Evidence for the Participation of a Cytosolic NADP+-Dependent Oxidoreductase in the Catabolism of γ-Hydroxybutyrate In Vivo

The concentration of gamma-hydroxybutyrate (GHB) in brain, kidney, and muscle as well as the clearance of [1-14C]GHB in plasma have been found to be altered by the administration of a number of metabolic intermediates and drugs that inhibit the NADP+-dependent oxidoreductase, "GHB dehydrogenase," an enzyme that catalyzes the oxidation of GHB to succinic semialdehyde. Administration of valproate, salicylate, and phenylacetate, all inhibitors of GHB dehydrogenase, significantly increased the concentration of GHB in brain; salicylate increased GHB concentration in kidney, and alpha-ketoisocaproate increased GHB levels in kidney and muscle. The half-life of [1-14C]GHB in plasma was decreased by D-glucuronate, a compound that stimulates the oxidation of GHB by this enzyme and was increased by a competitive substrate of the enzyme, L-gulonate. The results of these experiments suggest a role for GHB dehydrogenase in the regulation of tissue levels of endogenous GHB.     

Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ-Aminobutyric AcidA Receptors

A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.     

A High-Affinity, Na+-Dependent Uptake System for γ-Hydroxybutyrate in Membrane Vesicles Prepared from Rat Brain

Abstract: γ-Hydroxybutyrate (GHB) is a compound with numerous neuropharmacological properties. The discovery of its biosynthetic system, together with its endogenous repartition, have prompted its possible implication in neurotransmission. This role is also supported by the existence, reported here, of a high-affinity uptake system for GHB ( K _m= 46.4 μM)in both purified brain plasma membrane vesicles and in the crude mitochondrial fraction. GHB uptake is dependent on a Na⁺ gradient but is independent of the membrane electrical potential. Cl⁻ and K⁺ can also modulate the uptake. As an approach to determine the conformation required for GHB uptake, a series of related compounds, including aryl-or alkyl-derivatives, has been examined for ability to inhibit GHB uptake. The regional distribution of uptake is also indicative of its possible physiological role, since in striatum, an area where GHB has a known pharmacological effect on dopaminergic neurons, this uptake activity is the highest.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Transmembrane 36CI− Flux Measurements and Desensitization of the γ-Aminobutyric AcidA Receptor

Abstract: Some data on the concentration range of response and the concentration for half-response (EC₅₀) of γ-aminobutyric acid (GABA) for the GABA_A receptor are reviewed and compared. An analysis of the ³⁶CI⁻ flux assay demonstrates that both the EC₅₀ and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the ³⁶CI⁻ concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC₅₀ can be decreased by orders of magnitude by increasing the assay time. The EC₅₀ measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC₅₀ depends on the rate of receptor desensitization. The computer simulations showed that a GABA_A receptor with a half-response concentration of 100 μ M GABA can give ³⁶CI⁻ flux measurements with an EC₅₀ value 100-fold lower.     

γ-Aminobutyric Acid-Activated 36Cl- Influx: A Functional In Vitro Assay for CNS γ-Aminobutyric Acid Receptors of Insects

The effects of gamma-aminobutyric acid (GABA) on the uptake of 36Cl- into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 microM GABA (after 4-s incubation, then rapid quenching) the influx of 36Cl- was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 microM), but was not significantly affected by bicuculline (100 microM). Results of 36Cl- influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.     

Regulation of the γ-Aminobutyric AcidA Receptor by γ-Aminobutyric Acid Levels Within the Postsynaptic Cell

Synaptosomes and synaptoneurosomes were prepared from rat cerebral cortex. Comparison of the amino acid levels in the two types of organelles and of the effects of gabaculine thereon indicated that the neurosome portion of synaptoneurosomes constituted the major influencing component of the organelles. Administration to rats of inhibitors of gamma-aminobutyric acid (GABA) degradation, such as gabaculine and L-cycloserine, resulted in elevated GABA levels in synaptoneurosomes and a decrease in muscimol-stimulated Cl- up-take by the organelles. Addition of gabaculine directly to the incubation medium for the uptake assay had no effect on the Cl- transport. In contrast, administration to rats of isonicotinic acid hydrazide, an inhibitor of GABA synthesis, decreased the GABA level in synaptoneurosomes and increased the muscimol-stimulated Cl- uptake by the organelles. Although the evidence is not unequivocal, it does support the concept of GABA released from nerve endings being taken up by the postsynaptic cell, from where it exerts a regulatory influence on the functioning of the GABA receptor/ion channel complex.     

Generation of Two Forms of the γ-Aminobutyric AcidA Receptor γ-2-Subunit in Mice by Alternative Splicing

gamma-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The gamma 2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine gamma 2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids. LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the gamma 2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.     

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

Abstract: The localization of two forms of the γ subunit of G proteins, γ₃ and γ₁₂, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ₃ tended to decrease with age, whereas that of γ₁₂ in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ₃ was abundant in the neuropil, whereas γ₁₂ was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ₁₂-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ₁₂-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ₁₂ is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ₁₂ was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ₃ in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ₃ and γ₁₂ are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ₃ and γ₁₂ in the brain are related to the numbers and/or extent of maturation of these cells.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.     

Short and Long Form γ2 Subunits of the GABAA/Benzodiazepine Receptors

Abstract: Three novel antisera to the γ₂ subunit of the γ-aminobutyric acid_A (GABA_A) receptor/benzodiazepine receptor (GABA_AR/BZDR) complex have been made. Anti-γ_2S and anti-γ_2L are specific antibodies to synthetic peptides that recognize the γ_2S (short) and γ_2L (long) forms, respectively, of the γ₂ subunit. An antibody (anti-γ₂IL2) to staphylococcal protein A fusion protein of the large intracellular loop (γ₂IL) located between the putative transmembrane segments M3 and M4 of γ_2S recognizes both γ_2S and γ_2L subunits. The antibodies immunoprecipitated both the solubilized and affinity-purified GABA_AR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity-purified receptor from bovine cortex show that anti-γ_2S and anti-γ_2L recognize peptides of 45,000 and 47,000 M_r, respectively. Immunoprecipitation experiments indicate that γ_2S is more prevalent in hippocampus, whereas γ_2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of γ_2S and γ_2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both γ_2S and γ_2L subunits and 41–48% of the γ_2L subunit coexists with γ_2S in the same receptor complex. In cerebellum, in 27% of the clonazepam-sensitive and 39% of the clonazepam-insensitive BZDRs the γ_2S and γ_2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain α₆. In addition, almost all (93%) the clonazepam-insensitive BZDRs that contain γ_2L also contain a γ_2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain α₆, γ_2S, and γ_2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain α₆ subunit(s) have neither γ_2S nor γ_2L subunits. There are also species differences with respect to the relative abundance of γ_2S and γ_2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABA_AR/BZDR-mediated effects of ethanol intoxication involving cerebellar granule cells.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

Ca2+/Calmodulin-Dependent Protein Kinase II and Protein Kinase C Phosphorylate a Synthetic Peptide Corresponding to a Sequence That Is Specific for the γ2L Subunit of the GABAA Receptor

Abstract: The γ₂ subunit of the GABA_A receptor (GABA_A-R) is alternatively spliced. The long variant (γ_2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca²⁺/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ_2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K _m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ_2L subunit has been shown to be necessary for ethanol potentiation of the GABA_A-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Uptake of 13C-Tracer Arachidonate and γ-Linolenate by the Brain and Liver of the Suckling Rat Observed Using 13C-NMR

Arachidonic acid (AA; 20:4n-6) is one of the principal components of the phosphoglycerides in neural cell membranes. During the critical period of postnatal development in mammals, AA is supplied preformed, directly from the milk or derived from precursor fatty acids such as gamma-linolenic acid (GLA; 18:3n-6). In this study, 13C-NMR spectroscopy was applied to investigate the incorporation of [1-(13)C]AA and [3-(13)C]GLA into liver and brain lipids of 7-15-day-old rats. The main objective was to establish the importance of dietary GLA for tissue AA accretion relative to the contribution from preformed dietary AA. [1-(13)C]AA and [3-(13)C]GLA were injected into the stomach of 7-day-old rats as a mixture. 13C-NMR spectroscopy of lipid extracts revealed incorporation of [1-(13)C]AA and [5-(13)C]AA (the latter derived from metabolism of the injected [3-(13)C]GLA) into phosphoglycerides and triacylglycerols. Preformed AA was 10 (liver)-17 (brain) times more efficient in contributing to tissue AA than AA derived from precursor GLA. In separate experiments, NMR spectroscopy was used to assess uptake of [1-(13)C]AA directly in living rats and intact organs. Results showed that intact liver and brain contain an appreciable amount of NMR-detectable lipids. The in vivo/in vitro information obtained from organs provided details on the mobility and turnover of tissue lipids.     

Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

α2-Macroglobulin as a β-Amyloid Peptide-Binding Plasma Protein

Abstract: The β-amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α₂-macroglobulin (α₂M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds ¹²⁵I-Aβ_(1–42) with high affinity (apparent dissociation constant of 3.8 × 10^?10M). Approximately 1 mol of Aβ is bound per mole of α₂M. Both native and methylamine-activated α₂M bind ¹²⁵I-Aβ_(1–42). The binding of ¹²⁵I-Aβ_(1–42) to α₂M is enhanced by micromolar concentrations of Zn²⁺ (but not Ca²⁺) and is inhibited by noniodinated Aβ_(1–42) and Aβ_(1–40) but not by the reverse peptide Aβ_(40-1) or the cytokines interleukin 1β or interleukin 2. α₁-Antichymotrypsin, another plaque-associated protein, inhibits both the binding of ¹²⁵I-Aβ_(1–42) to α₂M as well as the degradation of ¹²⁵I-Aβ_(1–42) by proteinase-activated α₂M. Moreover, the binding of ¹²⁵I-Aβ_(1–42) to α₂M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α₂M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.     

The Effect of Antibodies to Gangliosides on Ca2+ Channel-Linked Release of γ-Aminobutyric Acid in Rat Brain Slices

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.     

The Family of Na+/Cl− Neurotransmitter Transporters

Abstract: The termination of neurotransmission is achieved by rapid uptake of the released neurotransmitter by specific high-affinity neurotransmitter transporters. Most of these transporters are encoded by a family of genes (Na⁺/Cl⁻ transporters) having a similar membrane topography of 12 transmembrane helices. An evolutionary tree revealed five distinct subfamilies: γ-aminobutyric acid transporters, monoamine transporters, amino acid transporters, "orphan" transporters, and the recently discovered bacterial transporters. The bacterial transporters that belong to this family may help to develop heterologous expression systems with the aim of solving the three-dimensional structure of these membrane proteins. Some of the neurotransmitter transporters have been implicated as important sites for drug action. Monoamine transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs. Localization of individual transporters in specific cells and brain areas is pertinent to understanding their contribution to neurotransmission and their potential as targets for drugs. The most important questions in the field include resolving the mechanism of neurotransmitter transport, the structure of the transporters, and the interaction of each transporter in complex neurological activities.