Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP-and YFP-labeled rat ␥-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na ؉ / Cl ؊ -dependent neurotransmitter transporters.It is widely accepted that tyrosine kinases and related receptors signal as dimers (1). Similarly, the oligomeric nature of voltage-dependent and ligand-gated ion channels is firmly established. In addition, over recent years it has been determined that other integral membrane proteins, which were originally thought to exist in monomeric form, actually form homo-and hetero-oligomers. For instance, this is true for several G protein-coupled receptors (2-5) and for a number of transporters, e.g. the erythrocyte glucose transporter-1 and the brain glutamate transporter (6, 7). The structural organization of transporters is likely to determine their function. This consideration is particularly relevant in understanding the transporters that mediate the re-uptake of neurotransmitters from the synaptic cleft (8). These proteins depend on the presence of Na ϩ and Cl Ϫ and generate a current during transport, i.e. they may share properties similar to ion channels (9 -12) that are known to be organized as oligomeric complexes. The human serotonin transporter (hSERT) 1 is a prototypic member of this family; its properties are of considerable clinical interest because the inhibitors are useful as antidepressants, and substrates that induce the reversal of transport (e.g. "ecstasy") are abused (13). The complexity of the transport reaction is suggestive of a higher level of organization, and recent biochemical experiments on the SERT of different species indicate that the transporter can, in principle, form oligomeric str...

Na؉ /Cl ؊ -dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat ␥-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Fö rster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane. Naϩ /Cl Ϫ -dependent neurotransmitter transporters (e.g. the transporters for dopamine, serotonin, or GABA) 1 retrieve neurotransmitters from the synaptic cleft into the presynaptic specialization (1). The medical relevance of these proteins is obvious; for instance, it has long been known that antidepressant drugs block the transporter for norepinephrine and serotonin (2). Likewise, tiagabine, an inhibitor of GABA transport, is used as an anticonvulsant in the treatment of epileptic seizures (3). Transporters support bidirectional flux of substrate, i.e. not only do they mediate influx of substrate but they also allow for non-exocytotic release of substrate (4). Compounds that induce reverse transport enjoy widespread popularity among illicit drug users; this is particularly true for amphetamine and its congeners, including ecstasy.Increasing evidence suggests that neurotransmitter transporters are oligomers (5, 6). Constitutive oligomerization has been visualized in intact cells by FRET microscopy (7); the oligomeric nature of neurotransmitter transporters has also been demonstrated by other, more disruptive, approaches, e.g. co-immunoprecipitation from detergent extracts and crosslinking (8 -10) and by freeze-fracture electron microscopy (11). However, the functional role of oligomer formation remains enigmatic. The hypothesis has been formulated that neurotransmitter transporters function in a manner analogous to ligand-gated ion channels, as binding of substrate induces an ion current (12-14). There is still an ongoing debate whether the GAT operates in a channel-like mode (15, 16) or according to the conservative "alternating ac...

Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.

Nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we have determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes), and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild type (WT) mice expressed (%): α3β4 (55), α3β4α5 (24), and α3β4β2 (21) nAChRs. nAChRs in β4 knockout (KO) mice were reduced to less than 15 % of controls and no longer contained the α5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between α5β4 KO and WT, suggesting that the reduced number of receptors in the KO did not impair transganglionic transmission. Deletions of α5 or β2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct α3β4 and α3β2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the α-conotoxins AuIB and MII. Our data - based on biochemical and functional experiments and several mouse KO models - clarifies and significantly extends previous observations on the function of nAChRs in heterologous system and the SCG.

Like all members of the Na؉ /Cl ؊ -dependent neurotransmitter transporter family, the rat ␥-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-⌬37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-⌬27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-⌬27 reached 50% of the wild type level. Additional extensions up to the position ؊3 (GAT1-⌬3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-⌬37 and GAT1-⌬32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif ( 569 VMI 571 ) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.Neurotransmission at synaptic junctions in the central nervous system is terminated by reuptake of the neurotransmitter into the synaptic ends (1). This is achieved by neurotransmitter transporters. GABA 1 is the major inhibitory neurotransmitter in the brain. There are three GABA transporters, referred to as GAT1, GAT2, and GAT3, which all belong to the Na ϩ /Cl Ϫ -dependent neurotransmitter transporter family. Members of this family share several features including a characteristic topology, i.e. intracellular amino and the carboxyl termini and a hydrophobic core composed of 12 transmembrane-spanning segments that are presumed to be predominantly ␣-helical. In the brain, GAT1 is the most widely distributed isoform. The transporter is of obvious therapeutic relevance: increases in synaptic GABA reduces excitability and prevents excessive neuronal firing. This action provides a rationale for the use of tiagabine in the treatment of epilepsy (2). In neurons, transporters must reach the presynaptic specialization. Thus, their biosynthetic pathway must also comprise mechanisms that afford the sorting and targeting to the axonal compartment and/or specific retention at perisynaptic sites (3). In addition, they undergo quality control in the endoplasmic reticulum (4) and posttranslational modification in the Golgi stacks. We recently demonstrated that neurotransmitter transporters such as GAT1 and the serotonin transporter form constitutive oligomers (5). If oligomerization of GAT1 is disrupted, the transporter is no longer expressed at the cell surface but is retained ...