment of the amino acid sequences of 63 sugar transporters or related proteins revealed 14 aromatic sites, including Tyr(446) of Gal2, that are conserved in >75% of these proteins. The importance of the remaining 13 conserved aromatic amino acids was examined individually by random mutagenesis using degenerate primers. Galactose transport-positive clones were identified by plate selection and subjected to DNA sequencing. For those transport-positive clones corresponding to Tyr(352), and Phe(504) mutants, all the amino acid substitutions comprised aromatic residues. The importance of the aromatic residues at these sites was further investigated by replacing them individually with each of the other 19 amino acids and measuring the galactose transport activity of the resulting mutants. Among both Tyr(352) and Phe(504) mutants, the other aromatic amino acids supported galactose transport; no other amino acids conferred high affinity transport activity. Thus, at least three aromatic sites are critical for galactose transport: one at the extracellular boundary of putative TM7 (Tyr(352)), one in the middle of putative TM10 (Tyr(446)), and one in the middle of putative TM12 (Phe(504)).     

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na(+)-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation.     

The N-terminal region of the transmembrane domain of human erythrocyte band 3. Residues critical for membrane insertion and transport activity

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.     

Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A

We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.     

Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport.

The Schizosaccharomyces pombe Tpr1 was isolated as suppressor of the Saccharomyces cerevisiae Delta trk1,2 potassium uptake deficient phenotype. Tpr1, for tetratrico peptide repeat, encodes a 1039 amino acid residues protein with several reiterated TPR units displaying significant homology to p150(TSP), a recently identified phosphoprotein of mouse, to S. cerevisiae CTR9 and to related sequences of human, Caenorhabditis elegans, Methanoccocus jannaschii and Arabidopsis thaliana. Expression of Tpr1 restored growth on 0.2 mM K(+) media, induced K(+) transport with a K(T) of 4.6 mM and resumed inward currents of -90 pA at -250 mV (pH 7.2) conducting K(+) and other alkali-metal ions. The tetratrico peptide repeat is a degenerate motif of 34 amino acids that is repeated several times within TPR-containing proteins and has been suggested to mediate protein-protein interactions. The sequence and putative binding properties of Tpr1 suggest the protein unlikely as transporter but involved in the enhancement of K(+) uptake via conventional carriers.     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Structural rearrangements at the translocation pore of the human glutamate transporter, EAAT1

Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-beta-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport.     

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.     

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.     

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.     

Functional characterization of high-affinity Na(+)/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na⁺/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is 70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a K_m for succinate of 4 µM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site. SLC13 gene family; citric acid cycle intermediate; lithium     

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.

     

Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat

In this study we examined the role of protein phosphorylation/dephosphorylation in the transport properties of the wheat ( Triticum aestivum ) root malate efflux transporter underlying Al resistance, TaALMT1. Pre-incubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurosporine) strongly inhibited both basal and Al³⁺-enhanced TaALMT1-mediated inward currents (malate efflux). Pre-incubation with phosphatase inhibitors (okadaic acid and cyclosporine A) resulted in a modest inhibition of the TaALMT1-mediated currents. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), enhanced TaALMT1-mediated inward currents. Since these observations suggest that TaALMT1 transport activity is regulated by PKC-mediated phosphorylation, we proceeded to modify candidate amino acids in the TaALMT1 protein in an effort to identify structural motifs underlying the process regulating phosphorylation. The transport properties of eight single point mutations (S56A, S183A, S324A, S337A, S351-352A, S384A, T323A and Y184F) generated in amino acid residues predicted to be phosphorylation sites and examined electrophysiologically. The basic transport properties of mutants S56A, S183A, S324A, S337A, S351-352A, T323A and Y184F were not altered relative to the wild-type TaALMT1. Likewise the sensitivity of these mutants to staurosporine resembled that observed for the wild-type transporter. However, the mutation S384A was noticeable, as in oocytes expressing this mutant protein TaALMT1-mediated basal and Al-enhanced currents were significantly inhibited, and the currents were insensitive to staurosporine or PMA. These findings indicate that S384 is an essential residue regulating TaALMT1 activity via direct protein phosphorylation, which precedes Al³⁺ enhancement of transport activity.     

Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (If or Ih) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brain If cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1-3. The transmembrane region of HAC4 was most homologous to partially cloned mouse If BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at -87.2 +/- 2.8 mV under control conditions and at -64.4 +/- 2.6 mV in the presence of intracellular 0.3 mM cAMP. The reversal potential was -34.2 +/- 0.9 mV in 140 mM Na+o and 5 mM K+o versus 10 mM Na+i and 145 mM K+i. These results indicate that HAC4 forms If in rabbit heart SA node.