PubMed Health⌕ Search

Biomedical subjects

M A Hediger

Publications and source records attributed to M A Hediger.

At least 91 records · Page 5Linked to original sources
PubMed1995

Neuronal high-affinity glutamate transport in the rat central nervous system.

EAAC1 is a neuronal and epithelial high affinity glutamate transporter previously cloned from rabbit intestine. Here we report the isolation of EAAC 1 from rat brain* and its expression in the central nervous system based on in situ hybridization. Strong signals were detected in brain, spinal cord and retina. Expression of EAAC1 was particularly strong in pyramidal cells of the cerebral cortex, pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, various thalamic nuclei and cells of certain retinal layers. EAAC1 was also expressed in non-glutamatergic neurons such as GABAergic cerebellar Purkinje cells and alpha-motor neurons of the spinal cord. We propose that EAAC1 is not only involved in the sequestration of glutamate at glutamatergic synapses and in protecting neurons from glutamate excitotoxicity, but also in the cellular metabolism involving glutamate.

Amino Acid Sequence↗
PubMed1995

Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter.

Active ion-coupled glutamate transport is of critical importance for excitatory synaptic transmission, normal cellular function, and epithelial amino acid metabolism. We previously reported the cloning of the rabbit intestinal high affinity glutamate transporter EAAC1 (Kanai, Y., and Hediger, M. A. (1992) Nature 360, 467-471), which is expressed in numerous tissues including intestine, kidney, liver, heart, and brain. Here, we report a detailed stoichiometric and kinetic analysis of EAAC1 expressed in Xenopus laevis oocytes. Uptake studies of 22Na+ and [14C]glutamate, in combination with measurements of intracellular pH with pH microelectrodes gave a glutamate to charge ratio of 1:1, a glutamate to Na+ ratio of 1:2, and a OH-/H+ to charge ratio of 1:1. Since transport is K+ dependent it can be concluded that EAAC1-mediated glutamate transport is coupled to the cotransport of 2 Na+ ions, the countertransport of one K+ ion and either the countertransport of one OH- ion or the cotransport of 1 H+ ion. We further demonstrate that under conditions where the electrochemical gradients for these ions are disrupted, EAAC1 runs in reverse, a transport mode which is of pathologic importance. 22Na+ uptake studies revealed that there is a low level of Na+ uptake in the absence of extracellular glutamate which appears to be analogous to the Na+ leak observed for the intestinal Na+/glucose cotransporter SGLT1. In voltage clamp studies, reducing extracellular Na+ from 100 to 10 mM strongly increased K0.5L-glutamate and decreased I(max). The data indicate that Na+ binding at the extracellular transporter surface becomes rate-limiting. Studies addressing the cooperativity of the substrate-binding sites indicate that there are two distinct Na(+)-binding sites with different affinities and that Na+ binding is modulated by extracellular glutamate. A hypothetical ordered kinetic transport model for EAAC1 is discussed.

ATP-Binding Cassette Transporters↗
PubMed1995

Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney.

Mammalian kidney is known to express a transport system specific for small peptides and pharmacologically active aminocephalosporins. This system is energized by a transmembrane electrochemical H+ gradient. Recently, a H(+)-coupled peptide transporter has been cloned from rabbit and human intestine (Fei et al. (1994) Nature 368, 563-566; Liang et al., J. Biol. Chem., in press). Functional studies have established that the renal peptide transport system is similar but not identical to its intestinal counterpart. Therefore, in an attempt to isolate the renal H+/peptide cotransporter cDNA, we screened a human kidney cDNA library with a probe derived from the rabbit intestinal H+/peptide cotransporter cDNA. This has resulted in the isolation of a positive clone with a 2190 bp long open reading frame. The predicted protein consists of 729 amino acids. Hydropathy analysis of the amino acid sequence indicates the presence of twelve putative transmembrane domains. The primary structure of this protein exhibits 50% identity and 70% similarity to the human intestinal H+/peptide cotransporter. Functional expression of the kidney cDNA in HeLa cells results in the induction of a H(+)-coupled transport system specific for small peptides and aminocephalosporins. Reverse transcription-coupled polymerase chain reaction demonstrates that the cloned transporter is expressed in human kidney but not in human intestine. This transporter, henceforth called PEPT 2, represents a new member in the growing family of H(+)-coupled transport systems in the mammalian plasma membrane.

Amino Acid Sequence↗
PubMed1995

Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization.

In mammalian small intestine, a H(+)-coupled peptide transporter is responsible for the absorption of small peptides arising from digestion of dietary proteins. Recently a cDNA clone encoding a H+/peptide cotransporter has been isolated from a rabbit intestinal cDNA library (Fei, Y.J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F.H., Romero, M.F., Singh, S.K., Boron, W. F., and Hediger, M. A. (1994) Nature 368, 563-566). Screening of a human intestinal cDNA library with a probe derived from the rabbit H+/peptide cotransporter cDNA resulted in the identification of a cDNA which when expressed in HeLa cells or in Xenopus laevis oocytes induced H(+)-dependent peptide transport activity. The predicted protein consists of 708 amino acids with 12 membrane-spanning domains and two putative sites for protein kinase C-dependent phosphorylation. The cDNA-induced transport process accepts dipeptides, tripeptides, and amino beta-lactam antibiotics but not free amino acids as substrates. The human H+/peptide cotransporter exhibits a high degree of homology (81% identity and 92% similarity) to the rabbit H+/peptide cotransporter. But surprisingly these transporters show only a weak homology to the H(+)-coupled peptide transport proteins present in bacteria and yeast. Chromosomal assignment studies with somatic cell hybrid analysis and in situ hybridization have located the gene encoding the cloned human H+/peptide cotransporter to chromosome 13 q33-->q34.

Amino Acid Sequence↗
PubMed1995

Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.

Amino Acid Sequence↗
PubMed1995

Cloning and regulation of expression of the rat kidney urea transporter (rUT2).

In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary-concentrating mechanism. Urea accumulation in the renal medulla is critical to the ability of the kidney to concentrate urine to an osmolality greater than systemic plasma. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of specialized phloretin-sensitive urea transporters. To study these transporters and their regulation of expression we isolated a cDNA which encodes the rat homologue (rUT2) of rabbit UT2 (You, G., C.P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M.A. Hediger, et al. Nature (Lond.). 1993. 365:844-847). Rat UT2 has 88% amino acid sequence identity to rabbit UT2 and 64% identity to the recently cloned human erythrocyte urea transporter, HUT11 (Olives, B., P. Neav, P. Bailly, M.A. Hediger, G. Rousselet, J.P. Cartron, and P. Ripoch J. Biol. Chem. 1994. 269:31649-31652). Analysis of rat kidney mRNA revealed two transcripts of size 2.9 and 4.0 kb which had spatially distinct distributions. Northern analysis and in situ hybridization showed that the 4.0-kb transcript was primarily responsive to changes in the protein content of the diet whereas the 2.9-kb transcript was responsive to changes in the hydration state of the animal. These studies reveal that the expression levels of the two rUT2 transcripts are modulated by different pathways to allow fluid and nitrogen balance to be regulated independently. Our data provide important insights into the regulation of the renal urea transporter UT2 and provide a basis on which to refine our understanding of the urinary concentrating mechanism and its regulation.

Amino Acid Sequence↗
PubMed1994

Cloning and functional expression of a urea transporter from human bone marrow cells.

A rapid passive urea transport has been previously described in the mammalian renal inner medullary collecting duct epithelial cells and in mammalian erythrocytes. Recently, a vasopressin-regulated urea transporter (UT2) has been cloned from a rabbit kidney medullary cDNA library (You, G., Smith, C. P., Kanai, Y., Lee, W. S., Stelzner, M., and Hediger, M. A. (1993) Nature 365, 844-847). We now report the cloning and characterization of a complementary DNA (HUT11) encoding an urea transporter isolated from a human bone marrow library. It encodes a 43,000-Da polypeptide of 391 amino acids that exhibited 63% sequence identity with the rabbit urea transporter and a similar membrane topology. HUT11 carries 2 putative glycosylation sites and 10 cysteines, of which only 7 are conserved at an equivalent position in UT2. HUT11 transcripts have been identified in human erythroid and renal tissues. Expression studies in Xenopus oocytes demonstrated that HUT11 mediates a facilitated urea transport that was inhibited, as described in mammalian erythrocytes, by very low concentrations of phloretin, p-chloromercuribenzene sulfonate, and urea analogues. No unidirectional movements of charged molecules, glycerol, or water were associated with HUT11 expression in oocytes. These findings suggest that HUT11 is most likely responsible for the facilitated urea transport in human red blood cells.

4-Chloromercuribenzenesulfonate↗
PubMed1994

The neuronal and epithelial human high affinity glutamate transporter. Insights into structure and mechanism of transport.

High affinity transport of glutamate across plasma membranes of brain neurons and epithelial is mediated by a Na(+)- and K(+)-coupled electrogenic transporter. Here we report the primary structure and functional characterization of the human high affinity glutamate transporter (HEAAC1). A unique characteristic of HEAAC1-mediated transport is that the affinity for glutamate and the maximal transport rate are strongly dependent on membrane potential. Our data provide new insights into individual steps of high affinity glutamate transport and show that the transport mechanism is distinct from that of the gamma-aminobutyric acid transporter GAT-1 and the Na+/glucose transporter SGLT1. Under voltage clamp condition, HEAAC1 mediated large substrate-evoked inward currents (up to 1 microA). The substrate specificity, stereospecificity, the Km value (30 +/- 3 microM at -60 mV) of the L-glutamate-evoked current, and Northern analysis all agree with previously reported characteristics of high affinity glutamate transport in brain. In contrast to SGLT1 and GAT-1, voltage jump studies of HEAAC1 yielded only minor relaxation currents. Classic inhibitors of brain glutamate uptake such as DL-threo-beta-hydroxyaspartate, L-trans-pyrrolidine 2,4,-dicarboxylic acid (PDC), and dihydrokainate were found to be either transport substrates or to have no significant effect on glutamate transport. We also found that the maximal transport rate for PDC was markedly reduced compared to that for L-glutamate. We propose that PDC most likely reduces the turnover rate of the transporter. A search of the sequence data bases revealed weak homology of HEAAC1 to the H(+)-coupled vesicular monoamine transporter, suggesting an evolutionary link between plasma membrane and vesicular transporters.

Adult↗
PubMed1994

Molecular cloning, primary structure, and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney.

Electrically silent Na(+)-(K+)-Cl- transporter systems are present in a wide variety of cells and serve diverse physiological functions. In chloride secretory and absorbing epithelia, these cotransporters provide the chloride entry mechanism crucial for transcellular chloride transport. We have isolated cDNAs encoding the two major electroneutral sodium-chloride transporters present in the mammalian kidney, the bumetanide-sensitive Na(+)-K(+)-Cl- symporter and thiazide-sensitive Na(+)-Cl- cotransporter, and have characterized their functional activity in Xenopus laevis oocytes. Despite their differing sensitivities to bumetanide and thiazides and their different requirements for potassium, these approximately 115-kDa proteins share significant sequence similarity (approximately 60%) and exhibit a topology featuring 12 potential membrane-spanning helices flanked by long non-hydrophobic domains at the NH2 and COOH termini. Northern blot analysis and in situ hybridization indicate that these transporters are expressed predominantly in kidney with an intrarenal distribution consistent with their recognized functional localization. These proteins establish a new family of Na(+)-(K+)-Cl- cotransporters.

Amino Acid Sequence↗
PubMed1994

The high affinity Na+/glucose cotransporter. Re-evaluation of function and distribution of expression.

We report the primary structure, functional characterization, and tissue distribution of the high affinity Na+/glucose cotransporter SGLT1 from rat kidney. Rat SGLT1 (665 amino acid residues) is 86-87% identical to SGLT1 from rabbit, pig, and human. High stringency Northern analysis demonstrated that SGLT1 is strongly expressed in small intestine and at lower levels in kidney, liver, and lung. In situ hybridization performed on kidney sections revealed that SGLT1 is predominantly present in S3 segments of the proximal tubule. In small intestine, SGLT1 message was located in cells of the lower two-thirds of intestinal villi. Expression of rat SGLT1 in Xenopus oocytes resulted in a large Na(+)-dependent uptake of [14C]-alpha-methyl-D-glucopyranoside (alpha MeGlc). Overall, the transport characteristics were similar to those of rabbit SGLT1. High affinity Na+/glucose cotransport in membrane vesicles was previously shown to be coupled to the cotransport of two Na+ ions (Turner, R. J., and Moran, A. (1982) J. Membr. Biol. 70, 37-45). Previous kinetic analysis of rat and rabbit SGLT1, however, demonstrated between second and first order dependence of sugar uptake on extracellular Na+ concentration, suggesting the existence of Na(+)-binding sites with different affinities. Here, we directly compared the initial rates of the alpha MeGlc uptake with alpha MeGlc-induced inward currents as an indicator of the Na+ flux. This analysis clearly revealed a Na+ to glucose coupling ratio of 2:1. In summary, our data provide important insights into the function and tissue distribution of the high affinity Na+/glucose cotransporter SGLT1 and clarify its role in the reabsorption mechanism of D-glucose in the kidney.

Amino Acid Sequence↗
PubMed1994

Expression cloning of a mammalian proton-coupled oligopeptide transporter.

In mammals, active transport of organic solutes across plasma membranes was thought to be primarily driven by the Na+ gradient. Here we report the cloning and functional characterization of a H(+)-coupled transporter of oligopeptides and peptide-derived antibiotics from rabbit small intestine. This new protein, named PepT1, displays an unusually broad substrate specificity. PepT1-mediated uptake is electrogenic, independent of extracellular Na+, K+ and Cl-, and of membrane potential. PepT1 messenger RNA was found in intestine, kidney and liver and in small amounts in brain. In the intestine, the PepT1 pathway constitutes a major mechanism for absorption of the products of protein digestion. To our knowledge, the PepT1 primary structure is the first reported for a proton-coupled organic solute transporter in vertebrates and represents an interesting evolutionary link between prokaryotic H(+)-coupled and vertebrate Na(+)-coupled transporters of organic solutes.

Amino Acid Sequence↗
PubMed1994

Expression of a rabbit renal ascorbic acid transporter in Xenopus laevis oocytes.

We examined the expression of renal ascorbic acid transporter(s) in Xenopus laevis oocytes after microinjection of cells with poly(A)+ RNA extracted from rabbit kidney cortex. Concomitant expression of the Na+-glucose cotransporter served as a control in these studies. Injection of poly(A)+ RNA into oocytes produced over a fivefold increase in the uptake of [14C]ascorbic acid (570 microM) compared with water-injected cells. Size fractionation of the kidney cortex mRNA by sucrose gradient revealed that the mRNA species that induced ascorbic acid transporter expression in oocytes was present in a fraction centered around 2.0 kilobases (kb) and had a size range of 1.8-3.1 kb. Injection of the active fraction into oocytes produced a > 40-fold increase in ascorbic acid uptake compared with water-injected controls. Expression of ascorbic acid transporter(s) was noticeable as early as 2 days after injection and was maximal after 7 days; it was also dependent on the amount of mRNA injected into oocytes. The induced uptake of [14C]ascorbic acid after injection of mRNA into oocytes was 1) Na+ dependent, as indicated by the almost complete lack of transport on removal of Na+ from the incubation medium; 2) significantly inhibited by unlabeled ascorbic acid and its structural analogue isoascorbic acid but not by D-glucose; and 3) saturable as a function of increasing the substrate concentration in the incubation medium (100-1,000 microM), with an apparent Km of 258 +/- 72.5 microM and a maximum velocity of 29.6 +/- 2.8 pmol.oocyte-1.2 h-1. These data demonstrate that X. laevis oocytes are a suitable system to functionally express the mammalian renal ascorbic acid transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

The major reabsorptive mechanism for D-glucose in the kidney is known to involve a low affinity high capacity Na+/glucose cotransporter, which is located in the early proximal convoluted tubule segment S1, and which has a Na+ to glucose coupling ratio of 1:1. Here we provide the first molecular evidence for this renal D-glucose reabsorptive mechanism. We report the characterization of a previously cloned human kidney cDNA that codes for a protein with 59% identity to the high affinity Na+/glucose cotransporter (SGLT1). Using expression studies with Xenopus laevis oocytes we demonstrate that this protein (termed SGLT2) mediates saturable Na(+)-dependent and phlorizin-sensitive transport of D-glucose and alpha-methyl-D-glucopyranoside (alpha MeGlc) with Km values of 1.6 mM for alpha MeGlc and approximately 250 to 300 mM for Na+, consistent with low affinity Na+/glucose cotransport. In contrast to SGLT1, SGLT2 does not transport D-galactose. By comparing the initial rate of [14C]-alpha MeGlc uptake with the Na(+)-influx calculated from alpha MeGlc-evoked inward currents, we show that the Na+ to glucose coupling ratio of SGLT2 is 1:1. Using combined in situ hybridization and immunocytochemistry with tubule segment specific marker antibodies, we demonstrate an extremely high level of SGLT2 message in proximal tubule S1 segments. This level of expression was also evident on Northern blots and likely confers the high capacity of this glucose transport system. We conclude that SGLT2 has properties characteristic of the renal low affinity high capacity Na+/glucose cotransporter as previously reported for perfused tubule preparations and brush border membrane vesicles. Knowledge of the structural and functional properties of this major renal Na+/glucose reabsorptive mechanism will advance our understanding of the pathophysiology of renal diseases such as familial renal glycosuria and diabetic renal disorders.

Animals↗
PubMed1994

Small intestine hexose transport in experimental diabetes. Increased transporter mRNA and protein expression in enterocytes.

The effect of insulinopenic diabetes on the expression of glucose transporters in the small intestine was investigated. Enterocytes were sequentially isolated from jejunum and ileum of normal fed rats, streptozotocin-diabetic rats, and diabetic rats treated with insulin. Facilitative glucose transporter (GLUT) 2, GLUT5, and sodium-dependent glucose transporter 1 protein content was increased from 1.5- to 6-fold in enterocytes isolated from diabetic animals in both jejunum and ileum. Insulin was able to reverse the increase in transporter protein expression seen after induction of diabetes. There was a four- to eightfold increase in the amount of enterocyte glucose transporter mRNA after diabetes with greater changes in sodium-dependent glucose transporter 1 and GLUT2 than in GLUT5 levels. In situ hybridization showed that after the induction of diabetes there was new hybridization in lower villus and crypt enterocytes that was reversed by insulin treatment. Thus, the increase in total hexose transport caused by diabetes is due to a premature expression of hexose transporters by enterocytes along the crypt-villus axis, causing a cumulative increase in enterocyte transporter protein during maturation. These changes are likely to represent an adaptive response by the organism to increase nutrient absorption in a perceived state of tissue starvation. These adaptive changes may lead to exacerbation of hyperglycemia in uncontrolled diabetes.

Amino Acid Sequence↗
PubMed1994

Structure, function and evolution of solute transporters in prokaryotes and eukaryotes.

In both prokaryotes and eukaryotes, transport systems of organic solutes can be classified as passive transporters, such as channels and facilitated transporters, and active transporters, which utilize diverse energy-coupling mechanisms. In the past decade, our understanding of the biochemistry and molecular biology of transporters from Escherichia coli has progressed significantly, whereas the analysis of mammalian transporters has initially been limited by the ability to purify membrane proteins. The recent development of methods to detect the activity of recombinant proteins in individual cells, however, has led to the cloning of several novel mammalian transporter cDNAs. One of the most useful expression cloning systems is Xenopus oocytes in conjunction with uptake studies and electrophysiological experiments. Overall, the sequence information and the functional data derived from many transporters has revealed unifying designs, similar energy-coupling mechanisms and common evolutionary origins. Here, I will provide a general survey of the known transport systems in bacteria, yeast, plants, insects and vertebrates and illustrate the different types of transport systems in mammals by discussing transporters recently studied in our laboratory.

Animals↗