Studies of molecular pharmacophore/receptor models for GABAA/benzodiazepine receptor subtypes: binding affinities of substituted beta-carbolines at recombinant alpha x beta 3 gamma 2 subtypes and quantitative structure-activity relationship studies via a comparative molecular field analysis.

Binding affinities of a series of 44 beta-carbolines with various substituents at the 3-, 4-, 6- and 7-positions are reported at 5 distinct recombinant GABAA/benzodiazepine receptor (BzR) subtypes [alpha x beta 3 gamma 2 (x = 1-3, 5, 6)]. Many of these ligands displayed better selectivity for the alpha 1 containing GABAA isoform. The most selective BCCT 2 and SPH 195 (17) displayed potent affinity (Ki = 0.72 and 7.2 nM for the alpha 1 beta 3 gamma 2 receptor subtype, respectively) and an overall selectivity of 20 and 23 fold, respectively, for the alpha 1 beta 3 gamma 2 receptor subtype. These are the most selective ligands in vitro for the alpha 1 containing GABAA/Bz receptor isoform reported to date to our knowledge. QSAR studies of these ligands for each receptor subtype have been carried out via a Comparative Molecular Field Analysis (CoMFA) and an included volume analysis. Geometries and charge distributions of these ligands have been optimized using ab initio methods (J. Med. Chem., 1992, 35, 4001-4010). Active conformations of flexible 3-alkoxylated beta-carbolines have been examined via a CoMFA approach. QSAR studies via CoMFA support the previous hypothesis that beta-carbolines with different intrinsic activities may follow an alternative alignment rule when they bind into the pharmacophore/receptor site of the BzR. Examination of binding affinities of beta-carbolines by this modeling strategy has established some of the differences, in particular, topologic differences between the lipophilic pockets in the alpha 1 beta 3 gamma 2, alpha 2 beta 3 gamma 2, alpha 3 beta 3 gamma 2, alpha 5 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 subtypes as well as some of the similarities among the pharmacophore/receptor models of these five distinct GABAA/Bz receptor subtypes.     

Effects of subunit types of the cloned GABAA receptor on the response to a neurosteroid.

Combinations of cloned GABAA receptor subtypes, having the subunit combinations alpha i + beta 1 or alpha i + beta 1 + gamma 2 (i = 1, 2, 3), were expressed in Xenopus oocytes. The endogenous steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiates GABA currents induced therein by GABA. This potentiation was greater in the alpha 1 + beta and alpha 3 + beta 1 than in the alpha 2 + beta 1 combinations. The presence of the gamma 2-subunit increased the steroid potency in alpha 1 + beta 1 and alpha 2 + beta 1, but the combination alpha 3 + beta 1 + gamma 2 became much less steroid-sensitive. It is concluded that the steroid modification of the GABAA receptor is strongly influenced by the alpha- and the gamma 2-subunit types.     

Studies of molecular pharmacophore/receptor models for GABAA/BzR subtypes: binding affinities of symmetrically substituted pyrazolo[4,3-c]quinolin-3-ones at recombinant alpha x beta 3 gamma 2 subtypes and quantitative structure-activity relationship studies via a comparative molecular field analysis.

A series of symmetrically substituted pyrazoloquinolinones was synthesized to probe the BzR binding site of different GABAA/Bz receptor subtypes. The affinities of the ligands for different BzR subtypes have been determined by radioligand binding assays on 5 distinct recombinant GABAA receptor isoforms [alpha x beta 3 gamma 2 (x = 1,2,3,5, or 6)]. Most of the ligands synthesized exhibited potent biological activity in vitro. Among them, 3 ligands exhibited enhanced affinity for the alpha 2 beta 3 gamma 2 subtype in comparison to the other subtypes, six ligands demonstrated higher affinity for the alpha 3 beta 3 gamma 2 subtype, while 2 ligands showed some enhanced affinity for the alpha 5 beta 3 gamma 2 subtype. The remainder of the ligands exhibited relatively higher affinities at the alpha 1 containing subtype. To map out the steric and electronic differences between the benzodiazepine binding subtypes, a QSAR analysis by the method of Comparative Molecular Field Analysis (CoMFA) of each receptor subtypes was carried out.     

Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination.

1. Human GABAA receptors containing different alpha and beta subunits with a gamma 2s subunit were expressed in Xenopus oocytes and the effects of pentobarbitone on these subunit combinations were examined by electrophysiological recording of GABA currents with the two-electrode voltage-clamp method. 2. Pentobarbitone has previously been shown to have three actions on GABAA receptors: a potentiation of GABA responses, a direct activation of GABAA receptors and, at high concentrations, a block of the GABA chloride channel. In this study pentobarbitone activity consisted of the above mentioned three components on all the subunit combinations tested. However, the affinities and efficacies varied with receptor subtype. 3. Potentiation of GABA by pentobarbitone occurred over the same concentration-range for all the subunits with affinities in the range of 20-35 microM. The degree of potentiation obtained, however, varied from 236% of GABA EC20 on alpha 1 beta 2 gamma 2s to 536% on alpha 6 beta 2 gamma 2s. 4. Examination of the direct effect of pentobarbitone revealed that the type of alpha subunit present determines both the degree of affinity and efficacy obtained. Receptors containing an alpha 6 subunit produced maximum direct responses to pentobarbitone larger than that obtainable with maximum GABA (150% to 170% of maximum GABA). The maximum direct pentobarbitone response obtainable with other alpha subunits ranged between 45% of maximum GABA for alpha 5 beta 2 gamma 2s to 82% for alpha 2 beta 2 gamma 2s. The affinity of the direct action of pentobarbitone on alpha 6 beta 2 gamma 2s was 58 microM compared to affinities for the other alpha subunits ranging from 139 microM on alpha 2 beta 2 gamma 2s to 528 microM on alpha 5 beta 2 gamma 2s. 5. The type of beta subunit present did not influence the direct action of pentobarbitone to the same extent as the alpha subunit. There were no significant differences between affinity or efficacy on oocytes expressing alpha 6 and gamma 2s with beta 1, beta 2 or beta 3. Affinities and efficacies on oocytes expressing alpha 1 and gamma 2s with beta 1, beta 2 or beta 3 were significantly different with pentobarbitone having a higher affinity and efficacy on alpha 1 beta 3 gamma 2s followed by alpha 1 beta 2 gamma 2s and then alpha 1 beta 1 gamma 2s. 6. The direct effect of pentobarbitone was blocked by picrotoxin but not by competitive antagonists, such as bicuculline or SR95531, indicating that the direct agonist activity of pentobarbitone was not mediated via the GABA binding site. 7. For the first time the influence of the various alpha and beta subunits on the effects of pentobarbitone were demonstrated. The results indicate that GABAA receptors containing alpha 6 subunits have both a higher affinity and efficacy for direct activation by pentobarbitone, and reveal that pentobarbitone binds to more than one site on the GABAA receptor, and these are dependent on receptor subunit composition.     

5-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-2,3,6,7-tetrahydro-1,4-thiazepines as compounds with high affinity at the benzodiazepine binding site on GABA(A) receptors

A series of thiazepines has been studied as new ligands for the benzodiazepine binding site of the GABAA receptor. Compounds with high affinity and weak selectivity regarding alpha beta3gamma2, alpha2beta3gamma2, alpha3beta3gamma2, and alpha5beta3gamma2 subtypes were found. The pharmacophore is discussed based on experimental and theoretical results. The thiazepine sulfur atom was found to be able to act as hydrogen bond acceptor.     

An N-terminal histidine is the primary determinant of alpha subunit-dependent Cu2+ sensitivity of alphabeta3gamma2L GABA(A) receptors

Copper (Cu2+) is a physiologically important cation and is released from nerve terminals. Cu2+ modulates GABAA receptor currents in an alpha subunit subtype-dependent manner; alpha1beta3gamma2L receptors are more sensitive to Cu2+ than alpha6beta3gamma2L receptors. We compared the effect of Cu2+ on alphabeta3gamma2L receptors containing each of the six alpha subtypes and generated alpha1/alpha6 chimeras and mutants to determine the functional domain(s) and specific residues responsible for alpha subtype-dependent differences in Cu2+ sensitivity. Whole-cell GABAA receptor currents were obtained from L929 fibroblasts coexpressing wild-type, chimeric and mutant alpha subunits with beta3 and gamma2L subunits. Maximal Cu2+ inhibition of alpha1beta3gamma2L and alpha2beta3gamma2L receptor currents was larger (52.2 +/- 3.0 and 59.0 +/- 2.5%, respectively) than maximal inhibition of alpha3beta3gamma2L, alpha4beta3gamma2L, alpha5beta3gamma2L, and alpha6beta3gamma2L receptor currents (22.6 +/- 3.1, 19.2 +/- 3.4, 20.2 +/- 4.8, and 21.2 +/- 3.6%, respectively). Receptors containing chimeric constructs with alpha1 subtype N-terminal sequence between residues 127 and 232 were inhibited by Cu2+ to an extent similar to those with alpha1 subtypes, suggesting that this N-terminal region (127-232) contains a major determinant for high Cu2+ sensitivity. alpha1 subtype residues V134, R135, and H141 in a VRAECPMH motif (VQAECPMH in the alpha2 subtype) conferred higher Cu2+ sensitivity, and the H141 residue was the major determinant in the motif. The beta3 subtype M2 domain residue H267, which is a major determinant of Zn2+ inhibition, and alpha6 subtype M2-M3 loop residue H273, which is responsible for the increased Zn2+ sensitivity of the alpha6 subtype, also seemed to contribute to Cu2+ inhibition. These data suggest that the N-terminal VR(Q)AECPMH motif in alpha1 and alpha2 subtypes is the major determinant of increased subtype-dependent inhibition by Cu2+, that residue H141 is the major determinant in that motif, and that Cu2+ may also interact with GABAA receptors at sites similar to or overlapping Zn2+ sites.     

Activity of chlormethiazole at human recombinant GABA(A) and NMDA receptors

1. Investigation into the modulatory effects of chlormethiazole at human recombinant gamma-aminobutyric acid A receptor (GABAA) and N-methyl-d-aspartate (NMDA) receptors was undertaken to gain insight into its mechanism of action and determine if the drug exhibited any subtype-selective activity. 2. Despite a structural similarity to the beta-subunit-selective compound loreclezole, chlormethiazole did not show any difference in maximum efficacy and only a slight difference in EC50 in its potentiating action at alpha1beta1gamma2 and alpha1beta2gamma2 GABAA receptor subtypes with preference for alpha1beta1gamma2. 3. Similar to the previously reported subtype-dependent activity of pentobarbital, chlormethiazole elicited a significantly greater degree of maximum potentiation on receptors lacking a gamma2 subunit, and also those receptors containing an alpha4 or alpha6 subunit. This also demonstrates that chlormethiazole does not act via the benzodiazepine binding site. 4. Unlike pentobarbital and propofol, chlormethiazole elicited only a slight direct GABAA receptor activation at concentrations up to 1 mm. In addition, the drug did not potentiate anaesthetic-mediated currents elicited by pentobarbital or propofol, suggesting that chlormethiazole may be acting via an anaesthetic binding site. 5. Chlormethiazole produced weak nonselective inhibition of human NMDA NR1a+NR2A and NR1a+NR2B receptors. IC50's were approximately 500 microm that likely exceed the therapeutic dose range for chlormethiazole, indicating that the primary mechanism of the compounds in vivo activity is via GABAA receptors.     

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.     

Preferential coassembly of alpha4 and delta subunits of the gamma-aminobutyric acidA receptor in rat thalamus.

Pharmacological study of rat thalamic gamma-aminobutyric acidA (GABAA) receptors revealed the presence of two distinct populations, namely, diazepam-sensitive and diazepam-insensitive [3H]Ro15-4513 binding sites accounting for 94 +/- 2% (1339 +/- 253 fmol/mg protein) and 6 +/- 2% (90 +/- 44 fmol/mg protein) of total sites, respectively. Thalamic diazepam-insensitive sites exhibited a pharmacology that was distinct from diazepam-sensitive sites but comparable to that of the alpha4beta3gamma2 subtype of the GABAA receptor stably expressed in L(tk-) cells. Immunoprecipitation experiments with a specific anti-alpha4-antiserum immunoprecipitated 20 and 7% of total thalamic [3H]muscimol and [3H]Ro15-4513 sites, respectively. Combinatorial immunoprecipitation using antisera against the alpha4, gamma2, and delta subunit revealed that alpha4delta- and alpha4gamma2-containing receptors account for 13 +/- 2 and 8 +/- 3% of [3H]muscimol sites from thalamus, respectively. It also indicated that all delta subunits coexist with an alpha4 subunit in this brain region. In conclusion, our results show that in rat thalamus both alpha4betagamma2 and alpha4betadelta subtypes are expressed but alpha4betadelta is the major alpha4-containing GABAA receptor population.     

Pharmacological study on recombinant human GABA-A receptor complex containing alpha5 (leucine155 to valine) combined with beta3gamma2s subunits.

AIM: To investigate the characterization of the pharmacology of recombinant human GABA-A receptor complex containing alpha5beta3gamma2s subunits, and clarify which amino acids are essential for binding affinity. METHODS: We constructed chimeric subunit of alpha5 (leucine mutated to valine in 155) by site-directed mutation, combined alpha5 or alpha5 (valine 155) with beta3gamma2s subunits by using the baculovirus-transfected Sf-9 insect cell expressing system. Fifteen commercially available effective compounds for the GABA binding recognition site were determined. RESULTS: We found that competitive antagonist SR95531 and inhibitor Thio-4-piol increased IC50 values about 3 folds in alpha5 (valine155) beta3gamma2s compare to alpha5 (leucine155) beta3gamma2s receptor subunit combinations; Other GABA-A receptor ligands have little difference. CONCLUSION: The amino acid residues in 155 is important for the binding affinity and efficacy of SR95531 and Thio-4-piol to GABA-A receptor combinations containing an alpha5 subunit.     

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes of cloned GABAA receptors.

1. In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2. The prototype compounds, U-85575 (12-chloro-5-(5-cyclopropyl-1'',2'',4''- oxadiazol-3''-yl)-2,3-dihydro-diimidazo [1,5-a;1,2-c]quinazoline), and U-92330 (5-acetyl-3-(5''-cyclopropyl-1'',2'',4''-oxadiazole-3''-yl)-7-chloro-4,5-d ihy dro [1,5-a]quinoxaline), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA-mediated Cl- currents in both subtypes were sensitive to Ro 15-1788, a classical benzodiazepine antagonist. 3. Minor changes in the ring substituents of the drugs reduced their affinity to the alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5-benzoyl group showed a lower affinity to the alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple structural features of steroids mediate subtype-selective effects on human alpha4beta3delta GABAA receptors

Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory gamma-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the delta subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional delta-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk-) cells stably transfected with human GABAA receptor alpha4beta3delta and alpha4beta3gamma2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the alpha4beta3delta receptor. 5beta-Pregnan-3beta-ol-20-one, 5beta-pregnane-3alpha,20beta-diol and 5beta-pregnane-3alpha,17alpha-diol-11,20-dione showed particularly high efficacy for alpha4beta3delta. No compounds were identified that simply inhibited responses at delta-containing receptors. However, 5beta-pregnane-3alpha,17alpha,20beta-triol, prednisolone 21-acetate, 4-pregnene-17alpha,20alpha-diol-3-one-20-acetate, 4-pregnen-20alpha-ol-3-one, and 5beta-pregnane-3alpha,17alpha,21-triol-20-one inhibited, though did not abolish, GABA responses at the alpha4beta3gamma2 subtype, while evoking modest-amplitude potentiation of alpha4beta3delta responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of delta-containing GABAA receptors.     

Receptor subtype-dependent positive and negative modulation of GABA(A) receptor function by niflumic acid,a nonsteroidal anti-inflammatory drug

In addition to blocking cyclooxygenases, members of the fenamate group of nonsteroidal anti-inflammatory drugs have been proposed to affect brain GABAA receptors. Using quantitative autoradiography with GABAA receptor-associated ionophore ligand [35S]t-butylbicyclophosphorothionate (TBPS) on rat brain sections, one of the fenamates, niflumate, at micromolar concentration was found to potentiate GABA actions in most brain areas, whereas being in the cerebellar granule cell layer an efficient antagonist similar to furosemide. With recombinant GABAA receptors expressed in Xenopus laevis oocytes, we found that niflumate potentiated 3 microM GABA responses up to 160% and shifted the GABA concentration-response curve to the left in alpha1beta2gamma2 receptors, the predominant GABAA receptor subtype in the brain. This effect needed the gamma2 subunit, because on alpha1beta2 receptors, niflumate exhibited solely an antagonistic effect at high concentrations. The potentiation was not abolished by the specific benzodiazepine site antagonist flumazenil. Niflumate acted as a potent antagonist of alpha6beta2 receptors (with or without gamma2 subunit) and of alphaXbeta2gamma2 receptors containing a chimeric alpha1 to alpha6 subunit, which suggests that niflumate antagonism is dependent on the same transmembrane domain 1- and 2-including fragment of the alpha6 subunit as furosemide antagonism. This antagonism was noncompetitive because the maximal GABA response, but not the potency, was reduced by niflumate. These data show receptor subtype-dependent positive and negative modulatory actions of niflumate on GABAA receptors at clinically relevant concentrations, and they suggest the existence of a novel positive modulatory site on alpha1beta2gamma2 receptors that is dependent on the gamma2 subunit but not associated with the benzodiazepine binding site.     

Thymol, a constituent of thyme essential oil, is a positive allosteric modulator of human GABA(A) receptors and a homo-oligomeric GABA receptor from Drosophila melanogaster

The GABA-modulating and GABA-mimetic activities of the monoterpenoid thymol were explored on human GABAA and Drosophila melanogaster homomeric RDLac GABA receptors expressed in Xenopus laevis oocytes, voltage-clamped at -60 mV. The site of action of thymol was also investigated. Thymol, 1-100 microm, resulted in a dose-dependent potentiation of the EC20 GABA response in oocytes injected with either alpha1beta3gamma2s GABAA subunit cDNAs or the RDLac subunit RNA. At 100 microm thymol, current amplitudes in response to GABA were 416+/-72 and 715+/-85% of controls, respectively. On both receptors, thymol, 100 microm, elicited small currents in the absence of GABA. The EC50 for GABA at alpha1beta3gamma2s GABAA receptors was reduced by 50 microm thymol from 15+/-3 to 4+/-1 microm, and the Hill slope changed from 1.35+/-0.14 to 1.04+/-0.16; there was little effect on the maximum GABA response. Thymol (1-100 microm) potentiation of responses to EC20 GABA for alpha1beta1gamma2s, alpha6beta3gamma2s and alpha1beta3gamma2s human GABAA receptors was almost identical, arguing against actions at benzodiazepine or loreclezole sites. Neither flumazenil, 3-hydroxymethyl-beta-carboline (3-HMC), nor 5alpha-pregnane-3alpha, 20alpha-diol (5alpha-pregnanediol) affected thymol potentiation of the GABA response at alpha1beta3gamma2s receptors, providing evidence against actions at the benzodiazepine/beta-carboline or steroid sites. Thymol stimulated the agonist actions of pentobarbital and propofol on alpha1beta3gamma2s receptors, consistent with a mode of action distinct from that of either compound. These data suggest that thymol potentiates GABAA receptors through a previously unidentified binding site.     

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate is a novel positive modulator of GABAA receptors

Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate (TG41) enhanced the binding both of gamma-aminobutyric acid (GABA) and of flunitrazepam to rat cerebral cortical membranes. Electrophysiological recordings from Xenopus oocytes expressing various recombinant GABA(A) receptor subtypes revealed that TG41 enhanced the function of all receptor subunit combinations tested. The potency of TG41 at receptors containing alpha1, beta2, and gamma2L subunits was greater than that of alphaxalone, etomidate, propofol, or pentobarbital. The potency of TG41 was also greater at receptors containing alpha1 or alpha2 subunits than at those containing alpha4 and it was markedly higher at receptors containing beta2 or beta3 subunits than at those containing beta1. This drug induced a reversible loss of the righting reflex in Xenopus tadpoles and it elicited hypnosis (5 mg/kg) after intravenous administration in rats. These results indicate that the pharmacological profile of TG41 is similar to that of general anesthetics which potentiate the activity of GABA(A) receptors containing the beta2 or beta3 subunit.     

The influence of nicotinic receptor subunit composition upon agonist, alpha-bungarotoxin and insecticide (imidacloprid) binding affinity

A series of cell lines stably expressing recombinant nicotinic acetylcholine receptors (nAChRs) has been established by transfection of mammalian (rat) and insect (Drosophila) nicotinic subunit cDNAs. By equilibrium radioligand binding, we have examined the influence of individual subunits upon the affinity of two nicotinic agonists (epibatidine and methylcarbamylcholine), an antagonist (the snake neurotoxin, alpha-bungarotoxin) and a recently developed chloronicotinyl insecticide (imidacloprid). Imidacloprid bound with very low affinity to the rat alpha4/beta2 nAChR but did so with high affinity to hybrid nAChRs containing Drosophila alpha subunits co-assembled with rat beta2. Of the subunit combinations examined, imidacloprid showed highest affinity binding to nAChRs containing the recently identified Drosophila alpha subunit, D alpha3, co-assembled with beta2. In contrast, no specific binding of imidacloprid was detected when D alpha3 was co-expressed with the mammalian neuronal beta4 subunit, or with the muscle-type (gamma or delta) subunits. However, despite the absence of imidacloprid binding to D alpha3/beta4, D alpha3/gamma or D alpha3/delta, these subunit combinations all exhibited high affinity binding of other nicotinic radioligands. Epibatidine showed substantially higher affinity binding to subunit combinations containing neuronal (beta2 or beta4) subunits than it did to combinations containing muscle-type (gamma or delta) subunits. In contrast, alpha-bungarotoxin bound with higher affinity to combinations containing muscle-type subunits. Our results demonstrate that both alpha and non-alpha subunits exert a profound influence upon the affinity of nicotinic ligands for recombinant nAChRs.     

GABA receptor subunit composition relative to insecticide potency and selectivity

Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.     

Expression and regulation of GABA receptors in the brain]

GABA receptors are classified into two receptor subtypes: GABAA and GABAB receptors. The GABAA receptor, one of the ionotropic type receptors, is formed by various subunits (alpha, beta, gamma and delta subunits) and constitutes the GABA-gated Cl- channel. The different combinations of these subunits are known to produce functionally heterogeneous GABAA receptors both pharmacologically and physiologically. On the other hand, GABAB receptor is known to be metabotropic type which is negatively coupled with adenylate cyclase and inositol phosphate turnover systems via inhibitory GTP binding protein.     

Antagonism of neurosteroid modulation of native gamma-aminobutyric acid receptors by (3alpha,5alpha)-17-phenylandrost-16-en-3-ol

Endogenous pregnane neurosteroids are allosteric modulators at gamma-aminobutyric acid type-A (GABAA) receptors at nanomolar concentrations. There is direct evidence for multiple distinct neurosteroid binding sites on GABAA receptors, dependent upon subunit composition and stoichiometry. This view is supported by the biphasic kinetics of various neuroactive steroids, enantioselectivity of some neurosteroids, selective mutation studies of recombinantly expressed receptors and the selectivity of the neurosteroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA) on 5alpha-pregnane steroid effects on recombinant GABAA receptors expressed in Xenopus oocytes and native receptors in dissociated neurons. However, it is unclear whether this antagonist action is present in a mature mammalian system. The present study evaluated the antagonist activity of 17PA on neurosteroid agonists both in vivo and in vitro by examining the effects of 17PA on 5alpha-pregnane-induced sedation in rats, native mature GABAA receptor ion channels utilizing the chloride flux assay and further studies in recombinant alpha1beta2gamma2 receptors. The data show that 17PA preferentially inhibits 3alpha,5alpha-THP vs. alphaxalone in vivo, preferentially inhibits 3alpha,5alpha-THDOC vs. alphaxalone potentiation of GABA-mediated Cl- uptake in adult cerebral cortical synaptoneurosomes, but shows no specificity for 3alpha,5alpha-THDOC vs. alphaxalone in recombinant alpha1beta2gamma2 receptors. These data provide further evidence of the specificity of 17PA and the heterogeneity of neurosteroid recognition sites on GABAA receptors in the CNS.     

Role of human GABA(A) receptor beta3 subunit in insecticide toxicity

The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.