Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

Transport of GABA at the Blood-CSF Interface

Abstract: The entry of GABA into cerebrospinal fluid (CSF) was studied in dogs anesthetized with pentobarbital and relaxed with suxamethonium. GABA was administered intravenously as a priming dose and subsequent maintenance infusion to compensate for the rapid elimination of the amino acid. Steady state concentrations of GABA in CSF were reached between 10 and 60 min after injection, the rate of entry tending to decrease with increasing plasma levels. During steady state conditions CSF concentrations showed great interin-dividual differences and varied between 0.03 and 5.1% of those in plasma. Probenecid and sodium valproate considerably enhanced the CSF/plasma concentration ratio of GABA. When GABA was directly injected into the liquor space, probenecid slowed down the elimination of GABA from CSF. The results suggest a transport of GABA into and out of CSF, the outward transport being inhibited by probenecid and sodium valproate.     

Functional Identification of the Hypoxanthine/Guanine Transporters YjcD and YgfQ and the Adenine Transporters PurP and YicO of Escherichia coli K-12

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N⁶-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.     

Iron and Porphyrin Trafficking in Heme Biogenesis

Iron is an essential element for diverse biological functions. In mammals, the majority of iron is enclosed within a single prosthetic group: heme. In metazoans, heme is synthesized via a highly conserved and coordinated pathway within the mitochondria. However, iron is acquired from the environment and subsequently assimilated into various cellular pathways, including heme synthesis. Both iron and heme are toxic but essential cofactors. How is iron transported from the extracellular milieu to the mitochondria? How are heme and heme intermediates coordinated with iron transport? Although recent studies have answered some questions, several pieces of this intriguing puzzle remain unsolved.     

Transport protein synthesis in non-growing yeast cells

Addition of a metabolizable substrate (glucose, ethanol and, to a degree, trehalose) to non-growing baker's yeast cells causes a boost of protein synthesis, reaching maximum rate 20 min after addition of glucose and 40–50 min after ethanol or trehalose addition. The synthesis involves that of transport proteins for various solutes which appear in the following sequence: H⁺, l-proline, sulfate, l-leucine, phosphate, α-methyl-d-glucoside, 2-aminoisobutyrate. With the exception of the phosphate transport system, the K_t of the synthesized systems is the same as before stimulation. Glucose is usually the best stimulant, but ethanol matches it in the case of sulfate and exceeds it in the case of proline. This may be connected with ethanol's stimulating the synthesis of transport proteins both in mitochondria and in the cytosol while glucose acts on cytosolic synthesis alone. The stimulation is often repressed by ammonium ions (leucine, proline, sulfate, H⁺), by antimycin (proline, trehalose, sulfate, H⁺), by iodoacetamide (all systems tested), and by anaerobic preincubation (leucine, proline, trehalose, sulfate). It is practically absent in a respiration-deficient petite mutant, only little depressed in the op₁ mutant lacking ADP/ATP exchange in mitochondria, but totally suppressed (with the exception of transport of phosphate) in a low-phosphorus strain. The addition of glucose causes a drop in intracellular inorganic monophosphate by 30%, diphosphate by 45%, ATP by 70%, in total amino acids by nearly 50%, in transmembrane potential (absolute value) by about 50%, an increase of high-molecular-weight polyphosphate by 65%, of total cAMP by more than 100%, in the endogenous respiration rate by more than 100%, and a change of intracellular pH from 6.80 to 7.05. Ethanol caused practically no change in ATP, total amino acids, endogenous respiration, intracellular pH or transmembrane potential; a slight decrease in inorganic monophosphate and diphosphate and a sizeable increase in high-molecular-weight polyphosphate. The synthesis of the various transport proteins thus appears to draw its energy from different sources and with different susceptibility to inhibitors. It is much more stimulated in facultatively aerobic species (Saccharomyces cerevisiae, Endomyces magnusii) than in strictly aerobic ones (Rhodotorula glutinis, Candida parapsilosis) where an inhibition of transport activity is often observed after preincubation with metabolizable substrates.     

Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction,oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the -configurated pyridine nucleotide and the -configurated form. Since NADH-linked dehydrogenases usually do not work with -NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.Abbreviation NADH -nicotinamide adenine dinucleotide reduced form     

Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

Regional Kinetic Constants for Blood–Brain Barrier Pyruvic Acid Transport in Conscious Rats by the Monocarboxylic Acid Carrier

The present investigation using labeled pyruvate describes the regional distribution and kinetics of the monocarboxylic acid carrier at the blood-brain barrier of conscious rats. The experimental procedure involved the arterial injection of a single bolus of 200 microliter containing [1-14C]pyruvate, [3H]water, and varying concentrations of unlabeled pyruvate into the common carotid via an indwelling externalized catheter. The hemisphere ipsi-lateral to the injection and rostral to the midbrain was removed and dissected into five regions. A kinetic analysis revealed no significant regional differences in Km values with an overall average of 1.37 mM. However, there was regional variation in the density of the monocarboxylic acid carrier as indicated by varied levels of the kinetic constant Vmax. The cortex showed the highest Vmax value of 0.42 +/- 0.08 mumol/min/g whereas values for the caudate/putamen, thalamus/hypothalamus, and remaining portion of hemisphere ranged significantly lower at 0.22-0.27 mumol/min/g. The Vmax for the hippocampus was intermediate at 0.37 +/- 0.12 mumol/min/g. The nonsaturable carrier described kinetically by KD had an overall average of 0.034 ml/min/g. The present study confirms quantitatively previous results suggesting a variable regional distribution of the monocarboxylic acid carrier.     

Coxal organs of Lithobius forficatus (Myriapoda,Chilopoda)

Summary In Lithobius forficatus each of the coxae of the four posterior trunk segments bear a pore field with several coxal pores. The surrounding single-layered epithelium is composed of four different cell types: the main epithelial cells having a fine-structural organization of transport cells with deep apical and basal folds of the cell surfaces and plasmalemma-mitochondrial complexes, junctional cells, exocrine glands, and the wall cells of the pore channel. The entire epithelium is separated from the hemolymph by an inner cellular sheath. It is assumed that the coxal organs participate in fluid uptake.