The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Tax1-binding protein 1 is expressed in the retina and interacts with the GABA(C) receptor rho1 subunit

Macromolecular signalling complexes that link neurotransmitter receptors to functionally and structurally associated proteins play an important role in the regulation of neurotransmission. Thus the identification of proteins binding to neurotransmitter receptors describes molecular mechanisms of synaptic signal transduction. To identify interacting proteins of GABA(C) (where GABA is gamma-aminobutyric acid) receptors in the retina, we used antibodies specific for GABA(C) receptor rho1-3 subunits. Analysis of immunoprecipitated proteins by MALDI-TOF MS (matrix-assisted laser-desorption ionization-time-of-flight MS) identified the liver regeneration-related protein 2 that is identical with amino acids 253-813 of the Tax1BP1 (Tax1-binding protein 1). A C-terminal region of Tax1BP1 bound to an intracellular domain of the rho1 subunit, but not to other subunits of GABA(C), GABA(A) or glycine receptors. Confocal laser-scanning microscopy demonstrated co-localization of Tax1BP1 and rho1 in clusters at the cell membrane of transfected cells. Furthermore, Tax1BP1 and GABA(C) receptors were co-expressed in both synaptic layers of the retina, indicating that Tax1BP1 is a component of GABA(C) receptor-containing signal complexes.     

Marlin-1, a novel RNA-binding protein associates with GABA receptors

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.     

Floxed allele for conditional inactivation of the GABAB(1) gene

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter GABA. GABA(B) receptors are broadly expressed in the nervous system. Their complete absence in mice causes premature lethality or--when mice are viable--epilepsy, impaired memory, hyperalgesia, hypothermia, and hyperactivity. A spatially and temporally restricted loss of GABA(B) function would allow addressing how the absence of GABA(B) receptors leads to these diverse phenotypes. To permit a conditional gene inactivation, we flanked critical exons of the GABA(B(1)) gene with lox511 sites. GABA(B(1)) (lox511/lox511) mice exhibit normal levels of GABA(B(1)) protein, are fertile, and do not display any behavioral phenotype. We crossed GABA(B(1)) (lox511/lox511) with Cre-deleter mice to produce mice with an unrestricted GABA(B) receptor elimination. These GABA(B(1)) (-/-) mice no longer synthesize GABA(B(1)) protein and exhibit the expected behavioral abnormalities. The conditional GABA(B(1)) allele described here is therefore suitable for generating mice with a site- and time-specific loss of GABA(B) function.     

Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions

Gamma-aminobutyric acid (GABA) binding to GABA(A) receptors (GABA(A)Rs) triggers conformational movements in the alpha(1) and beta(2) pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABA(A)R channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABA(A)R pre-M1 regions. Alpha(1)beta(2) GABA(A)Rs containing cysteine substitutions in the pre-M1 alpha(1) (K219C, K221C) and beta(2) (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC(50) values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto alpha(1)K219C and alpha(1)K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For alpha(1)K221Cbeta(2) receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For alpha(1)beta(2)K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.     

Endoplasmic Reticulum Sorting and Kinesin-1 Command the Targeting of Axonal GABA(B) Receptors

In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABA(B)Rs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABA(B)Rs are obligatory heteromers constituted by two subunits, GABA(B)R1 and GABA(B)R2. GABA(B)R1a and GABA(B)R1b are the most abundant subunit variants. GABA(B)R1b is located in the somatodendritic domain whereas GABA(B)R1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABA(B)R1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABA(B)Rs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABA(B)R1a operate in a pre-Golgi compartment. In the axon GABA(B)R1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABA(B)R1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABA(B)Rs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.     

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.     

GABAB receptor association with the PDZ scaffold Mupp1 alters receptor stability and function

gamma-Aminobutyric acid, type B (GABA(B)) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABA(B) receptor (GABA(B)R) functionality, we screened the GABA(B)R2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABA(B)R2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA(B)R2 in cells. Disruption of the GABA(B)R2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABA(B) receptor signaling. The effects of mutating the GABA(B)R2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABA(B) receptor stability and signaling can be modulated via GABA(B)R2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABA(B) receptors in the central nervous system.     

Dominant role of GABAB2 and Gbetagamma for GABAB receptor-mediated-ERK1/2/CREB pathway in cerebellar neurons

gamma-aminobutyric acid type B (GABA(B)) receptor is an allosteric complex made of two subunits, GABA(B1) and GABA(B2). GABA(B2) plays a major role in the coupling to G protein whereas GABA(B1) binds GABA. It has been shown that GABA(B) receptor activates ERK(1/2) in neurons of the central nervous system, but the molecular mechanisms underlying this event are poorly characterized. Here, we demonstrate that activation of GABA(B) receptor by either GABA or the selective agonist baclofen induces ERK(1/2) phosphorylation in cultured cerebellar granule neurons. We also show that CGP7930, a positive allosteric regulator specific of GABA(B2), alone can induce the phosphorylation of ERK(1/2). PTX, a G(i/o) inhibitor, abolishes both baclofen and CGP7930-mediated-ERK(1/2) phosphorylation. Moreover, both baclofen and CGP7930 induce ERK-dependent CREB phosphorylation. Furthermore, by using LY294002, a PI-3 kinase inhibitor, and a C-term of GRK-2 that has been reported to sequester Gbetagamma subunits, we demonstrate the role of Gbetagamma in GABA(B) receptor-mediated-ERK(1/2) phosphorylation. In conclusion, the activation of GABA(B) receptor leads to ERK(1/2) phosphorylation via the coupling of GABA(B2) to G(i/o) and by releasing Gbetagamma subunits which in turn induce the activation of CREB. These findings suggest a role of GABA(B) receptor in long-term change in the central nervous system.     

Inhibitors of the γ-aminobutyric acid transporter 1 (GAT1) do not reveal a channel mode of conduction

We expressed the γ-aminobutyric acid (GABA) transporter GAT1 (SLC6A1) in Xenopus laevis oocytes and performed GABA uptake experiments under voltage clamp at different membrane potentials as well as in the presence of the specific GAT1 inhibitors SKF-89976A and NO-711. In the absence of the inhibitors, GAT1 mediated the inward translocation of 2 net positive charges across the plasma membrane for every GABA molecule transported into the cell. This 2:1 charge flux/GABA flux ratio was the same over a wide range of membrane potentials from −110 mV to +10 mV. Moreover, when GABA-evoked (500 μM) currents were measured at −50 and −90 mV, neither SKF-89976A (5 and 25 μM) nor NO-711 (2 μM) altered the 2:1 charge flux/GABA flux ratio. The results are not consistent with previous hypotheses that (i) GABA evokes an uncoupled channel-mediated current in GAT1, and (ii) GAT1 inhibitors block the putative uncoupled current gated by GABA. Rather, the results suggest tight coupling of GAT1-mediated charge flux and GABA flux.     

Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits.

To investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABA(A) receptors) in the presence of various compounds, we expressed receptors composed of alpha(1) and beta(1) subunits by injecting cells with the cRNAs synthesized from cloned bovine GABA(A) receptor cDNAs and measured the electrical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABA(A) receptor-mediated response was quantitatively analyzed using a simple model with the assumption that the receptors have two identical binding sites for GABA molecules with a dissociation constant of K(1), and one potentiation site for the compound with a dissociation constant of K(p), and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K(1) to K(1p). The estimated K(p) and K(1p) were dependent on the functional groups and the chain length of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABA(A) receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These compounds accelerated the decay of the response, possibly due to desensitization of the receptors, which was also analyzed on the basis of the model.     

The murine GABA(B) receptor 1: cDNA cloning, tissue distribution, structure of the Gabbr1 gene, and mapping to chromosome 17

GABA (gamma-aminobutyric acid) is a major inhibitory neurotransmitter in the central nervous system (CNS) which activates both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptor systems. We identified two alternatively spliced cDNA variants of the murine GABA(B) receptor 1 that are predominantly expressed in the CNS. Deduced protein structures are highly homologous to the previously characterized rat and human receptors. Comparison of the genomic structures of mouse and human revealed that alternative splicing occurred at the same position, whereas the mouse gene has an additional 5' exon. Radiation hybrid mapping, combined with database searches, indicated that the GABA(B) receptor gene (Gabbr1) is located on mouse chromosome 17, adjacent to the marker D17Mit24 in a region homologous to human chromosome 6p21.3.     

GABA receptor rho1 subunit interacts with a novel splice variant of the glycine transporter, GLYT-1

Ionotropic gamma-aminobutyric acid (GABA(A) and GABA(C)) receptors mediate fast synaptic inhibition in the central nervous system. GABA(C) receptors are expressed predominantly in the retina on bipolar cell axon terminals, and are thought to mediate feedback inhibition from GABAergic amacrine cells. Utilizing the yeast two-hybrid system, we previously identified MAP1B as a binding partner of the GABA(C) receptor rho1 subunit. Here we describe the isolation of an additional rho1 interacting protein: a novel C-terminal variant of the glycine transporter GLYT-1. We show that GLYT-1 exists as four alternatively spliced mRNAs which encode proteins expressing one of two possible intracellullar N- and C-terminal domains. Variants containing the novel C terminus efficiently transport glycine when expressed in COS cells, but with unusual kinetics. We have confirmed the interaction between the novel C terminus and rho1 subunit and demonstrated binding in heterologous cells. This interaction may be crucial for the integration of GABAergic and glycinergic neurotransmission in the retina.     

Enhanced learning and memory in GAT1 heterozygous mice

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The termination of GABA transmission is through the action of a family of membrane proteins, called GABA transporters (GAT1-4). It is well established that GABA system is involved in the modulation of memory. Our previous study showed that homozygous GAT1(-/-) mice exhibited impaired hippocampus-dependent learning and memory. To evaluate the impact of endogenous reduced GABA reuptake on mice cognitive behaviors, the ability of learning and memory of heterozygous GAT1(+/-) mice was detected by the passive avoidance paradigm and Morris water maze. The hole board paradigm was also used to measure changes in anxiety-related behavior or exploratory behavior in such mice. As one form of synaptic plasticity, long-term potentiation was recorded in the mouse hippocampal CA1 area. We found that GAT1(+/-) mice displayed increased learning and memory, decreased anxiety-like behaviors, and highest synaptic plasticity compared with wild-type and homozygous GAT1(-/-) mice. Our results suggest that a moderate reduction in GAT1 activity causes the enhancement of learning and memory in mice.     

AtGAT1, a high affinity transporter for gamma-aminobutyric acid in Arabidopsis thaliana

Functional characterization of Arabidopsis thaliana GAT1 in heterologous expression systems, i.e. Saccharomyces cerevisiae and Xenopus laevis oocytes, revealed that AtGAT1 (At1g08230) codes for an H(+)-driven, high affinity gamma-aminobutyric acid (GABA) transporter. In addition to GABA, other omega-aminofatty acids and butylamine are recognized. In contrast to the most closely related proteins of the proline transporter family, proline and glycine betaine are not transported by AtGAT1. AtGAT1 does not share sequence similarity with any of the non-plant GABA transporters described so far, and analyses of substrate selectivity and kinetic properties showed that AtGAT1-mediated transport is similar but distinct from that of mammalian, bacterial, and S. cerevisiae GABA transporters. Consistent with a role in GABA uptake into cells, transient expression of AtGAT1/green fluorescent protein fusion proteins in tobacco protoplasts revealed localization at the plasma membrane. In planta, AtGAT1 expression was highest in flowers and under conditions of elevated GABA concentrations such as wounding or senescence.