Glutamine Synthetase Stability and Subcellular Distribution in Astrocytes Are Regulated by γ-Aminobutyric Type B Receptors

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABA_BRs) are expressed by astrocytes within the mammalian brain. GABA_BRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABA_BR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABA_BR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABA_BR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABA_BRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABA_BRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.     

Proteasomal Degradation of Eukaryotic Elongation Factor-2 Kinase (EF2K) Is Regulated by cAMP-PKA Signaling and the SCFβTRCP Ubiquitin E3 Ligase

Protein translation and degradation are critical for proper protein homeostasis, yet it remains unclear how these processes are dynamically regulated, or how they may directly balance or synergize with each other. An important translational control mechanism is the Ca²⁺/calmodulin-dependent phosphorylation of eukaryotic elongation factor-2 (eEF-2) by eukaryotic elongation factor-2 kinase (EF2K), which inhibits elongation of nascent polypeptide chains during translation. We previously described a reduction of EF2K activity in PC12 cells treated with NGF or forskolin. Here, we show that both forskolin- and IGF-1-mediated reductions of EF2K activity in PC12 cells are due to decreased EF2K protein levels, and this is attenuated by application of the proteasome inhibitor, MG132. We further demonstrate that proteasome-mediated degradation of EF2K occurs in response to A2A-type adenosine receptor stimulation, and that activation of protein kinase A (PKA) or phospho-mimetic mutation of the previously characterized PKA site, Ser-499, were sufficient to induce EF2K turnover in PC12 cells. A similar EF2K degradation mechanism was observed in primary neurons and HEK cells. Expression of a dominant-negative form of Cul1 in HEK cells demonstrated that EF2K levels are regulated by an SCF-type ubiquitin E3 ligase. Specifically, EF2K binds to the F-box proteins, βTRCP1 and βTRCP2, and βTRCP regulates EF2K levels and polyubiquitylation. We propose that the proteasomal degradation of EF2K provides a mechanistic link between activity-dependent protein synthesis and degradation.     

CD3ζ-Chain Expression of Human T Lymphocytes Is Regulated by TNF via Src-like Adaptor Protein-Dependent Proteasomal Degradation

Decreased expression of the TCR ζ-chain has been reported in several autoimmune, inflammatory, and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. Although ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is less clear. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data have reported that SLAP regulates immunoreceptor signaling. We have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. TNF treatment of human T lymphocytes (15-40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose-dependent manner (p < 0.05) and decreases activation-induced IL-2 expression (p < 0.01). Although blocking of the lysosomal compartment fails to restore TNF-induced CD3 ζ-chain downregulation, inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalization of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p < 0.05 and p < 0.01, respectively), whereas TNF-induced ζ-chain downregulation was inhibited by gene silencing of SLAP with small interfering RNA. SLAP levels of the CD4(+) T lymphocytes isolated from patients with rheumatoid arthritis were more than 2-fold higher than that of the healthy donors' (p < 0.05); moreover, TNF treatment did not alter the SLAP expression of the CD4(+) cells of anti-TNF therapy-treated patients. Our present data suggest that TNF modulates T cell activation during inflammatory processes by regulating the amount of CD3 ζ-chain expression via a SLAP-dependent mechanism. These data provide evidence for SLAP-dependent regulation of CD3 ζ-chain in the fine control of TCR signaling.     

Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit

We have found that the large intracellular loop of the γ2 GABA_A receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABA_ARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABA_AR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABA_AR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABA_AR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABA_AR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABA_AR subunit promoting GABA_AR degradation.     

Presynaptic Targeting of ��4��2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1��

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre- and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs)⁵ regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of α2–α10 and β2–β4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The α4β2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for α4β2 AChRs in nicotine addiction. α4β2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7, 8). Furthermore, β2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). α4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing α4β2 AChRs hypersensitive to nicotine demonstrate that α4β2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12). High resolution ultrastructural studies show that α4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13), and a sequence motif has been identified within the α4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14). However, the mechanisms underlying the targeting and clustering of α4β2 AChRs to presynaptic sites in neurons remain elusive.Recently, bi-directional interactions between neurexins and neuroligins have been shown to promote synapse assembly and maturation by fostering pre- and postsynaptic differentiation (reviewed in Refs. 15–17). The neurexins are encoded by three genes corresponding to neurexins I–III (18, 19), each encoding longer α-neurexins and shorter β-neurexins, because of differential promoter use. Neurexins recruit N- and P/Q-type calcium channels via scaffolding proteins, including calmodulin-associated serine/threonine kinase (20), to active zones of presynaptic terminals (21, 22). Recently, α-neurexins were shown to specifically induce GABAergic postsynaptic differentiation (23). Neuroligins, postsynaptic binding partners of neurexins, cluster N-methyl-d-aspartate receptors and GABA_A receptors by recruiting the scaffolding proteins PSD-95 (post-synaptic density 95) and gephyrin, respectively (24, 25). Interestingly, neurexins and neuroligins also modulate the postsynaptic clustering of α3-containing AChRs in chick ciliary ganglia (26, 27). In this study, using multiple experimental strategies, we provide evidence for the formation of complexes between neurexin-1β and α4β2 AChRs and a role for neurexin in the targeting of α4β2 AChRs to presynaptic terminals of neurons.     

Potentiation of Neuronal Nicotinic Receptors by 17β-Estradiol: Roles of the Carboxy-Terminal and the Amino-Terminal Extracellular Domains

The endogenous steroid 17β-estradiol (βEST) potentiates activation of neuronal nicotinic receptors containing α4 subunits. Previous work has shown that the final 4 residues of the α4 subunit are required for potentiation. However, receptors containing the α2 subunit are not potentiated although it has these 4 residues, and only one amino acid difference in the C-terminal tail (FLAGMI vs. WLAGMI). Previous work had indicated that the tryptophan residue was involved in binding an analog of βEST, but not in potentiation by βEST. To determine the structural basis for the loss of potentiation we analyzed data from chimeric subunits, which indicated that the major factor underlying the difference between α2 and α4 is the tryptophan/phenylalanine difference, while the N-terminal extracellular domain is a less significant factor. When the tryptophan in α4 was mutated, both phenylalanine and tyrosine conferred lower potentiation while lysine and leucine did not. The reduction reflected a reduced maximal magnitude of potentiation, indicating that the tryptophan is involved in transduction of steroid effects. The regions of the α4 N-terminal extracellular domain involved in potentiation lie near the agonist-binding pocket, rather than close to the membrane or the C-terminal tail, and appear to be involved in transduction rather than binding. These observations indicate that the C-terminal region is involved in both steroid binding (AGMI residues) and transduction (W). The role of the N-terminus appears to be independent of the C-terminal tryptophan and likely reflects an influence on conformational changes caused during channel activation by agonist and potentiation by estradiol.     

Increasing the Receptor Tyrosine Kinase EphB2 Prevents Amyloid-β-induced Depletion of Cell Surface Glutamate Receptors by a Mechanism That Requires the PDZ-binding Motif of EphB2 and Neuronal Activity

Diverse lines of evidence suggest that amyloid-β (Aβ) peptides causally contribute to the pathogenesis of Alzheimer disease (AD), the most frequent neurodegenerative disorder. However, the mechanisms by which Aβ impairs neuronal functions remain to be fully elucidated. Previous studies showed that soluble Aβ oligomers interfere with synaptic functions by depleting NMDA-type glutamate receptors (NMDARs) from the neuronal surface and that overexpression of the receptor tyrosine kinase EphB2 can counteract this process. Through pharmacological treatments and biochemical analyses of primary neuronal cultures expressing wild-type or mutant forms of EphB2, we demonstrate that this protective effect of EphB2 depends on its PDZ-binding motif and the presence of neuronal activity but not on its kinase activity. We further present evidence that the protective effect of EphB2 may be mediated by the AMPA-type glutamate receptor subunit GluA2, which can become associated with the PDZ-binding motif of EphB2 through PDZ domain-containing proteins and can promote the retention of NMDARs in the membrane. In addition, we show that the Aβ-induced depletion of surface NMDARs does not depend on several factors that have been implicated in the pathogenesis of Aβ-induced neuronal dysfunction, including aberrant neuronal activity, tau, prion protein (PrP^C), and EphB2 itself. Thus, although EphB2 does not appear to be directly involved in the Aβ-induced depletion of NMDARs, increasing its expression may counteract this pathogenic process through a neuronal activity- and PDZ-dependent regulation of AMPA-type glutamate receptors.     

Antiparallel Conformation of Knob and Hole Aglycosylated Half-Antibody Homodimers Is Mediated by a CH2–CH3 Hydrophobic Interaction

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating “knob” and “hole” heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric “knob” and “hole” Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K_D of < 1 nM. Homodimers of the “knob” and “hole” mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.     

Inhibition of α-Synuclein Fibrillization by Dopamine Is Mediated by Interactions with Five C-Terminal Residues and with E83 in the NAC Region

The interplay between dopamine and α-synuclein (AS) plays a central role in Parkinson''s disease (PD). PD results primarily from a severe and selective devastation of dopaminergic neurons in substantia nigra pars compacta. The neuropathological hallmark of the disease is the presence of intraneuronal proteinaceous inclusions known as Lewy bodies within the surviving neurons, enriched in filamentous AS. In vitro, dopamine inhibits AS fibril formation, but the molecular determinants of this inhibition remain obscure. Here we use molecular dynamic (MD) simulations to investigate the binding of dopamine and several of its derivatives onto conformers representative of an NMR ensemble of AS structures in aqueous solution. Within the limitations inherent to MD simulations of unstructured proteins, our calculations suggest that the ligands bind to the ¹²⁵YEMPS¹²⁹ region, consistent with experimental findings. The ligands are further stabilized by long-range electrostatic interactions with glutamate 83 (E83) in the NAC region. These results suggest that by forming these interactions with AS, dopamine may affect AS aggregation and fibrillization properties. To test this hypothesis, we investigated in vitro the effects of dopamine on the aggregation of mutants designed to alter or abolish these interactions. We found that point mutations in the ¹²⁵YEMPS¹²⁹ region do not affect AS aggregation, which is consistent with the fact that dopamine interacts non-specifically with this region. In contrast, and consistent with our modeling studies, the replacement of glutamate by alanine at position 83 (E83A) abolishes the ability of dopamine to inhibit AS fibrillization.     

Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation

GABA-gated chloride channels (GABA_ARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABA_ARs on the cell surface to better understand the trafficking of GABA_ARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABA_AR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABA_ARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABA_AR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABA_ARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

Activation of γ-Aminobutyrate Production by Chloroplastic H2O2 Is Associated with the Oxidative Stress Response

A series of N-substituted aryl and alkyl carbamates (RNHCOOR′; R: aryl, alkyl; R′: aryl, alkyl) was prepared and screened for inhibitory activity toward the germination of oat seeds. The activity of each compound was compared with that of chlorpropham (isopropyl 3-chlorocarbanilate). Some of the synthetic carbamates possessing the N-(phenylthio)methyl group, PhSCH₂NHCOOR´, showed inhibitory activity close or comparable to that of chlorpropham.     

Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k_pol is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.     

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.     

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABA_A). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families.