Bicuculline-insensitive GABA Research Articles

An Historical Perspective on Gabaergic Drugs

In 1950, γ-aminobutyric acid (GABA) was discovered in the brain and in 1967 it was recognized as an inhibitory neurotransmitter. The discovery of the benzodiazepines Librium® (launched in 1960) and Valium® by Sternbach initiated huge research activities resulting in 50 marketed drugs. In 1975, Haefely found that GABA is involved in the actions of benzodiazepines. The baclofen-sensitive, bicuculline-insensitive GABA(B) receptor was discovered by Bowery in 1980, and the baclofen-insensitive, bicuculline-insensitive GABA(C) receptor by Johnston in 1984. Barnard & Seeburg reported the cloning of the GABA(A) receptor in 1987, Cutting the GABA(C) receptor in 1991 and Bettler the GABA(B1a) and GABA(B1b) receptors in 1997. Six groups cloned the GABA(B2) receptor in 1998/1999 showing that the GABA(B) receptor functions as a heterodimer with GABA(B1b)/GABA(B2) mediating postsynaptic inhibition and GABA(B1a)/GABA(B2) mediating presynaptic inhibition. Möhler and McKernan dissected the pharmacology of the benzodiazepine-receptor subtypes. Antagonists and positive allosteric modulators of GABA(B) receptors were discovered in 1987 and 2001, respectively. GABA transporter inhibitor, tiagabine, was launched in 1996, a GABA aminotransferase inhibitor, vigabatrin, in 1998 and a glutamic acid decarboxylase activator, pregabalin, in 2004. Most recently, brain-penetrating GABA(C)-receptor antagonists were reported in 2009.

Insect GABA Receptors: Splicing, Editing, and Targeting by Antiparasitics and Insecticides

Ionotropic GABA receptors are abundant in both vertebrate and invertebrate nervous systems, where they mediate rapid, mostly inhibitory synaptic transmission. A GABA-gated chloride channel subunit from Drosophila melanogaster [Resistant to Dieldrin (RDL)] has been cloned, functionally expressed, and found to exhibit many aspects of the pharmacology of native, bicuculline-insensitive insect GABA receptors. RDL is the target of the commercially important insecticide fipronil. A point mutation in the channel-lining region of the RDL molecule is known to underlie most cases of resistance to insecticides acting on GABA receptors. RDL is widely distributed throughout the insect nervous system, but the subunit composition of RDL-containing in native receptors is unknown. It is possible that in some instances, RDL coexpresses with glutamate-gated chloride channel subunits. Other ionotropic receptor subunits (LCCH3 and GRD) form GABA-gated cation channels when heterologously expressed. Interest in RDL as a model ligandgated anion channel has been enhanced by the recent discovery of pre-mRNA A-to-I editing, which, together with alternative splicing, adds to the functional diversity of this GABA receptor subunit.

Expression of GABA receptor rho subunits in rat brain.

The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

Identification of GABAA Receptor Subunits in Rat Retina: Cloning of the Rat GABAA Receptor ρ2‐Subunit cDNA

We identified GABAA receptor subunits in rat retina using PCR. The high degree of conservation among previously described members of ligand-gated anion channels in transmembrane domains was used to design degenerate sense and antisense oligonucleotides. These oligonucleotides were used as primers for PCR, which was applied to the rat retina cDNA. Analysis of clones derived from the PCR amplification identified the GABAA alpha 1, beta 1, beta 3, and gamma 2 subunits and the glycine alpha 1 subunit. In addition, two clones closely related to the human GABAA rho-subunit class were obtained. Molecular cloning revealed one of them as the rat counterpart of the human rho 2 subunit. Northern blot analysis demonstrated the expression of mRNAs for rho subunits in retina. These results further support the hypothesis that bicuculline-insensitive GABA channels in rat retina are comprised of rho subunits.

Expression of GABA Receptor ρ1 and ρ2 Subunits in the Retina and Brain of the Rat

We have investigated the distribution of GABA receptor rho 1 and rho 2 subunits in the rat central nervous system. Cloning of rat rho 1 and rho 2 cDNA fragments revealed similarities to the corresponding human sequences of 99% (rho 1) and 88% (rho 2) at the protein level. Whereas the human rho 2 subunit has no consensus sequence for phosphorylation by protein kinase C, the cytoplasmic loop of the rat sequence contains two such sites. Use of the polymerase chain reaction with reverse-transcribed total RNA (RT-PCR) from different brain tissues revealed that transcript for the rho 1 subunit was present in the retina only. The rho 2 mRNA was detected in all brain regions, with the highest level of expression in the retina. In situ hybridization of retinal sections revealed that rho 1 and rho 2 transcripts are present in the inner nuclear layer. RT-PCR and in situ hybridization of isolated retinal cells showed that both rho subunits are present in rod bipolar cells. Since these cells express bicuculline-insensitive GABA receptors, our results further support the idea that rho subunits are part of the GABAc receptor.

Differential pharmacology of GABA A and GABA C receptors on rat retinal bipolar cells

GABA A and GABA C receptors were studied on cultured or freshly isolated rat retinal bipolar cells. The cells displayed GABA-induced whole-cell currents, which were only partially blocked by high concentrations (100 μM) of the GABA A reeptor antagonist bicuculline. The bicuculline-resistant (GABA C) component was insensitive to the GABA A receptor modulators flunitrazepam (1 μM) and pentobarbital (50 μM). The bicuculline-sensitive portion of the current was strongly augmented by both drugs, indicating that it was mediated by conventional GABA A receptors. The GABA C and GABA A receptor subtypes displayed a 7-fold difference in their binding affinity for GABA, the EC 50 values being 4.2 μM and 27.1 μM, respectively. The Hill coefficient was ∼ 2 for both receptors. The bicuculline-insensitive GABA C receptors were markedly blocked by 100 μM picrotoxinin, 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide (SR-95531) and γ-hexachlorocyclohexane, drugs known to be antagonists of GABA A receptors. Examination of single-channel currents indicated main-state conductances of 7.9 pS and 29.6 pS for GABA C and GABA A receptors, respectively. The pore diameter of open GABA C receptor channels was 5.1 Å, i.e. close to the value of 5.6 Å reported for the GABA A receptor. These results demonstrate that rod bipolar cells possess two populations of pharmacologically distinct GABA receptors, GABA A and novel-type GABA C receptors, which might subserve different physiological functions in controlling visual transduction in the retina.

Gaba B-mediated modulation of the voltage-gated Ca 2+ channels

1. 1. The amino acid, γ-aminobutyric acid (GABA), activates two different receptor types (Bowery et al., 1980; reviewed by Ogata, 1990a). 2. 2. GABA A receptors are bicuculline-sensitive and are coupled to Cl - channels, while activation of bicuculline-insensitive GABA B receptors has been implicated in the modulation of Ca 2+ (Dunlap and Fischbach, 1981) and K + (Gahwiler and Brown, 1985; Inoue et al., 1985a,b; reviewed by Ogata, 1990b) channels. 3. 3. Baclofen is a specific agonist for GABA B receptors (Bowery et al., 1980). In rat sensory neurones, baclofen suppresses the membrane Ca 2+ current ( I Ca) by a mechanism involving a partussis toxin-sensitive G protein (Holz et al., 1986; Scott and Dolphin, 1986). 4. 4. It has been shown that the inhibitory effect of baclofen is more potent on the early portion of I Ca than on the later portion and consequently the rate of I Ca activation is slowed (Deisz and Lux, 1985; Dolphin and Scott, 1986). 5. 5. The mechanisms underlying these GABA B-mediated modulation of I Ca is not fully understood. This article reviews the inhibitory action of baclofen on I Ca in sensory neurones.

Synthesis, pharmacology and X-ray studies of baclofen analogues

Baclofen (β- p-chlorophenyl-GABA) is the reference selective agonist for the bicuculline-insensitive GABA B receptor. The search for new compounds having a high affinity for the GABA B receptor is very important to clarify structural requirements. In that sense, we report the synthesis, binding studies and X-ray determinations of various 3-heteroaromatic γ-aminobutyric acids. Biochemical investigations concerning their abilities to displace [ 3H] muscimol (GABA A) and [ 3H] baclofen (GABA B) in binding studies showed that the 4-amino-3-(5-methoxybenzo[b]furan-2-yl)butanoic acid 6a ( IC 50 = 22.16 μM/ R (−) [ 3H] baclofen; IC 50 = 5.6 μM/ RS [ 3H] baclofen) has a specific affinity for the GABA B receptor. The crystal structure of compounds 6a and 6b associated with computer graphics molecular superimpositions allows some structural requirements for GABA B receptor ligands to be proposed.

The cholinergic system of the forebrain modulates activity of GABA-ergic interneurons responsible for presynaptic regulation in the neocortex of kittens.

1. The GABA-ergic synaptic transmission begins to function at the end of the first week of postnatal life and is mediated predominantly through the bicuculline-insensitive GABA receptors. 2. Conditioning stimulation of the cholinergic system of the forebrain induces a selective inhibition of the sensory reaction of neurons of the neocortex. 3. The inhibitory influence of the cholinergic system is mediated by GABA-ergic neurons and is realized through bicuculline-insensitive GABA receptors.

Bicuculline-insensitive GABA binding to catfish neuronal membranes

GABA receptor binding to mammalian neuronal membranes has been classified into at least 2 subtypes—GABA A and GABA B binding sites. In catfish brain GABA A receptor sites have previously been demonstrated. Evidence is now presented that under appropriate conditions which rule out GABA A receptor binding, [ 3H]GABA binds to membranes prepared from catfish brain. This binding is bicuculline-insensitive but differs enough from mammalian GABA B binding to cast some doubt on the idea that GABA B receptors exist in catfish brain. Specific binding was detected that was saturable and exhibited a dissociation constant of 4μM. (±)Baclofen, a potent inhibitor in rat brain, was a weak inhibitor, producing a maximum of 43% inhibition. This inhibitory effect could be enhanced, however, in the presence of 320 μM isoguvacine. [ 3H]GABA binding was unaffected by bicuculline. Thus bicuculline-insensitive GABA binding sites exist in catfish brain but they differ in a number of ways from the GABA B receptor site found in mammals. Furthermore, a third [ 3H]GABA binding site appears to exist that is both baclofen- and bicuculline-insensitive, yet is inhibited by high concentrations of isoguvacine, a known GABA A agonist.

γ-Aminobutyric acid A (GABA A) receptors modulate [ 3H]GABA release from isolated neuronal growth cones in the rat

Potassium-induced release of γ-[ 3H]aminobutyric acid ([ 3H]GABA) from a growth cone-enriched fraction isolated from neonatal rat forebrain was inhibited by the GABA mimetic muscimol in a dosedependent manner (IC 50 15 nM). The GABA antagonist bicuculline completely reversed the effect of muscimol. Bicuculline alone slightly potentiated the K +-induced release of [ 3H]GABA. Baclofen, a proposed selective agonist for a bicuculline-insensitive GABA B receptor, was found to cause only a slight reduction in the K +-induced release of [ 3H]GABA. These results are compatible with the presence of a negative feedback mechanism mediated by GABA A receptors for controlling [ 3H]GABA release from growth cones of the developing rat forebrain.

Securinine alkaloids: A new class of GABA receptor antagonist

Experiments were undertaken to determine the site of action of securinine and related convulsant indolizidines. All of these compounds induced tonic seizures in mice, with CD 50 values ranging from 11 to 87 mg/kg. The CD 50 for bicuculline was found to be 8 mg/kg. Equilibrium binding assays revealed that securinine and dihydrosecurinine inhibit [ 3H]GABA binding to rat brain membranes with an IC 50 of approximately 50 μM, which is some 7 times less potent than bicuculline. Allosecurinine and virosecurinine have IC 50 values greater than 1 mM. Both dihydrosecurinine and securinine inhibited GABA-stimulated benzodiazepine binding in rat brain membranes, though they were somewhat weaker than bicuculline in this respect. Other binding assays revealed that securinine and its analogs were inactive as inhibitors of bicuculline-insensitive GABA binding, benzodiazepine, cholinergic muscarinic, and β-adrenergic receptor binding. In addition, while thiocyanate ion increased the apparent binding potency of bicuculline 10-fold, it had little effect on that of securinine. Extracellular electrophysiological studies on neurons in the cat spinal cord indicated that securinine and dihydrosecurinine blocked the inhibitory action of GABA while having no effect on that of glycine. Allo- and virosecurinine were much less active as GABA receptor antagonists in this test. These results suggest that, like bicuculline, securinine and dihydrosecurinine are selective antagonists of GABA recognition sites on mammalian central neurons.

Bicuculline-insensitive GABA receptors: Studies on the binding of (−)-baclofen to rat cerebellar membranes

The binding of [ 3H](−)-baclofen to synaptic membranes prepared from rat cerebellum was studied. Consistent with pharmacological data that (−)-baclofen is the more active stereoisomer, studies on the binding of [ 3H](−)-baclofen showed increased specific binding, a higher affinity K d and a lower B max than equivalent studies using [ 3 H](±)- baclofen . Divalent metal ions (mercury, lead, calcium and zinc) inhibited the binding of [ 3H](−)-baclofen. The effects of some conformationally restricted analogues of γ-aminobutyric acid (GABA) on [ 3H](−)-baclofen binding indicated that GABA interacts with (−)-baclofen-sensitive binding sites (GABA B) in extended rather than folded conformations, and that folded analogues of GABA may interact with a class of binding site (GABA C?) insensitive to (−)-baclofen and bicuculline.

Modulation of adenylate cyclase activity by GABA B receptors

Radioligand binding to bicuculline-insensitive GABA B receptors has been shown to be modulated by GTP. In many systems this reflects a link with adenylate cyclase. Consequently the effects of GABA B receptor activation on basal and stimulated cAMP accumulation in brain slices and adenylate cyclase activity in membrane fragments has been studied. In brain slices, GABA, (−) baclofen but not (+) baclofen potentiated the response to stimulation of adenylate cyclase by catecholamines but depressed that to direct cyclase stimulation by forskolin. In membrane fragments activation of GABA B receptors inhibited forskolin activation of the cyclase but appeared not to influence the response to catecholamine stimulation. Little effect on basal adenylate cyclase activity was observed in either slices or membranes. These data indicate GABA B receptors are linked to adenylate cyclase and modulation of this enzyme may be responsible for some of the effects of GABA B receptor activation.

Bicuculline insensitive effects of baclofen

The action of bicuculline on the effect of baclofen and muscimol were investigated in behavioural, biochemical and binding studies in rats. Both (+/-)-baclofen (5 mg kg-1 i.p.) and muscimol (1.4 mg kg-1 i.p.) decreased motor activity and aggressiveness. In investigated doses the drugs mentioned raised DOPAC concentration in the rat striatum. (+)-Bicuculline (1.25 mg kg-1 s.c.) potentiated the behavioural effects of baclofen but failed to alter significantly those of muscimol. The DOPAC rise caused by baclofen and muscimol was not substantially affected by bicuculline. 3H-GABA binding to bicuculline insensitive GABA sites was determined in the rat striatal tissue. (+)-Bicuculline was used to suppress bicuculline sensitive GABA binding. Two GABA binding sites were revealed in the presence of calcium ions of which the low affinity site (KD = 447 nM, B max = 109 pmol g-1 protein) was affected by bicuculline whereas the high affinity site (KD = 8 nM, B max = 24 pmol g-1 protein) was not. Muscimol, GABA and (+/-)-baclofen bound in vitro to these GABA sites with IC50, 44, 49 and 431 nM respectively. The involvement of bicuculline insensitive GABA sites in the effects of (+/-)-baclofen on behaviour and dopamine metabolism is suggested.

Fenibut binding with bicuculline-insensitive GABA receptors in the rat brain

(+/-) Fenibut beta-phenyl-GABA) was not able to displace 3H-GABA in Na+ independent GABA binding (IC50 greater than 250 microM). Nevertheless, (+/-) fenibut and (+/-) baclofen effectively displaced 3H-GABA in Ca2+ dependent GABA binding in the presence of 50 microM (+) bicuculline. (+/-) Fenibut was less potent in this respect. It is suggested that fenibut may act via bicuculline-insensitive GABA receptors.

Bicuculline-insensitive gaba receptors on peripheral autonomic nerve terminals

The action of γ-aminobutyric acid (GABA) and related compounds on rat isolated atria and mouse and guinea-pig isolated vas deferens has been studied. GABA depressed the evoked but not basal release of [ 3H]noradrenaline from atria (IC 50 4 μM) and reduced the twitch responses of the vas deferens (IC 50 3 μM) in a dose-dependent manner. These depressant effects were not prevented by recognised GABA antagonists such as bicuculline and picrotoxin. Numerous GABA analogues, in particular 3-aminopropanesulphonic acid, failed to mimic the action of GABA. However, β-p-chlorophenyl GABA (baclofen) was stereospecifically active. Other related β-substituted derivatives were also active but to a lesser degree than GABA. Pretreatment of the vas deferens with the neuronal GABA uptake inhibitors 2,4-diaminobutyric acid or cis-3-aminocyclohexanecarboxylic acid potentiated the action of GABA. These data suggest the presence of a bicuculline-insensitive GABA receptor on autonomic nerve terminals. Preliminary observations indicate a lack of chloride ion dependence in the action of GABA at this site.