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B I Kanner

Publications and source records attributed to B I Kanner.

At least 37 records · Page 2Linked to original sources
PubMed1995

Glutamate 404 is involved in the substrate discrimination of GLT-1, a (Na+ + K+)-coupled glutamate transporter from rat brain.

Sodium-coupled glutamate transporters, located in the plasma membrane of nerve terminals and glial processes, serve to keep its extracellular glutamate concentration below extracellular levels. Moreover, they help in conjunction with diffusion to terminate the transmitter's action in synaptic transmission. We have investigated the role of negatively charged amino acid residues of GLT-1, a cloned (Na+ + K+)-coupled glutamate transporter from rat brain. Using site-directed mutagenesis we modified these negative residues, which are located in hydrophobic surroundings and are highly conserved within the glutamate transporter family. Out of five residues meeting these criteria, three, aspartate 398, glutamate 404, and aspartate 470, are critical for heterologously expressed glutamate transport. This defective transport cannot be attributed to the mere requirement of a negative charge at these positions. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of all mutant transporters indicates that their expression levels are similar to that of wild type. No cryptic activity was revealed by reconstitution experiments aimed to monitor the activity of transporter molecules not located in the plasma membrane. Significantly, whereas all of the mutants at the glutamate 404 position exhibit impaired transport of glutamate, they possess considerable transport of D- and L-aspartate, up to 80% of wild type values. Binding of glutamate is not impaired in these mutants. Our observations indicate that the glutamate 404 residue may be located in the vicinity of the glutamate-aspartate permeation pathway.

ATP-Binding Cassette Transporters↗
PubMed1994

The number of amino acid residues in hydrophilic loops connecting transmembrane domains of the GABA transporter GAT-1 is critical for its function.

Transporter proteins consist of multiple transmembrane domains connected by hydrophillic loops. As the importance of these loops in transport processes is poorly understood, we have studied this question using the cDNA coding for GAT-1, a Na+/Cl(-)-coupled gamma-aminobutyric acid transporter from rat brain. Deletions of randomly picked non-conserved single amino acids in the loops connecting helices 7 and 8 or 8 and 9 result in inactive transport upon expression in HeLa cells. However, transporters where these amino acids are replaced with glycine retain significant activity. The expression level of the inactive mutant transporters was similar to that of the wild-type, but one of these, delta Val-348, appears to be defectively targetted to the plasma membrane. Our data are compatible with the idea that a minimal length of the loops is required, presumably to enable the transmembrane domains to interact optimally with each other.

Amino Acid Sequence↗
PubMed1994

Histidine 326 is critical for the function of GLT-1, a (Na+ + K+)-coupled glutamate transporter from rat brain.

Removal of glutamate from the synaptic cleft is carried out by transporter molecules located in presynaptic nerve terminals and fine glial processes surrounding the cleft. Three such transporters, which are approximately 55% identical to each other, have recently been cloned. They catalyze electrogenic transport of this neurotransmitter, which is coupled to the fluxes of three ions: sodium, potassium, and protons (or hydroxyl). One of these transporters, GLT-1, contains 573 amino acids and 6-10 putative membrane-spanning alpha-helices. These helices contain only two positively charged amino acid residues (lysine 298 and histidine 326) that are fully conserved in the glutamate transporters and two related neutral amino acid transporters. Using site-directed mutagenesis we have investigated the role of these residues, each of which was replaced by small hydrophilic as well as by positively charged amino acids. Expression of all replacement mutants at the histidine 326 position reveals that they are severely impaired in sodium-dependent glutamate transport. On the other hand, mutations at lysine 298 retain significant activity, especially if a positively charged amino acid replaces the lysine. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of all mutant transporters indicates that their expression levels are similar to those of wild type. Reconstitution experiments, aimed to reveal the activity of transporter molecules not located in the plasma membrane, indicate that the lowered activity of the K298T and K298N transporters in intact cells is partly due to a targeting defect. Histidine residue 326 appears to be required for the intrinsic activity of the transporter. As histidine residues have been implicated in the mechanism of H+ transport in several systems, we propose that histidine 326 may be involved in the proton translocation accompanying sodium- and potassium-coupled glutamate transport.

Amino Acid Transport System X-AG↗
PubMed1994

Localization of the glutamate transporter GLT-1 in rat and macaque monkey retinae.

Antibodies directed against a glutamate transporter (GLT-1) purified from rat brain were applied to cryostat sections of rat and macaque monkey retinae. In the brain, GLT-1 expression is found mainly in astrocytes, and therefore it has been suggested that GLT-1 may be a glutamate transporter specific to glial cells. However, in the rat retina, cones and two distinct cone bipolar cell types were strongly immunoreactive. In the monkey retina, flat midget bipolars and one diffuse bipolar cell type (DB2)), were found to be labelled. Müller cells or astrocytes, the neuroglial cells of rat and monkey retinae, were not GLT-1-immunoreactive.

Amino Acid Transport System X-AG↗
PubMed1994

Identification of tryptophan residues critical for the function and targeting of the gamma-aminobutyric acid transporter (subtype A).

The gamma-aminobutyric acid transporter is localized in nerve terminals. It catalyzes coupled electrogenic translocation of the neurotransmitter with two or three sodium ions and one chloride ion. The transporter contains 599 amino acids and 12 putative membrane spanning alpha-helices. It is the first described member of a neurotransmitter transporter superfamily. Using site-directed mutagenesis we have investigated the role of all 10 tryptophan residues predicted to reside in these helices. All 10 have been changed to serine as well as to leucine residues. Expression of mutant cDNAs in which the tryptophans, located in positions 68, 222, and 230, are replaced by either of these two amino acids reveals that they are severely impaired in gamma-aminobutyric acid transport. Mutants in which a phenylalanine or a tyrosine residue is introduced, at either position 68 or 230, are active. On the other hand, at the 222 position replacement of the tryptophan by the aromatic amino acids results in inactive transport. After prelabeling of the proteins with [35S]methionine, immunoprecipitation of mutant transporters indicates that their expression levels are similar to those of the wild type. Reconstitution experiments, aimed to reveal the activity of transporter molecules not apparent in the plasma membrane, indicate that the lack of activity of the W230S transporter in intact cells is by and large due to its inefficient targeting to the plasma membrane. Tryptophan residues 68 and 222 appear to be required for the intrinsic activity of the transporter. Based on several observations, including one that tryptophan residue 222 is conserved in all amino acid transporter members of the superfamily, but not in those transporting biogenic amines, we hypothesize that the pi electrons of this tryptophan could be involved in the binding of the amino group of these neurotransmitters.

Amino Acid Sequence↗
PubMed1994

Sodium-coupled neurotransmitter transport: structure, function and regulation.

The removal of neurotransmitters by their transporters--located in the plasma membranes of nerve terminals and glial cells--plays an important role in the termination of synaptic transmission. In the last 3 years, many neurotransmitter transporters have been cloned. Structurally and functionally they can be divided into two groups: glutamate transporters, of which to date three have been cloned, couple the flow of glutamate to that of sodium and potassium. The second group of transporters includes those for GABA, glycine, taurine, norepinephrine, dopamine and serotonin. They are sodium- and chloride-dependent, but do not require potassium for function. One of these, the GABAA transporter, encoded by GAT-1, is perhaps the best characterized. It has been purified and reconstituted and has a molecular mass of around 80 kDa, of which 10-15 kDa is sugar. Amino and carboxyl termini (around 50 amino acids each) are not required for function. The transporter is protected against proteolysis at multiple sites by GABA, provided that the two cosubstrates--sodium and chloride--are present. Several amino acid residues that are critical for function have been identified in the GABA transporter. These include arginine-69 and tryptophan-222 located in the first and fourth putative transmembrane helices, respectively. The first is possibly involved in the binding of chloride. The tryptophan appears to serve as a binding site for the amino group of GABA.

Animals↗
PubMed1993

Phosphorylation and modulation of brain glutamate transporters by protein kinase C.

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.

Amino Acid Transport System X-AG↗
PubMed1993

The substrates of a sodium- and chloride-coupled gamma-aminobutyric acid transporter protect multiple sites throughout the protein against proteolytic cleavage.

Fragments of the (Na(+) + Cl-)-coupled GABAA transporter were produced by proteolysis of membrane vesicles and reconstituted preparations from rat brain. The former were digested with Pronase, the latter with trypsin. Fragments with different apparent molecular masses were recognized by sequence-directed antibodies raised against this transporter. When GABA was present in the digestion medium, the generation of these fragments was almost entirely blocked. At the same time, the neurotransmitter largely prevented the loss of activity caused by the protease. The effect was specific for GABA; protection was not afforded by other neurotransmitters. It was only observed when the two cosubstrates, sodium and chloride, were present on the same side of the membrane as GABA. The results indicate that the transporter may exist in two conformations. In the absence of one or more of the substrates, multiple sites located throughout the transporter are accessible to the proteases. In the presence of all three substrates--conditions favoring the formation of the translocation complex--the conformation is changed such that these sites become inaccessible to protease action.

Amino Acid Sequence↗
PubMed1993

Hippocampal gamma-aminobutyric acid and benzodiazepine receptors after early phenobarbital exposure.

Mice were exposed to phenobarbital (PhB) prenatally (PreB offspring) by feeding their mothers 3 g/kg PhB in milled food on gestation days 9-18, or neonatally by directly injecting pups of intact mothers with daily dose of 50 mg PhB on postnatal days 2-21 (NeoB offspring). At age 22 or 50 days, the offspring were tested for gamma-aminobutyric acid (GABA) up take in the hippocampus and in the rest of the brain. In addition, [3H]muscimol and [3H]flunitrazepam binding in the hippocampus and cortex were measured in the offspring at age 22 and 50 days. Long-term decrease in GABA uptake was found in the NeoB group. A 23% decrease was found in 22-day-old mice (P < 0.001) and a 22% decrease in 50-day-old mice (P < 0.05). In addition, there was a 22% decrease in GABA uptake in the brain of 22-day-old PreB mice (P < 0.05). An increase of 52% in [3H]muscimol binding (P < 0.001) and 45% (P < 0.001) in [3H]flunitrazepam binding were measured in the hippocampus in the 22-day-old NeoB mice; no differences were found in affinity. The differences were short-term and could no longer be detected at age 50 days. No differences were found in the cortex; unlike NeoB, PreB mice did not differ from controls. The results suggest upregulation of the GABAergic system in early PhB exposed mice.

Animals↗
PubMed1993

Glutamate transporters from brain. A novel neurotransmitter transporter family.

The removal of neurotransmitters by their transporters in presynaptic nerve terminals and glial cells plays an important role in the termination of synaptic transmission. Many neurotransmitter transporters, which are sodium- and chloride-coupled, have been cloned and shown to constitute a large superfamily. Glutamate is the major excitatory neurotransmitter in the central nervous system. If not efficiently removed, it causes death of neuronal cells. Its transporter couples the flow of glutamate to that of sodium and potassium. Recently three different but related glutamate transporters have been cloned, which have no significant homology to the members of the superfamily.

Amino Acid Sequence↗
PubMed1993

Identification of domains of a cloned rat brain GABA transporter which are not required for its functional expression.

The sodium and chloride coupled gamma-aminobutyric acid (GABA) transporter purified from rat brain, belongs to a superfamily of neurotransmitter transporters. They are involved in the termination of synaptic transmission and are predicted to have 12 membrane spanning alpha-helices with both amino- and carboxyl-termini oriented toward the cytoplasm. In order to define the domains not required for functional expression, we have constructed and expressed a series of deletion mutants in GAT-1, the cDNA clone encoding for the transporter. Transporters truncated at either end until just a few amino-acids distance from the beginning of helix 1 and the end of helix 12, retain their ability to catalyze sodium and chloride-dependent GABA transport.

Amino Acid Sequence↗
PubMed1993

Only one of the charged amino acids located in the transmembrane alpha-helices of the gamma-aminobutyric acid transporter (subtype A) is essential for its activity.

The gamma-aminobutyric acid (GABA) transporter (subtype A) is located in nerve terminals and catalyses coupled electrogenic uptake of the neurotransmitter with two or three sodium and one chloride ions. It contains 599 amino acids and 12 putative membrane spanning alpha-helices and is the first described member of a neurotransmitter transporter superfamily. The membrane domain contains 5 charged amino acids which are basically conserved. Using site-directed mutagenesis, we show that only one of them, arginine 69, is absolutely essential for activity. It is located in a highly conserved region encompassing parts of helices 1 and 2. The three other positively charged amino acids and the only negative charged one, glutamate 467, are not critical. These results suggest that the translocation pathway of the sodium ions through the membrane does not involve charged amino acid residues and underline the importance of the highly conserved stretch between amino acids 66 and 86.

Base Sequence↗
PubMed1992

Cloning and expression of a rat brain L-glutamate transporter.

Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Amino Acid Sequence↗
PubMed1992

A monoclonal antibody against a Na(+)-L-glutamate cotransporter from rat brain.

A monoclonal mouse IgM antibody (Z8E9) was raised against the Na(+)-L-glutamate cotransporter from rat brain. In a preparation of brain plasma membrane vesicles, Z8E9 binds specifically to a polypeptide with an apparent molecular weight of 70,000 and inhibits Na+ gradient-dependent L-glutamate cotransport (up to 50%) in brain membrane vesicles. In the membrane vesicles, the antibody does not alter the membrane permeability for Na+ and K+ nor the Na+ gradient-dependent uptake of gamma-aminobutyric acid. Kinetic experiments showed that Z8E9 does not alter the K0.5 values for L-glutamate and Na+ activation of L-glutamate transport. However, an apparent cooperativity observed for L-glutamate activation was increased, and the Vmax of L-glutamate transport was decreased. Immunostaining of rat cerebellum identified antigenic sites of Z8E9 in Golgi epithelial cells and astrocytes (by light and electron microscopy), whereas no labeling at nerve terminals was detected. The data suggest that a component of a Na(+)-L-glutamate cotransporter subtype has been identified that is specific for glia cells in brain.

Amino Acid Transport System X-AG↗
PubMed1992

Comparative analysis of sodium-dependent L-glutamate transport of synaptosomal and astroglial membrane vesicles from mouse cortex.

Uptake of [3H]L-glutamate into membrane vesicles prepared from either mouse cortical astrocyte cultures or synaptosomes was found to be an electrogenic sodium- and potassium-dependent transport process with saturable uptake kinetics. Pharmacological differences were revealed by using a variety of substrate analogues. L-trans-PDC inhibited the synaptosomal glutamate transport 2-4-fold stronger than the astroglial uptake. The substrate analogues DL-threo-beta-hydroxy-aspartate, DL-aspartate-beta-hydroxamate, L-aspartate and D-aspartate inhibited glutamate transport of astroglial and neuronal membrane vesicles in a distinctive manner, whereas D-glutamate, quisqualate and dihydrokainate had no effect in either case. Immunoblotting and immunocytochemical labeling with antibodies against the rat brain glutamate transporter revealed the selective reaction of a band at about 75 kDa mol. wt. and a specific pattern of astrocyte immunostaining.

Amino Acid Transport System X-AG↗
PubMed1992

Neuronal and glial gamma-aminobutyric acid+ transporters are distinct proteins.

In the central nervous system, two subtypes of sodium- and chloride-coupled GABA transporter exist. One is sensitive to ACHC, the other to beta-alanine. They are thought to be of neuronal and glial origin, respectively. GABA transport in membrane vesicles derived from astroglial cells was found to be sodium- and chloride-dependent, electrogenic and much more sensitive to beta-alanine than to ACHC. Immunoblotting with antibodies directed against a variety of sequences of the ACHC-sensitive transporter indicated that none of these epitopes was shared by the glial transporter.

Amino Acids↗