NH4 + transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

NH₄ ⁺ transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [¹⁴C]methylammonium ion (¹⁴CH₃NH^{3 +}) into the intact cells. ¹⁴CH₃NH₃ ⁺ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH₄Cl instead of glutamate. Vibrio ABE-1 did not utilize CH₃NH₃ ⁺ as a carbon or nitrogen source. NH₄Cl and nonradiolabeled CH₃NH₃ ⁺ completely inhibited ¹⁴CH₃NH₃ ⁺ uptake. These results indicate that ¹⁴CH₃NH₃ ⁺ uptake in this bacterium is mediated via an NH₄ ⁺ transport system and not by a specific carrier for CH₃NH₃ ⁺. The respiratory substrate succinate was required to drive ¹⁴CH₃NH₃ ⁺ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H⁺ and/or Na⁺ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated ¹⁴CH₃NH₃ ⁺ uptake. The ¹⁴CH₃NH₃ ⁺ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0° and 15°C, and the apparent K _m value for CH₃NH₃ ⁺ of the uptake did not change significantly over the temperature range from 0° to 25°C. Thus, the NH₄ ⁺ transport system of this bacterium was highly active at low temperatures. Received: August 1, 1998 / Accepted: October 8, 1998     

Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural studies

Mutations in the human SLC26A4/Pendrin polypeptide (hPDS) cause Pendred Syndrome /DFNB4, syndromic deafness with enlargement of the vestibular aqueduct and low-penetrance goiter. Here we present data on cloning, protein overexpression and purification, refolding, and biophysical characterization of the recombinant hPDS STAS domain lacking its intrinsic variable sequence (STAS-ΔIVS). We report a reproducible protein refolding protocol enabling milligram scale expression and purification of uniformly ¹⁵N- and ¹³C^/15N-enriched hPDS STAS-ΔIVS domain suitable for structural characterization by solution NMR. Circular dichroism, one-dimensional ¹H, two-dimensional ¹H–¹⁵N HSQC, and ¹H–¹³C HSQC NMR spectra confirmed the well-folded state of purified hPDS STAS-ΔIVS in solution. Heteronuclear NMR chemical shift perturbation of select STAS-ΔIVS residues by GDP was observed at fast-to-intermediate NMR time scales. Intrinsic tryptophan fluorescence quench experiments demonstrated GDP binding to hPDS STAS-ΔIVS with K_d of 178 μM. These results are useful for structure/function characterization of hPDS STAS, the cytoplasmic subdomain of the congenital deafness protein, pendrin, as well as for studies of other mammalian STAS domains.     

Analysis of the SLC4A1 gene in three Mexican patients with hereditary spherocytosis: Report of a novel mutation

We analyzed the SLC4A1 gene in three Mexican patients with Hereditary Spherocytosis (HS). The promoter and all 20 exons were investigated through heteroduplex analysis and DNA sequencing. No DNA changes were detected in one of the three patients. Two well-known polymorphisms, Memphis I and the Diego-a blood group, were detected in another one. In the third, the HS phenotype could be explained by the novel 1885_1888dupCCGG mutation found in heterozygosis. This frameshift mutation is predicted to result in a truncated and unstable protein lacking normal functions.     

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification

1. Download high-res image (265KB)
2. Download full-size image

     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3

Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP⁺ uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP⁺ transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general.     

Regulation and induction of CYP3A11, CYP3A13 and CYP3A25 in C57BL/6J mouse liver

This study reports that dexamethasone (DEX) significantly induces CYP3A11, CYP3A13 and CYP3A25 mRNA expression in male and female 4 days, 3 weeks and 18 weeks old C57BL/6J mice. Furthermore, CYP3A activity, as measured by erythromycin-N-demethylation, is also significantly increased. PXR, RXRalpha and CAR are known to be involved in the induction of CYP3As. Here we report nuclear receptors PXR and RXRalpha but not CAR demonstrate gender- and age-dependent expression. Also, treatment of C57BL/6J mice with DEX induces PXR but not RXRalpha or CAR. In summary, we demonstrate DEX is not only able to up-regulate CYP3A expression and activity, but also the nuclear receptor PXR through which it may exert this effect. Furthermore, the gender- and age-dependent pattern of basal PXR and RXRalpha expression is similar to the 3 CYP3As analysed.     

Regulation of nitrate uptake into Chara corallina cells via NH+4 stimulation of NO−3 efflux

Abstract. Net NO₃ uptake by NO⁻₃ deficient Chara cells was used to calculate [NO⁻₃]_c assuming that the cytoplasm occupies 10% total volume and that nitrate reduction and storage are negligible (i.e. maximum [NO⁻₃]_c was calculated). A linear relationship was found between NO⁻₃ efflux and [NO⁻₃]_c. There was an initial burst of NO⁻₃ efflux when NH⁺₄ was added, followed by a slower efflux rate which matched influx rate such that net NO⁻₃ uptake was zero. Over 50% of NO⁻₃ that had been taken up in 2 h was lost within the first 5 min of NH⁺₄ addition. The Nernst equation was used to predict the direction of the electrochemical driving force for NO⁻₃ entry. Under the experimental conditions used NO⁻₃ efflux is actively transported. The differential involvement of both NO⁻₃ influx and NO⁻₃ efflux in the regulation of NO⁻₃ uptake is discussed and a model is proposed to account for these results which envisages discrete NO⁻₃ influx and NO⁻₃ efflux carriers.     

Molecular and phylogenetic characterization of a novel putative membrane transporter (SLC10A7), conserved in vertebrates and bacteria

The 'Solute Carrier Family SLC10' consists of six annotated members in humans, comprising two bile acid carriers (SLC10A1 and SLC10A2), one steroid sulfate transporter (SLC10A6), and three orphan carriers (SLC10A3 to SLC10A5). In this study we report molecular characterization and expression analysis of a novel member of the SLC10 family, SLC10A7, previously known as C4orf13. SLC10A7 proteins consist of 340-343 amino acids in humans, mice, rats, and frogs and show an overall amino acid sequence identity of >85%. SLC10A7 genes comprise 12 coding exons and show broad tissue expression pattern. When expressed in Xenopus laevis oocytes and HEK293 cells, SLC10A7 was detected in the plasma membrane but revealed no transport activity for bile acids and steroid sulfates. By immunofluorescence analysis of dual hemagglutinin (HA)- and FLAG-labeled SLC10A7 proteins in HEK293 cells, we established a topology of 10 transmembrane domains with an intracellular cis orientation of the N-terminal and C-terminal ends. This topology pattern is clearly different from the seven-transmembrane domain topology of the other SLC10 members but similar to hitherto uncharacterized non-vertebrate SLC10A7-related proteins. In contrast to the established SLC10 members, which are restricted to the taxonomic branch of vertebrates, SLC10A7-related proteins exist also in yeasts, plants, and bacteria, making SLC10A7 taxonomically the most widespread member of this carrier family. Vertebrate and bacterial SLC10A7 proteins exhibit >20% sequence identity, which is higher than the sequence identity of SLC10A7 to any other member of the SLC10 carrier family.     

Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site

The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na⁺ and K⁺ conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3–5 and TM8 that should function as an anion exchanger and a cation leak.     

A Substrate Access Tunnel in the Cytosolic Domain Is Not an Essential Feature of the Solute Carrier 4 (SLC4) Family of Bicarbonate Transporters

Anion exchanger 1 (AE1; Band 3; SLC4A1) is the founding member of the solute carrier 4 (SLC4) family of bicarbonate transporters that includes chloride/bicarbonate AEs and Na⁺-bicarbonate co-transporters (NBCs). These membrane proteins consist of an amino-terminal cytosolic domain involved in protein interactions and a carboxyl-terminal membrane domain that carries out the transport function. Mutation of a conserved arginine residue (R298S) in the cytosolic domain of NBCe1 (SLC4A4) is linked to proximal renal tubular acidosis and results in impaired transport function, suggesting that the cytosolic domain plays a role in substrate permeation. Introduction of single and double mutations at the equivalent arginine (Arg²⁸³) and at an interacting glutamate (Glu⁸⁵) in the cytosolic domain of human AE1 (cdAE1) had no effect on the cell surface expression or the transport activity of AE1 expressed in HEK-293 cells. In addition, the membrane domain of AE1 (mdAE1) efficiently mediated anion transport. A 2.1-Å resolution crystal structure of cdΔ54AE1 (residues 55–356 of cdAE1) lacking the amino-terminal and carboxyl-terminal disordered regions, produced at physiological pH, revealed an extensive hydrogen-bonded network involving Arg²⁸³ and Glu⁸⁵. Mutations at these residues affected the pH-dependent conformational changes and stability of cdΔ54AE1. As these structural alterations did not impair functional expression of AE1, the cytosolic and membrane domains operate independently. A substrate access tunnel within the cytosolic domain is not present in AE1 and therefore is not an essential feature of the SLC4 family of bicarbonate transporters.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

HCV NS3/4A蛋白酶抑制剂细胞筛选模型

丙型肝炎病毒(hepatitis C virus,HCV)持续感染可导致慢性丙型肝炎,并可发展为肝硬化和肝细胞癌。HCV NS3/4A蛋白酶在HCV复制和致病机制中起着非常关键的作用,已成为HCV研究热点。由于候选药物分子必须具有易进入细胞、稳定性好等特征,建立一种细胞水平上酶活性的测定系统对于筛选抗NS3/4A药物无疑有着重要意义。目前已有多种NS3/4A蛋白酶筛选系统开发出来,本文将对此作一综述。     

丙型肝炎病毒NS3/4A丝氨酸蛋白酶瞬时表达小鼠模型的建立

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶体内活性评价模型。方法:利用NS4A/B是NS3/4A丝氨酸蛋白酶作用底物的特性,构建融合基因NS3/NS4A/B-SEAP,底物片段NS4A/B插在NS3/4A和人分泌性碱性磷酸酶(SEAP)之间,融合基因表达后SEAP的分泌依赖于有活性的NS3/4A在NS4A/B位点的切割。将含融合基因的质粒NS3/4A(△4AB)SEAP通过水动力转染技术转染到小鼠体内,检测小鼠血清中SEAP的活性,高活性的SEAP是该评价体系成立的证据。结果与结论:在瞬时表达NS3/4A的小鼠血清中检测到了高活性的SEAP,建立了可用于评价抗NS3/4A的小鼠体内瞬时模型。     

The Axin/TNKS complex interacts with KIF3A and is required for insulin-stimulated GLUT4 translocation

Insulin-stimulated glucose uptake by the glucose transporter GLUT4 plays a central role in whole-body glucose homeostasis, dysregulation of which leads to type 2 diabetes. However, the molecular components and mechanisms regulating insulin-stimulated glucose uptake remain largely unclear. Here, we demonstrate that Axin interacts with the ADP-ribosylase tankyrase 2 (TNKS2) and the kinesin motor protein KIF3A, forming a ternary complex crucial for GLUT4 translocation in response to insulin. Specific knockdown of the individual components of the complex attenuated insulin-stimulated GLUT4 translocation to the plasma membrane. Importantly, TNKS2^−/− mice exhibit reduced insulin sensitivity and higher blood glucose levels when re-fed after fasting. Mechanistically, we demonstrate that in the absence of insulin, Axin, TNKS and KIF3A are co-localized with GLUT4 on the trans-Golgi network. Insulin treatment suppresses the ADP-ribosylase activity of TNKS, leading to a reduction in ADP ribosylation and ubiquitination of both Axin and TNKS, and a concurrent stabilization of the complex. Inhibition of Akt, the major effector kinase of insulin signaling, abrogates the insulin-mediated complex stabilization. We have thus elucidated a new protein complex that is directly associated with the motor protein kinesin in insulin-stimulated GLUT4 translocation.     

Association of a serotonin transporter gene (SLC6A4) 5-HTTLPR polymorphism with body mass index categories but not type 2 diabetes mellitus in Mexicans

The serotonergic system has been hypothesized to contribute to the biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI) categories. We investigate a possible association of 5-HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter gene (SLC6A4) with the development of T2DM and/or higher BMI by analyzing a sample of 138 individuals diagnosed with T2DM and 172 unrelated controls from the Mexican general population. In the total sample genotypes were distributed according to Hardy-Weinberg equilibrium, and S allele frequency was 0.58. There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Mexican population sample (p = 0.12). Nevertheless, logistic regression analysis of the L allele and increased BMI disclosed an association, after adjusting for age, sex and T2DM (p = 0.02, OR 1.74, 95% CI: 1.079–2.808).