Species dependent dual modulation of the benzodiazepine/GABA receptor chloride channel by dihydroergosine

Dihydroergosine (50 and 100 mg/kg) enhanced the incidence of bicuculline (3 mg/kg)-induced convulsions in female rats, while 100 mg/kg of dihydroergosine given to female mice made 45% convulsive dose of bicuculline (2.5 mg/kg) to be subconvulsive. The same dose of dihydroergosine enhanced in mice the latency of bicuculline (4 mg/kg)-induced convulsions. Although, in in vitro experiments dihydroergosine showed very weak ability to prevent the binding of 3H-muscimol, the drug was able to diminish and to augment the IC50 of bicuculline and GABA when added to crude synaptosomal pellet of the rat and mouse brain respectively. Lower concentrations of dihydroergosine stimulated and higher inhibited 3H-TBOB binding to the crude synaptosomal pellet of the rat brain. In the preparation of mouse brain dihydroergosine produced only inhibition of 3H-TBOB binding. Only slight quantitative differences were observed in bicuculline-induced stimulation and in GABA- and diazepam-induced inhibition of 3H-TBOB binding between the two species. The results suggest that the opposite species-dependent effects of dihydroergosine on bicuculline-induced convulsions are due to the ability of this drug to modulate species-dependently the benzodiazepine/GABA receptor chloride channel complex.     

Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters

It was suggested in a recent report by Phillips et al. [J. Neurochem. 43, 479-486 (1984)] that the low-affinity binding of [3H]imipramine in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 microM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data). (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.     

In vivo labeling of the central GABA uptake carrier with 3H-Tiagabine.

The in vivo binding of 3H-Tiagabine to the central GABA uptake carrier in mouse brain was characterized. 3H-Tiagabine in vivo bound to a single class of binding sites with a Kd = 72.5 nM and a Bmax = 640 pmol/g tissue. 3H-Tiagabine binding in vivo was regionally distributed within the CNS, and showed a good correlation with 3H-Tiagabine binding in vitro. Pharmacological characterization of 3H-Tiagabine binding in vivo revealed a binding site exhibiting specificity for GABA uptake inhibitors. Experiments examining the in vivo receptor occupancy of the GABA uptake carrier for a series of GABA uptake inhibitors revealed that 20-30% of the GABA uptake sites were occupied at the ED50 for inhibiting DMCM-induced clonic convulsions, while a 50-62% receptor occupancy in vivo was needed to inhibit rotarod performance. These data suggest that 3H-Tiagabine in vivo binding may be a useful method for assessing GABA uptake inhibitor penetration into the CNS, and may be a useful tool for studying the physiological regulation of the GABA uptake carrier.     

Identification of gamma-aminobutyric acid and its binding sites in the rat ovary

Gamma-aminobutyric acid (GABA), GABA synthesizing enzyme and GABA binding sites were measured in rat ovaries. The concentration of GABA in the ovary (0.56 μg/mg protein) was less than that in the brain (1.2–3.4 μg/mg protein), but was six-fold higher than any other non-neuronal tissue examined. Glutamate decarboxylase, the GABA synthesizing enzyme was also found in high concentrations in whole ovarian homogenate but not in enriched ovarian granulosa cells, testis, anterior pituitary or muscles. Furthermore, high affinity (Kd = 15–21 nM), specific GABA binding sites were identified in the ovaries by specific [³H]muscimol binding and the majority of GABA binding sites were associated with the granulosa cells. These data suggest a possible role of GABA in the regulation of ovarian functions.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Action of analogues on γ-aminobutyric acid recognition sites in rat brain

With a view to finding potential GABA-mimetics, the effects of a number of structural analogues of GABA were studied on three parameters associated with GABA neural transmission of rat brain. These were (1) the binding of [³H]GABA to its receptor, (2) the binding of [³H]GABA to its transporter (sodium-dependent binding), and (3) the activity of GABA aminotransferase. Thirteen of the 21 compounds tested competitively inhibited both the low and the high affinity GABA receptor binding components. The most potent inhibitors were morpholinopropane sulphonic acid (MOPS) and aminoethylthiosulphonic acid (AETS). All of the compounds were markedly less effective in inhibiting the high affinity GABA receptor binding system than the low affinity system. The effect of each of the inhibitors was measured on [³H]diazepam receptor binding. Only 6-(morpholinomethyl)kojic acid, kojic amine, 1-piperidinepropane sulphonic acid and 4(4′-azido-benzoimidylamino)butanoic acid (ABBA) were able to induce a stimulation of binding. Four of the inhibitors of [³H]GABA binding were able to appreciably reduce GABA-induced enhancement of diazepam binding. These were N-(2-nitro,4-azidophenyl)aminopropane sulphonic acid, 8-amino-1-napthalene sulphonic acid, narcotine-N-oxide and 5-phenyl-2-pyrrolepropionic acid. These results demonstrate that MOPS and AETS are good inhibitors of GABA receptor binding although there is no other evidence that they might be agonists since they have no effect on diazepam receptor binding. Based on their ability to block GABA-induced stimulation of diazepam binding ABBA, 8-amino-1-naphthalene sulphonic acid and 5-phenyl-2-pyrrolepropionic acid may possess antagonistic properties. ABBA was the only compound to inhibit sodium-dependent [³H]GABA binding. None of the compounds had an effect on the activity of GABA aminotransferase. From this study at least two analogues, MOPS and AETS, have emerged that hold potential as GABA-mimetics. Also, the three GABA recognition sites of rat brain have been shown to possess marked pharmacological differences.     

Characterization of gamma-aminobutyric acid-benzodiazepine receptor complexes by protection against inactivation by group-specific reagents

Abstract: The chemical topography of the γ-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino- and tyrosyl-selective reagents and the protection from it by direct and allosteric ligands of the GABA-BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p -diazobenzenesulfonic acid (DSA), tetrani-tromethane (TNM), and N -acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABA_A site. TNM, like DSA, selectively decreased the number of the low-affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low-affinity sites. TNM pre-treatment also abolished the muscimol enhancement of [³H]diazepam binding, which suggests that the low-affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal-5-phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABA_A receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [³H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [³H]diazepam binding by N -bromosuccinimide and N -acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.     

Effect of taurine on a benzodiazepine-GABA-chloride ionophore receptor complex in rat brain membranes

Taurine at 10 mM had no effect on basal binding of [³H]diazepam to the membranes, while it significantly inhibited a GABA-stimulated binding of [³H]diazepam in cerebral cortex, hippocampus, but not in cerebellum. The inhibition by taurine in the presence of GABA (1M to 1 mM) was not competitive. At low concentrations (0.04 to 0.2 nM) the binding of [³H]propyl--carboline-3-carboxylate, a ligand exhibiting higher affinity for type I than type II benzodiazepine receptors, was not enhanced by GABA, while the binding of higher concentrations (0.5 nM) was. This GABA enhancement of [³H]propyl--carboline-3-carboxylate binding was also selectively blocked by taurine. Pentobarbital increased the binding of [³H]diazepam in a medium containing chloride and this effect was potentiated by taurine at 1–10 mM. These findings may be relevant to the modulatory role of taurine in the central nervous system.     

GABAA type binding sites on membranes of spermatozoa

The binding of [3H] gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABAA type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa.     

Enhancement of γ-Aminobutyric Acid Binding by the Anxiolytic β-Carbolines ZK 93423 and ZK 91296

The effects of two anxiolytic beta-carboline derivatives, ZK 93423 and ZK 91296, on the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to brain membrane preparations from rat cerebral cortex were examined. ZK 93423 concentration-dependently enhanced the specific binding of [3H]GABA, with a maximal increase of 45% above control at a 50 microM concentration. A less pronounced increase was induced by diazepam and by the partial agonist ZK 91296. Scatchard plot analysis revealed that the effect of ZK 93423 was due to an increase in the total number of high- and low-affinity GABA binding sites. The action of ZK 93423 was mediated by benzodiazepine recognition sites since it was blocked by the benzodiazepine antagonists Ro 15-1788 and ZK 93426 at concentrations that failed to modify [3H]GABA binding on their own. Moreover the stimulatory effect of ZK 93423 on [3H]GABA binding was also blocked by the beta-carboline inverse agonist ethyl beta-carboline-3-carboxylate. These results are consistent with the view that ZK 93423 and ZK 91296, similarly to benzodiazepines, exert their pharmacological effects by enhancing the GABAergic transmission at the level of the GABA/benzodiazepine receptor complex.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Dual Action of Pentobarbitone on GABA Binding: Role of Binding Site Integrity

The effects of pentobarbitone on the binding of gamma-aminobutyric acid (GABA) to crude synaptosomal rat brain membranes were studied. In extensively washed P2 membranes, pentobarbitone had a biphasic action: at concentrations ranging between 12.5 and 500 microM, pentobarbitone enhanced GABA binding in a concentration-dependent manner; at concentrations greater than 500 microM, this enhancement was progressively reversed towards control levels of GABA binding. The effect of pentobarbitone seen at higher concentrations may reflect a GABA-mimetic action, since similar concentrations enhanced diazepam binding to washed P2 membranes, an effect antagonized by bicuculline methochloride and picrotoxinin. When washed P2 membranes were incubated in 0.5% Triton X-100 (30 min at 37 degrees C), the enhancement of GABA binding by low concentrations of pentobarbitone was abolished, while at higher concentrations GABA binding was progressively inhibited, suggesting that the GABA-mimetic action is retained. When washed P2 membranes were subjected to high-frequency homogenization, the biphasic dose-response relationship for pentobarbitone was markedly shifted to the right. The choice of membrane preparation appears to be a critical factor in examining drug-receptor interactions in vitro, at least for those involving GABA and the barbiturates.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

GABAA Receptor Populations Bind Agonists and Antagonists Differentially and with Opposite Affinities

Pretreatment of synaptosomal membranes with a diazo-coupling reagent and the presence of Cl- ions were used to differentiate high- and low-affinity populations of postsynaptic gamma-aminobutyric acid (GABAA) receptors. The super-low-affinity GABAA receptors were characterized by the enhancing effect of GABA on [3H]diazepam binding. The GABA antagonists 2-(3-carboxypropyl)-3-amino-4-methyl-6-phenylpyridazinium chloride (SR 95103) and 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135) shifted and suppressed the dose-response curve of GABA on diazepam binding. SR 95103 displaced the lower affinity [3H]GABA binding with higher potency. Dissociation of the binding of the antagonist 2-(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) was polyphasic. Displacing potencies of SR 95531 and GABA were examined on the major (85%) rapid and minor slower phases of dissociation separated kinetically. The slower phase corresponded to higher affinity binding of SR 95531 which was displaced by GABA with about 10 times less potency. Photoaffinity labeling with muscimol decreased the number of [3H]muscimol binding sites by 27%. It decreased the displacing potency of GABA by 72%, but not that of bicuculline methiodide. These findings can be explained by a preferential binding of antagonists to hydrophobic accessory sites around low-affinity GABAA receptors.     

GABA-benzodiazepine interactions: physiological, pharmacological and developmental aspects

Many of the pharmacological actions of the benzodiazepines can be attributed to their actions on gamma-aminobutyric acid (GABA) systms in the brain. Electrophysiological studies on dorsal raphe neurons indicate that the benzodiazepines act postsynaptically to potentiate GABAergic inhibition in this midbrain nucleus. Direct binding studies have shown that both in vitro and in vivo binding of [3H]diazepam to a specific high affinity benzodiazepine binding site in cerebral cortical tissue are enhanced by the direct in vitro addition of GABA and GABA agonists or by pretreatment of animals with GABA analogs and agents that elevate GABA levels in brain. Ontogenic development of [3H]diazepam binding in brain parallels the development of the sodium-independent [3H]GABA binding. The ability of GABA to enhance benzodiazepine binding is present throughout development and inversely related to age. These data suggest that there is a functionally significant interaction between the benzodiazepines and GABA throughout development and at maturity. A model is proposed to relate these interactions to conformational changes in a benzodiazepine/GABA/Cl- ionophore complex.     

γ-AMINOBUTYRIC ACID RECEPTOR BINDING and UPTAKE IN MEMBRANE FRACTIONS OF CRAYFISH MUSCLE

Abstract— The uptake and binding of [³H]GABA and the binding of [³H]muscimol were measured in cell-free fractions of crayfish muscle. The uptake of GABA was saturable, of high affinity ( K _m= 0.5μ m ), and inhibited by low concentrations of compounds believed to block GABA uptake specifically, such as nipecotic acid and 2,4,diaminobutyric acid. The GABA uptake activity was localized to sucrose gradient fractions enriched in sarcolemma as demonstrated by marker enzymes and electron microscopy. The binding of the potent GABAergic agonist muscimol was also localized to the sarcolemma. The binding was saturable, of high affinity (K _D = 9 n m ), and inhibited by GABA (K ₁ = 125 n m ) and by low concentrations of receptor-specific GABA analogues, such as isoguvacine, imidazole acetic acid, and 3-aminopropane sulfonic acid. The rank order for inhibition by GABA analogues of [³H]muscimol binding sites correlated very well with activity on GABA synapses in invertebrates, consistent with specific postsynaptic receptor labeling.     

Na(+)- and Cl(-)-dependent [3H]GABA binding to catfish brain particles

The uptake of [3H]GABA by homogenates of catfish brain was previously shown to be temperature-sensitive and sodium-dependent, and to display saturation kinetics. The present study is a continuation of this work and was undertaken to characterize the initial binding of [3H]GABA to its transport system. [3H]GABA binding to catfish brain particles at 4 degrees C displayed saturability and was totally dependent on both Na+ and Cl-, the optimum concentrations of which were 150 mM and 75 mM, respectively. The effects of a number of drugs on binding were established. Unlabelled GABA was the most potent inhibitor (IC50 = 3.2 microM). The structural analogues nipecotic acid and guvacine were also strongly inhibitory. Interestingly, verapamil, a Ca2+ channel blocker, also inhibited [3H]GABA binding (IC50 = 38 microM). Harmaline, known to compete for Na+ binding in other transport systems, did not appear to influence Na+ binding but was effective at displacing [3H]GABA. These results suggest that the interaction of GABA with its carrier is similar to that found in the mammalian nervous system and is further evidence that GABA is involved in neurotransmission in catfish brain.     

Evidence for Feedback Regulation of Glutamate Decarboxylase by γ-Aminobutyric Acid

Abstract: The concentration of γ-aminobutyric acid (GABA) in the human ovary and the capacity of a membrane preparation from the same organ to bind [³H]GABA specifically were examined. The GABA concentration in the ovary was found to be 214 ± 66 nmol/g frozen tissue (mean ± SEM of six independent determinations). Moreover, a single population of high-affinity GABA binding sites has been identified in the ovarian membranes. The apparent dissociation constant ( K _d) and maximum binding capacity ( B _max) were 38.3 n M and 676 fmol/mg protein, respectively. The specific binding of [³H]GABA was displaced by muscimol, unlabelled GABA, or (+)bicuculline, but was unaffected by (±)baclofen and picrotoxin. The present results show that GABA and an extremely high density of GABA_A receptor binding sites are present in the human ovary, indicating a physiological significance of this amino acid in the female reproductive system.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes.

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 micrograms/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd = 3.34 +/- 0.36 nM without tagetone and Kd,t = 5.86 +/- 0.86 nM with tagetone; p < 0.05, two tailed Student's t-test) without affecting maximal binding (Bmax = 488 +/- 24 fmoles/mg protein, and Bmax,t = 500 +/- 25 fmoles/mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 microM with and without tagetone respectively). GABA was able to decrease merocyanine delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 microM. Tagetone, at a concentration of 50 micrograms/ml and in the presence of GABA 30 microM or 60 microM, enhanced the ability of GABA alone on decreasing delta A570-610. Tagetone alone did not change delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine delta A570-610 value are mainly exerted at the membrane potential level (delta psi m). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding: on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

A Role for Loop F in Modulating GABA Binding Affinity in the GABA(A) Receptor

The brain's major inhibitory neuroreceptor is the ligand-gated ion channel γ-aminobutyric acid (GABA) type A receptor (GABAR). GABARs exist in a variety of different subunit combinations that act to modulate the physiological behavior of GABAR by altering its pharmacological profile, as well as its affinity for GABA. While the α(1)β(2)γ(2) subtype is one of the most prevalent GABARs, the less populous α(6)β(3)δ subtype has much higher GABA sensitivity. Previous studies identified residues crucial for GABA binding; however, the specific molecular differences responsible for this diverse sensitivity are not known. Furthermore, the role of loop F is a divisive subject, with conflicting evidence for ligand binding function. Using homology modeling, ligand docking, and molecular dynamics simulations, we investigated the GABA binding sites of the two receptor subtypes. Simulations identified seven residues that consistently interacted with GABA in both subtypes: αF65, αR132, βL99, βE155, βR/K196, βY205, and βR207. Residue substitution at position β196 (arginine in α(6)β(3)δ, lysine in α(1)β(2)γ(2)) resulted in a shift in GABA binding. However, the major difference between the two binding sites was the magnitude of loop F involvement, with a greater contribution in the α(6)β(3)δ receptor. Free energy calculations confirm that the α(6)β(3)δ binding pocket has an increased affinity for GABA. Thus, the possible role for loop F across the GABAR family is to modulate GABA affinity.