Human Gamma-aminobutyric Acid Type Research Articles

Carboetomidate

Etomidate is a sedative hypnotic that is often used in critically ill patients because it provides superior hemodynamic stability. However, it also binds with high affinity to 11beta-hydroxylase, potently suppressing the synthesis of steroids by the adrenal gland that are necessary for survival. The authors report the results of studies to define the pharmacology of (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), a pyrrole analog of etomidate specifically designed not to bind with high affinity to 11beta-hydroxylase. The hypnotic potency of carboetomidate was defined in tadpoles and rats using loss of righting reflex assays. Its ability to enhance wild-type alpha1beta2gamma2l and etomidate-insensitive mutant alpha1beta2M286Wgamma2l human gamma-aminobutyric acid type A receptor activities was assessed using electrophysiologic techniques. Its potency for inhibiting in vitro cortisol synthesis was defined using a human adrenocortical cell assay. Its effects on in vivo hemodynamic and adrenocortical function were defined in rats. Carboetomidate was a potent hypnotic in tadpoles and rats. It increased currents mediated by wild-type but not etomidate-insensitive mutant gamma-aminobutyric acid type A receptors. Carboetomidate was a three orders of magnitude less-potent inhibitor of in vitro cortisol synthesis by adrenocortical cells than was etomidate. In rats, carboetomidate caused minimal hemodynamic changes and did not suppress adrenocortical function at hypnotic doses. Carboetomidate is an etomidate analog that retains many beneficial properties of etomidate, but it is dramatically less potent as an inhibitor of adrenocortical steroid synthesis. Carboetomidate is a promising new sedative hypnotic for potential use in critically ill patients in whom adrenocortical suppression is undesirable.

Single- and Multilocus Allelic Variants within the GABA B Receptor Subunit 2 ( GABAB2) Gene Are Significantly Associated with Nicotine Dependence

Twelve single-nucleotide polymorphisms (SNPs) in the human gamma-aminobutyric acid type B (GABA(B)) receptor subunit 2 gene (GABAB2) were tested for association with nicotine dependence (ND) in an extensively phenotyped cohort of 1,276 smokers and nonsmokers, representing approximately 404 nuclear families of African American (AA) or European American (EA) origin. The GABAB2 gene encodes a subunit of the GABA(B) receptor for GABA, an inhibitory neurotransmitter involved in the regulation of many physiological and psychological processes in the brain. The gene is located within a region of chromosome 9q22 that showed a "suggestive" linkage to ND. Individual SNP analysis performed using the PBAT-GEE program indicated that two SNPs in the AAs and four SNPs in the EAs were significantly associated with ND. Haplotype analysis using the Family-Based Association Test revealed that, even after Bonferroni correction, the haplotype C-C-G of rs2491397-rs2184026-rs3750344 had a significant positive association with ND in both the pooled and the AA samples. In the EAs, we identified two haplotypes, C-A-C-A and T-A-T-A, formed by SNPs rs1435252-rs378042-rs2779562-rs3750344, that showed a highly significant negative and positive association with ND, respectively. In summary, our findings provide evidence of a significant association of GABAB2 variants with ND, implying that this gene plays an important role in the etiology of this drug addiction.

Effects of the plant alkaloid tetrandrine on human nicotinic acetylcholine receptors.

Functional effects of the well-characterized antagonist of L-type Ca(2+) channels tetrandrine on recombinant human gamma-aminobutyric acid type A (GABA(A)) (alpha1beta2gamma2s) receptor or human alpha7, alpha4beta2, alpha1beta1deltagamma and alpha1beta1delta epsilon nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage clamp. Tetrandrine inhibited the function of acetylcholine nicotinic receptors, but it had no effect on GABA(A) receptors. Potency of inhibition was influenced by the receptor subtype and the rank order was alpha4beta2>alpha7>alpha1beta1deltagamma congruent with alpha1beta1delta epsilon. Functional inhibition of alpha4beta2 and alpha1beta1deltagamma receptors was noncompetitive, but only inhibition of alpha1beta1deltagamma receptors was voltage-dependent. Binding of 125I-alpha-bungarotoxin to alpha1beta1deltagamma or 3H-cytisine to alpha4beta2 receptors was also inhibited by tetrandrine, but inhibition was noncompetitive and required concentrations higher than those needed to inhibit receptor function. Inhibition of both alpha7 receptor function and binding of 125I-alpha-bungarotoxin to alpha7 receptor were mixed competitive/noncompetitive and occurred at a similar concentration range.

Association analysis of an (AC)n repeat polymorphism in the GABA(B) receptor gene and schizophrenia.

The human gamma-aminobutyric acid type B (GABA(B)) receptor gene is a candidate gene for schizophrenia due to its chromosomal location and neurobiologic roles. In the present study, association analyses of genetic polymorphisms of the GABA(B) receptor gene with schizophrenia were carried out in 102 unrelated schizophrenic patients and 100 healthy controls, using a polymerase chain reaction-based, single-strand conformational polymorphism analysis. Although the Ala20Val and Gly489Ser mutations were not found in our samples, we found a novel polymorphism of (AC)n dinucleotide repeats located approximately 1.6 kb upstream from the translational start site. No significant difference in allele frequencies was found between controls and patients with schizophrenia (P = 0.0587) using the Monte Carlo method. Significant differences were found between controls and patients with continuous-course schizophrenia (P = 0.0019), and between controls and patients with a positive family history of psychoses (P = 0.0015). These differences, however, were not significant after Bonferroni correction. These data did not support our hypothesis that polymorphisms of the GABA(B) receptor gene may confer vulnerability for schizophrenia.

Allosteric modulators affect the efficacy of partial agonists for recombinant GABA(A) receptors.

Different alpha subunits of human gamma-aminobutyric acid type A (GABA(A)) receptors were transiently expressed together with beta(3) and gamma(2) subunits in Xenopus oocytes to examine the interactions of various GABA(A) agonists and representative allosteric modulators. Chloride currents elicited by agonists were measured using two electrode voltage clamp electrophysiology. Where compounds behaved as full agonists, i.e. GABA on all subtypes and 4,5,6, 7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) on alpha2beta(3)gamma(2) GABA(A) receptors, agonist concentration-response curves were shifted to the left by the benzodiazepine full agonist chlordiazepoxide and the anticonvulsant loreclezole, or to the right by the inverse agonist 6, 7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (DMCM), with no effect on the maximal currents (I(max)). In contrast, maximal responses for different partial GABA(A) agonists on all benzodiazepine-sensitive alpha(x)beta(3)gamma(2) GABA(A) receptors were enhanced by chlordiazepoxide. I(max) values for piperidine-4-sulphonic acid (P4S) on alpha(1)beta(3)gamma(2), THIP on alpha(3)beta(3)gamma(2), and 5-(4-piperidyl)isothiazol-3-ol (thio-4-PIOL) on alpha(2)beta(3)gamma(2) and alpha(5)beta(3)gamma(2) GABA(A) receptors were increased by chlordiazepoxide, while that for P4S on alpha(1)beta(3)gamma(2) receptors was decreased by DMCM. The I(max) values for partial agonists were also enhanced by pentobarbitone, the neurosteroid allopregnanolone and loreclezole irrespective of receptor subtype or the nature of the partial agonist. In the light of models of ligand-gated ion channel receptor activation we suggest two possible mechanisms of action for the effects of allosteric modulators on partial agonist receptor activation: either selective modulation of agonist affinity for the open/closed state, or direct modulation of the gating process itself.

Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels.

gamma-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

Functional Modulation of Human Recombinant γ-Aminobutyric Acid Type A Receptor by Docosahexaenoic Acid

Human gamma-aminobutyric acid type A (GABAA) receptors were expressed in the baculovirus/Sf-9 insect cell expression system using recombinant cDNA of alpha1beta2gamma2s subunits. The effect of unsaturated fatty acids on GABAA receptor complexes was investigated electrophysiologically using conventional whole cell recording under voltage clamp. Three distinct effects of docosahexaenoic acid (DHA) on the GABA responses were observed. First, DHA, at a concentration of 10(-7) M or greater, accelerated the desensitization after the peak of the GABA-induced current. Second, DHA (10(-6) M) potentiated the peak amplitude of GABA response. This potentiation by DHA was inhibited in the presence of Zn2+ (10(-5) M); Cu2+ and Ni2+ mimicked the action of Zn2+. Zn2+ (10(-5) M) did not block the GABA response on alpha1beta2gamma2s receptor complexes. Third, DHA, at a concentration of 3 x 10(-6) M or higher, gradually suppressed the peak amplitude of GABA response. A protein kinase A inhibitor, a protein kinase C inhibitor, and a Ca2+ chelator did not modify the effects of DHA on GABA-induced chloride ion current. Six unsaturated fatty acids other than DHA were examined. Arachidonic acid mimicked the effect of DHA while e.g. oleic acid had no effect. The inhibition of the GABA response in the presence of DHA was also observed in cells expressing GABAA receptors of alpha1 and beta2 subunit combinations. The data show that the gamma subunit is essential for DHA and arachidonic acid to potentiate the GABA-induced Cl- channel activity and to affect the desensitization kinetics of the GABAA receptor.

Differences in Agonist/Antagonist Binding Affinity and Receptor Transduction Using Recombinant Human γ-Aminobutyric Acid Type A Receptors

Using human gamma-aminobutyric acid type A (GABAA) receptor subunit combinations, expressed in cell lines and Xenopus laevis oocytes, the pharmacology of a number of ligands interacting directly with the GABA recognition site has been studied in [3H]muscimol binding and electrophysiologically. The binding affinity of GABAA agonist and antagonist ligands showed small but statistically significant dependence on the subunit composition of receptors that include gamma 2 and different alpha and beta subunits. The potency of antagonist ligands was largely independent of receptor subunit composition, whereas the composition of receptors expressed in oocytes strongly influenced the EC50 value of agonists. An apparent reciprocal correlation between subunits favoring agonist binding and antagonist binding, respectively, was observed. Whereas antagonists showed comparable potencies in binding and functional studies, the potency of agonists in binding studies was generally two to three orders of magnitude higher than the agonist potencies measured electrophysiologically. 5-(4-Piperidyl)isothiazol-3-ol, which behaves as a low efficacy partial agonist at GABAA receptors in cultured cortical neurons, showed no efficacy in oocytes, but produced pure antagonist effects with a binding/functional affinity ratio between those observed for the agonists and antagonists. It is concluded that the GABAA receptor mechanisms transducing binding into physiological response, but not the binding per se, is dependent on the receptor subunit composition.

A single amino acid of the human gamma-aminobutyric acid type A receptor gamma 2 subunit determines benzodiazepine efficacy

Incorporation of the gamma 2 subunit into gamma-aminobutyric acid type A (GABAA) receptors is required for the expression of benzodiazepine pharmacology, but the regions of the subunit responsible for benzodiazepine actions have not been defined. Using mutagenesis, we identified a single amino acid of the gamma 2 subunit of the human GABAA receptor that profoundly alters the nature of this pharmacology. When threonine 142 was mutated to serine, the benzodiazepine receptor antagonist, flumazenil, and the weak inverse agonist, Ro 15-4513, both acted as potent partial agonists. Further, potentiation of GABA responses by diazepam, alprazolam, clonazepam, or flunitrazepam doubled in receptors containing the Ser-142 gamma 2 subunit. In contrast, responses to the Type I benzodiazepine receptor selective ligands, zolpidem, alpidem, and CL218,872, were roughly halved. This change in pharmacology appears to occur at a stage following ligand binding, i.e. the mutation affects benzodiazepine efficacy. There was no effect on GABA affinity or efficacy or pentobarbital, Ro 5-4864, or alphaxalone modulation of GABA responses. These findings demonstrate that very minor changes in receptor structure can profoundly affect the efficacy of receptor ligands. Thus, agonism is determined not only by the structure of the drug, but also by the structure of the receptor, or protein complex, with which it interacts.