Gamma-aminobutyric acid type B receptors with specific heterodimer composition and postsynaptic actions in hippocampal neurons are targets of anticonvulsant gabapentin action.

Gamma-aminobutyric acid (GABA) activates two qualitatively different inhibitory mechanisms through ionotropic GABA(A) multisubunit chloride channel receptors and metabotropic GABA(B) G protein-coupled receptors. Evidence suggests that pharmacologically distinct GABA(B) receptor subtypes mediate presynaptic inhibition of neurotransmitter release by reducing Ca2+ conductance, and postsynaptic inhibition of neuronal excitability by activating inwardly rectifying K+ (Kir) conductance. However, the cloning of GABA(B) gb1 and gb2 receptor genes and identification of the functional GABA(B) gb1-gb2 receptor heterodimer have so far failed to substantiate the existence of pharmacologically distinct receptor subtypes. The anticonvulsant, antihyperalgesic, and anxiolytic agent gabapentin (Neurontin) is a 3-alkylated GABA analog with an unknown mechanism of action. Here we report that gabapentin is an agonist at the GABA(B) gb1a-gb2 heterodimer coupled to Kir 3.1/3.2 inwardly rectifying K+ channels in Xenopus laevis oocytes. Gabapentin was practically inactive at the human gb1b-gb2 heterodimer, a novel human gb1c-gb2 heterodimer and did not block GABA agonism at these heterodimer subtypes. Gabapentin was not an agonist at recombinant GABA(A) receptors as well. In CA1 pyramidal neurons of rat hippocampal slices, gabapentin activated postsynaptic K+ currents, probably via the gb1a-gb2 heterodimer coupled to inward rectifiers, but did not presynaptically depress monosynaptic GABA(A) inhibitory postsynaptic currents. Gabapentin is the first GABA(B) receptor subtype-selective agonist identified providing proof of pharmacologically and physiologically distinct receptor subtypes. This selective agonism of postsynaptic GABA(B) receptor subtypes by gabapentin in hippocampal neurons may be its key therapeutic advantage as an anticonvulsant.

Gbetagamma subunit combinations differentially modulate receptor and effector coupling in vivo.

In vitro, little specificity is seen for modulation of effectors by different combinations of Gbetagamma subunits from heterotrimeric G proteins. Here, we demonstrate that the coupling of specific combinations of Gbetagamma subunits to different receptors leads to a differential ability to modulate effectors in vivo. We have shown that the beta(1)AR and beta(2)AR can activate homomultimers of the human inwardly rectifying potassium channel Kir 3.2 when coexpressed in Xenopus oocytes, and that this requires a functional mammalian Gs heterotrimer. Modulation was independent of cAMP production, suggesting a membrane-delimited mechanism. To analyze further the importance of different Gbetagamma combinations, we have tested the facilitation of Kir 3.2 activation by betaAR mediated by different Gbetagamma subunits. The subunits tested were Gbeta(1,5) and Ggamma(1,2,7,11). These experiments demonstrated significant variation between the ability of the Gbetagamma combinations to activate the channels after receptor stimulation. This was in marked contrast to the situation in vitro where little specificity for binding of a Kir 3.1 C-terminal GST fusion protein by different Gbetagamma combinations was detected. More importantly, neither receptor, although homologous both structurally and functionally, shared the same preference for Gbetagamma subunits. In the presence of beta(1)AR, Gbeta(5)gamma(1) and Gbeta(5)gamma(11) activated Kir 3.2 to the greatest extent, while for the beta(2)AR, Gbeta(1)gamma(7), Gbeta(1)gamma(11,) and Gbeta(5)gamma(2) produced the greatest responses. Interestingly, no preference was seen in the ability of different Gbetagamma subunits to facilitate receptor-stimulated GTPase activity of the Gsalpha. These results suggest that it is not the receptor/G protein alpha subunit interaction or the Gbetagamma/effector interaction that is altered by Gbetagamma, but rather that the ability of the receptor to interact productively with the Gbetagamma subunit directly and/or the G protein/effector complex is dependent on the specific G protein heterotrimer associated with the receptor.

State-dependent barium block of wild-type and inactivation-deficient HERG channels in Xenopus oocytes.

The effects of Ba2+ on current resulting from the heterologous expression of the human ether-à-go-go related gene (HERG) (IHERG) was studied with two-electrode voltage clamp techniques in Xenopus oocytes. Ba2+ produced time- and voltage-dependent block of IHERG. Significant inhibition was seen at concentrations as low as 1 microM. Inhibition was greatest at step potentials between -40 and 0 mV; at more positive potentials, inhibition decreased in association with time-dependent unblocking of channels. An inactivation-attenuated mutant of HERG (S631A) was prepared and expressed in Xenopus oocytes. Ba2+ block of S631A differed from that of HERG in that extensive unblocking was no longer seen at positive potentials and the voltage dependence of step current block was greatly attenuated. A mathematical model was applied to analyse quantitatively the inhibitory effects of Ba2+ on IHERG. The model suggested similar voltage-dependent affinity of Ba2+ for the open and closed states, along with absence of binding to the inactivated state, and accounted well for Ba2+ effects on both wild-type and S631A channels. We conclude that Ba2+ potently inhibits IHERG in a characteristic state-dependent fashion, with strong unblocking at positive potentials related to the presence of an intact C-type inactivation mechanism.

Coexpression of full-length gamma-aminobutyric acid(B) (GABA(B)) receptors with truncated receptors and metabotropic glutamate receptor 4 supports the GABA(B) heterodimer as the functional receptor.

Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.

Stable association of G proteins with beta 2AR is independent of the state of receptor activation.

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.

Identification of a GABAB receptor subunit, gb2, required for functional GABAB receptor activity.

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Co-expression of human Kir3 subunits can yield channels with different functional properties.

To date, no comprehensive study has been done on all combinations of the human homologues of the Kir3.0 channel family, and the human homologue of Kir3.3 has not yet been identified. To obtain support for the contention that most of the functional data on non-human Kir3.0 channels can be extrapolated to human channels, we have cloned the human homologues of the Kir3.0 family, including the yet unidentified human Kir3.3, and the human Kir4.1. The expression pattern of these channels in various human brain areas and peripheral tissues, analysed by Northern blot analysis, allows for the existence of various homomeric and heteromeric forms of human Kir3.0 channels. Expression studies of all possible combinations in Xenopus oocytes indicated that in homomeric Kir3.2c and heteromeric Kir3.1/3.2c channels mediate, in our studies, inward currents with largest amplitude of any other Kir3.0 channel combinations, followed by heteromeric Kir3.1/3.4 and homomeric Kir4.1 channels. Channel combinations which include Kir3.3 are detrimental to the formation of functional channels. The co-expression experiments with different Kir channel subunits indicate the selective formation of certain channel combinations, suggesting that channel specificity is not solely dependent on spatial and temporal regulation of Kir subunit expression.

Gene cloning, DNA sequencing, and expression of thermostable beta-mannanase from Bacillus stearothermophilus.

A DNA genomic library constructed from Bacillus stearothermophilus, a gram-positive, facultative thermophilic aerobe that secretes a thermostable beta-mannanase, was screened for mannan hydrolytic activity. Recombinant beta-mannanase activity was detected on the basis of the clearing of halos around Escherichia coli colonies grown on a dye-labelled substrate, Remazol brilliant blue-locust bean gum. The nucleotide sequence of the mannanase gene, manF, corresponded to an open reading frame of 2,085 bp that codes for a 32-amino-acid signal peptide and a mature protein with a molecular mass of 76,089 Da. From sequence analysis, ManF belongs to glycosyl hydrolase family 5 and exhibits higher similarity to eukaryotic than to bacterial mannanases. The manF coding sequence was subcloned into the pH6EX3 expression plasmid and expressed in E. coli as a recombinant fusion protein containing a hexahistidine N-terminal sequence. The fusion protein has thermostability similar to the native enzyme and was purified by Ni2+ affinity chromatography. The values for the kinetic parameters Vmax and Km were 384 U/mg and 2.4 mg/ml, respectively, for the recombinant mannanase and were comparable to those of the native enzyme.

Functional rescue of a constitutively desensitized beta2AR through receptor dimerization.

We have recently demonstrated that wild-type beta2-adrenergic receptors (beta2AR) form homodimers and that disruption of receptor dimerization inhibits signalling via Gs [Hebert, Moffett, Morello, Loisel, Bichet, Barret and Bouvier (1996) J. Biol. Chem. 271, 16384-16392]. Here taking advantage of the altered functional properties of a non-palmitoylated, constitutively desensitized mutant beta2AR (C341Gbeta2AR), we sought to study whether physical interactions between mutant and wild-type beta2AR expressed in Sf9 cells could occur and have functional consequences. Using metabolic labelling with [3H]palmitate and co-immunoprecipitation we demonstrated the existence of heterodimerization between wild-type and C341Gbeta2AR. Furthermore, we show that, in co-expression experiments, wild-type receptors have a dominant positive effect resulting in the functional complementation of C341Gbeta2AR. Indeed, when expressed alone, the mutant C341G receptor displays altered functional characteristics in that (1) the response of the receptor to agonist is reduced as compared to the wild-type receptor and (2) the desensitization of the receptor in response to prolonged exposure to agonist is minimal. In contrast, when C341G and the wild-type beta2AR were expressed together, both the response to agonist and subsequent desensitization (at a constant level of total receptor) were equivalent to the wild-type beta2AR expressed alone. This dominant positive effect was also seen when C341G was co-expressed with a second receptor mutant in which the two protein kinase A phosphorylation sites (S261, 262, 345, 346A beta2AR) were mutated. Taken together these data suggest that intermolecular interactions between receptors may have both functional and structural implications for G-protein-mediated signalling.