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The structure and function of sodium-coupled neurotransmitter transporters
294
S-18 Transporter Systems in the Brain
The structure and function of sodium-coupled neurotransmitter transporters
B.I. Kanner Department (~/"Biochcmivlrv, ttadassah Medical School, The Hebrew University, .lcrusalem, Israel Key words. GABA and L-glutamate transporters: Coupled transport: Purification: Reconstitution: Molecular cloning; Sitedirected mutagenesis
Summary GABA and L-glutamate transporters are involved in terminating synaptic transmission by these transmitters. These sodium-coupled transporters have been purified, reconstituted and cloned. They are members of novel and distinct fanfilies. Their mechanism is being elucidated using site-directed mutagenesis and other approaches. The overall process of synaptic transmission may be divided into three stages: (a) release of transmitter into the synaptic cleft; (b) its interaction with postsynaptic receptors; (c) removal of the transmitter from the synapse. This removal, which enables termination of the signal in most cases, occurs through their reuptake back to the presynaptic terminal or into glial elements in a sodium dependent process. This process assures both constant and high levels of neurotransmitters in the neuron and low concentrations in the cleft. GABA is accumulated by electrogenic co-transport with sodium and chloride. Also the L-glutamate transporter from rat brain is sodium coupled. Although chloride is not required, the influx of L-glutamate is absolutely dependent oil internal potassium. It is of interest to note that influx of GABA is not dependent on internal potassium. L-Glutamate is cotransported with sodium and alter translocation of these two species, potassium is translocated the other way enabling reorientation of the binding sites for sodium and L-glutamate. The whole process is electrogenic. Using the reconstitution methodology which makes it possible to reconstitute many samples simultaneously and rapidly, both the GABA (Radian et al., 1986) and the L-glutamate transporter (Danbolt et al., 1990) have been purified to an apparent homogeneity. Both are glycoproteins and both have an apparent molecular weight of 70 80 kDa. The two transporters retain all the properties observed in membrane vesicles. We have recently cloned and expressed a GABA transporter from rat brain in a collaborative effort with the laboratories o1" H.A. Lester and N. Nelson (Guastella et al., 1990). Rat brain GABA transporter protein, purified as described (Radian et al.. 1986), was subjected to cyanogen bromide degradation, and several of the resulting fragments were sequenced. The sequence of the longest peptide (QPSEDIVRPENG) was used to design oligonucleotide probes. Since sucrose density RNA fractionation had shown that GABA transporter mRNA was in the 4~5 kb size range, a 2-ZAPII rat brain cDNA library containing inserts of 4 kb and greater was screened with conventional plaque hybridization techniques. Two plaques screened as positives through successive platings, mRNA was synthesized in vitro from each clone and tested for its ability to express functional GABA transporters in Xenopus oocytes. One clone tested positive in the oocyte assay. It was selected for detailed characterization and designated GAT-1 (GABA transporter 1). Oocytes injected with GAT-I synthetic mRNA accumuhtted [3H]GABA 50-100 fold over control levels. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium- and chloride-dependent, and is pharmacologically similar to neuron-specific plasma membrane GABA transporters. GAT-I expression in rat brain was also examined by probing polyadenylated RNA from cerebrum, cerebellum and brain stem with nick-translated GAT-1. A single band of about 4.2 kb was visualized in each brain sample, which agrees with RNA fractionation experiments: no bands were detectable in liver mRNA. The GAT-I protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kDa. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-I is not homologous to any previously identified proteins. Therefore, GAT-I appears to be a member of a previously uncharacterized family of transport molecules. As a matter of fact it appears to be the first identified member of a superfamily of neurotransmitter transporters. The membrane domain of GAT-1 contains five charged amino acids which are basically conserved. Using site-directed
295 mutagenesis we show that only one of them - argenine 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. Using the antibodies raised against the glutamate transporter, the immunocytochemical localization of the transporter was studied at the light and electron microscopic level in rat central nervous system. In all regions examined (including cerebral cortex, caudate-putamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial ceils rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared nonimmunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us (Danbolt et al., 1992). Using an antibody against the glial L-glutamate transporter from rat brain, we have isolated a complementary DNA clone (pT7-GLT-1) encoding this transporter. Expression of pT7-GLT-I in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and exhibits the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane c~-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. Therefore, GLT-l appears to be a member of a previously uncharacterized family of transport molecules (Pines et al., 1992). References Danbolt, N.C., Pines, G. and Kanner, B.I. (1990) Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain. Biochemistry 29, 6734~6740. Danbolt, N.C., Storm-Mathisen, J. and Kanner, B.I. (1992) A [ N a - + K+]-coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience 51,295-310. Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M.C., Davidson, N.C., Lester, H.A. and Kanner, B.I. (1990) Cloning and expression of a rat brain GABA transporter. Science 249, 1303 1306. Pines, G., Danbolt, N.C., Bjoras, M., Zhang, Y., Bendahan, A., Eide, L., Koepsell, H., Storm-Mathisen, J,, Seeberg, E. and Kanner, B.I. (1992) Nature 360, 464~467. Radian, R., Bendahan, A. and Kanner, B.I. (1986) Purification and identification of the functional sodium- and chloridecoupled 7-aminobutyric acid transport glycoprotein from rat brain. J. Biol. Chem. 261, 15437-15441.
Molecular characterization of the serotonin transporter
D. Graham and S.Z. Langer Synth~qabo Recherche (L.E.R,S.), 92220 Bagneux, France Key words." Serotonin transporter; Litoxetine; Antidepressant drugs; Depression
The sodium-ion coupled transport system for serotonin or serotonin transporter functions to reduce extracellular concentrations of this biogenic amine. The localization of this serotonin transporter at presynaptic serotonergic nerve terminals, for example, provides a neurotransmitter inactivation mechanism to reduce synaptic cleft concentrations of released serotonin. In addition, the presence of the serotonin transporter in other cell types such as platelets and mast cells permits the concentration and storage of serotonin for subsequent secretion. Moreover, uptake of serotonin by this transporter in fact represents a secondary active transport process in that the driving force for active neurotransmitter uptake stems from the electrochemical gradients for sodium and potassium ions created across the cellular plasma membranes by the functioning of the N a - K ~/ATPase pump. Molecular pharmacology studies Initial information on the serotonin transporter arose from experiments on radiolabelled serotonin uptake into
S-18 Transporter Systems in the Brain
The structure and function of sodium-coupled neurotransmitter transporters
B.I. Kanner Department (~/"Biochcmivlrv, ttadassah Medical School, The Hebrew University, .lcrusalem, Israel Key words. GABA and L-glutamate transporters: Coupled transport: Purification: Reconstitution: Molecular cloning; Sitedirected mutagenesis
Summary GABA and L-glutamate transporters are involved in terminating synaptic transmission by these transmitters. These sodium-coupled transporters have been purified, reconstituted and cloned. They are members of novel and distinct fanfilies. Their mechanism is being elucidated using site-directed mutagenesis and other approaches. The overall process of synaptic transmission may be divided into three stages: (a) release of transmitter into the synaptic cleft; (b) its interaction with postsynaptic receptors; (c) removal of the transmitter from the synapse. This removal, which enables termination of the signal in most cases, occurs through their reuptake back to the presynaptic terminal or into glial elements in a sodium dependent process. This process assures both constant and high levels of neurotransmitters in the neuron and low concentrations in the cleft. GABA is accumulated by electrogenic co-transport with sodium and chloride. Also the L-glutamate transporter from rat brain is sodium coupled. Although chloride is not required, the influx of L-glutamate is absolutely dependent oil internal potassium. It is of interest to note that influx of GABA is not dependent on internal potassium. L-Glutamate is cotransported with sodium and alter translocation of these two species, potassium is translocated the other way enabling reorientation of the binding sites for sodium and L-glutamate. The whole process is electrogenic. Using the reconstitution methodology which makes it possible to reconstitute many samples simultaneously and rapidly, both the GABA (Radian et al., 1986) and the L-glutamate transporter (Danbolt et al., 1990) have been purified to an apparent homogeneity. Both are glycoproteins and both have an apparent molecular weight of 70 80 kDa. The two transporters retain all the properties observed in membrane vesicles. We have recently cloned and expressed a GABA transporter from rat brain in a collaborative effort with the laboratories o1" H.A. Lester and N. Nelson (Guastella et al., 1990). Rat brain GABA transporter protein, purified as described (Radian et al.. 1986), was subjected to cyanogen bromide degradation, and several of the resulting fragments were sequenced. The sequence of the longest peptide (QPSEDIVRPENG) was used to design oligonucleotide probes. Since sucrose density RNA fractionation had shown that GABA transporter mRNA was in the 4~5 kb size range, a 2-ZAPII rat brain cDNA library containing inserts of 4 kb and greater was screened with conventional plaque hybridization techniques. Two plaques screened as positives through successive platings, mRNA was synthesized in vitro from each clone and tested for its ability to express functional GABA transporters in Xenopus oocytes. One clone tested positive in the oocyte assay. It was selected for detailed characterization and designated GAT-1 (GABA transporter 1). Oocytes injected with GAT-I synthetic mRNA accumuhtted [3H]GABA 50-100 fold over control levels. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium- and chloride-dependent, and is pharmacologically similar to neuron-specific plasma membrane GABA transporters. GAT-I expression in rat brain was also examined by probing polyadenylated RNA from cerebrum, cerebellum and brain stem with nick-translated GAT-1. A single band of about 4.2 kb was visualized in each brain sample, which agrees with RNA fractionation experiments: no bands were detectable in liver mRNA. The GAT-I protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kDa. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-I is not homologous to any previously identified proteins. Therefore, GAT-I appears to be a member of a previously uncharacterized family of transport molecules. As a matter of fact it appears to be the first identified member of a superfamily of neurotransmitter transporters. The membrane domain of GAT-1 contains five charged amino acids which are basically conserved. Using site-directed
295 mutagenesis we show that only one of them - argenine 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. Using the antibodies raised against the glutamate transporter, the immunocytochemical localization of the transporter was studied at the light and electron microscopic level in rat central nervous system. In all regions examined (including cerebral cortex, caudate-putamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial ceils rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared nonimmunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us (Danbolt et al., 1992). Using an antibody against the glial L-glutamate transporter from rat brain, we have isolated a complementary DNA clone (pT7-GLT-1) encoding this transporter. Expression of pT7-GLT-I in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and exhibits the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane c~-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. Therefore, GLT-l appears to be a member of a previously uncharacterized family of transport molecules (Pines et al., 1992). References Danbolt, N.C., Pines, G. and Kanner, B.I. (1990) Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain. Biochemistry 29, 6734~6740. Danbolt, N.C., Storm-Mathisen, J. and Kanner, B.I. (1992) A [ N a - + K+]-coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience 51,295-310. Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M.C., Davidson, N.C., Lester, H.A. and Kanner, B.I. (1990) Cloning and expression of a rat brain GABA transporter. Science 249, 1303 1306. Pines, G., Danbolt, N.C., Bjoras, M., Zhang, Y., Bendahan, A., Eide, L., Koepsell, H., Storm-Mathisen, J,, Seeberg, E. and Kanner, B.I. (1992) Nature 360, 464~467. Radian, R., Bendahan, A. and Kanner, B.I. (1986) Purification and identification of the functional sodium- and chloridecoupled 7-aminobutyric acid transport glycoprotein from rat brain. J. Biol. Chem. 261, 15437-15441.
Molecular characterization of the serotonin transporter
D. Graham and S.Z. Langer Synth~qabo Recherche (L.E.R,S.), 92220 Bagneux, France Key words." Serotonin transporter; Litoxetine; Antidepressant drugs; Depression
The sodium-ion coupled transport system for serotonin or serotonin transporter functions to reduce extracellular concentrations of this biogenic amine. The localization of this serotonin transporter at presynaptic serotonergic nerve terminals, for example, provides a neurotransmitter inactivation mechanism to reduce synaptic cleft concentrations of released serotonin. In addition, the presence of the serotonin transporter in other cell types such as platelets and mast cells permits the concentration and storage of serotonin for subsequent secretion. Moreover, uptake of serotonin by this transporter in fact represents a secondary active transport process in that the driving force for active neurotransmitter uptake stems from the electrochemical gradients for sodium and potassium ions created across the cellular plasma membranes by the functioning of the N a - K ~/ATPase pump. Molecular pharmacology studies Initial information on the serotonin transporter arose from experiments on radiolabelled serotonin uptake into