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.     

Multiple actions of propofol on alphabetagamma and alphabetadelta GABAA receptors

GABAA receptors are predominantly composed of alphabetagamma and alphabetadelta isoforms in the brain. It has been proposed that alphabetagamma receptors mediate phasic inhibition, whereas alphabetadelta receptors mediate tonic inhibition. Propofol (2,6-di-isopropylphenol), a widely used anesthetic drug, exerts its effect primarily by modulating GABAA receptors; however, the effects of propofol on the kinetic properties of alphabetagamma and alphabetadelta receptors are uncertain. We transfected human embryonic kidney (HEK293T) cells with cDNAs encoding rat alpha1, alpha6, beta3, gamma2L, or delta subunits and performed whole-cell patch-clamp recordings to explore this issue. Propofol (3 microM) increased GABA concentration-response curve maximal currents similarly for both alpha1beta3gamma2L and alpha6beta3gamma2L receptors, but propofol increased those for alpha1beta3delta and alpha6beta3delta receptors differently, the increase being greater for alpha1beta3delta than for alpha6beta3delta receptors. Propofol (10 microM) produced similar alterations in alpha1beta3gamma2L and alpha6beta3gamma2L receptor currents when using a preapplication protocol; peak currents were not altered, desensitization was reduced, and deactivation was prolonged. Propofol enhanced peak currents for both alpha1beta3delta and alpha6beta3delta receptors, but the enhancement was greater for alpha1beta3delta receptors. Desensitization of these two isoforms was not modified by propofol. Propofol did not alter the deactivation rate of alpha1beta3delta receptor currents but did slow deactivation of alpha6beta3delta receptor currents. The findings that propofol reduced desensitization and prolonged deactivation of gamma2L subunit-containing receptors and enhanced peak currents or prolonged deactivation of delta subunit-containing receptors suggest that propofol enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain.     

Pentobarbital differentially modulates alpha1beta3delta and alpha1beta3gamma2L GABAA receptor currents

GABAA receptors are modulated by a variety of compounds, including the neurosteroids and barbiturates. Although the effects of barbiturates on alphabetagamma isoforms, thought to dominate phasic (synaptic) GABAergic inhibition, have been extensively studied, the effects of pentobarbital on kinetic properties of alphabetadelta GABAA receptors, thought to mediate tonic (extra- or perisynaptic) inhibition, are unknown. Using ultrafast drug delivery and single channel recording techniques, we demonstrate isoform-specific pentobarbital modulation of low-efficacy, minimally desensitizing alpha1beta3 currents and high-efficacy, rapidly desensitizing alpha1beta3gamma2L currents. Specifically, with saturating concentrations of GABA, pentobarbital substantially potentiated peak alpha1beta3delta receptor currents but failed to potentiate peak alpha1beta3gamma2L receptor currents. Also, pentobarbital had opposite effects on the desensitization of alpha1beta3delta (increased) and alpha1beta3gamma2L (decreased) receptor currents evoked by saturating GABA. Pentobarbital increased steady-state alpha1beta3delta receptor single channel open duration primarily by introducing a longer duration open state, whereas for alpha1beta3gamma2L receptor channels, pentobarbital increased mean open duration by increasing the proportion and duration of the longest open state. The data support previous suggestions that GABA may be a partial agonist at alphabetadelta isoforms, which may render them particularly sensitive to allosteric modulation. The remarkable increase in gating efficacy of alpha1beta3delta receptors suggests that alphabetadelta isoforms, and by inference tonic forms of inhibition, may be important targets for barbiturates.     

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.     

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.     

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.     

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)     

Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors

Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.     

Influence of recombinant gamma-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of gamma-aminobutyric acid-gated Cl- currents.

gamma-Aminobutyric acid (GABA)-activated Cl- currents in neonatal rat cortical neurons and in cultured cells engineered for the expression of specific molecular forms of the GABAA receptor alpha, beta, and gamma subunits, were recorded with the patch-clamp technique in the whole-cell configuration. The effects of various allosteric modulators of GABAA receptors were determined. Diazepam and clonazepam showed greater efficacy as positive modulators of GABA-elicited currents in alpha 2 beta 1 gamma 2 or alpha 3 beta 1 gamma 2 receptors than in alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Alpidem was more efficacious at alpha 1 beta 1 gamma 2 or alpha 2 beta 1 gamma 2 receptors than at alpha 1 beta 1 gamma 2 or alpha 5 beta 1 gamma 2 receptors or in cortical neurons. Conversely, zolpidem was equally efficacious for all these receptors except for alpha 5 beta 1 gamma 2. Both imidazopyridines (alpidem and zolpidem) were virtually ineffective at modulating the GABA response of alpha 5 beta 1 gamma 2 receptors and in almost all the receptors assembled from alpha 1, alpha 2, alpha 3 or alpha 5 subunits together with beta 1 and gamma 1 subunits. The beta-carboline derivatives methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and methyl-beta-carboline-3-carboxylate (beta-CCM) elicited a positive allosteric modulation of alpha 1 beta 1 gamma 1 or alpha 2 beta 1 gamma 1 receptors, whereas they acted as negative allosteric modulators at nearly all other receptors tested, as they do in cortical neurons. Although the positive allosteric modulation by beta-carbolines never exceeded a doubling of the GABA response, DMCM was more efficacious at alpha 1 beta 1 gamma 1 receptors and beta-CCM was more efficacious at alpha 2 beta 1 gamma 1 receptors. DMCM was inactive at alpha 3 beta 1 gamma 1 receptors, whereas beta-CCM was virtually inactive at alpha 5 beta 1 gamma 1 receptors. The benzodiazepine 4'-chlorodiazepam, which is a negative modulator resistent to flumazenil inhibition, acted at all the various GABAA receptors that contained a gamma subunit.     

Interaction of beta-carboline inverse agonists for the benzodiazepine site with another site on GABAA receptors.

1. We examined the effects of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-aminobutyric acid (GABA)-induced Cl-currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl- currents were measured in the whole cell configuration of patch clamp techniques. 2. DMCM at low concentrations (< 0.5 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 3. Ro 15-1788 (a classical benzodiazepine antagonist) had no effect on Cl- current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl- current enhancement by pentobarbitone or by 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4. Among beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.     

Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes.

1. A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human alpha 3 beta 2 gamma 2L) or invertebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recombinant GABA receptors has been made using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. 2. Oocytes preinjected with the appropriate cRNAs responded to bath applied GABA with a concentration-dependent inward current. EC50 values of 102 +/- 18 microM; 152 +/- 10 microM and 9.8 +/- 1.7 microM were determined for human alpha 3, beta 1 gamma 2L, Rdl splice variant and the Rdl receptors respectively. 3. Pentobarbitone enhanced GABA-evoked currents mediated by either the mammalian or invertebrate receptors. Utilizing the appropriate GABA EC10, the EC50 for potentiation was estimated to be 45 +/- 1 microM, 312 +/- 8 microM and 837 +/- 25 microM for human alpha 3, beta 1 gamma 2L, Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC10 concentration of GABA where this latter response = 1) at the mammalian receptor (10.2 +/- 1 fold) was greater that at either the Rdl splice variant (5.5 +/- 1.3 fold) or Rdl (7.9 +/- 0.8 fold) receptors. 4. Pentobarbitone directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 1.2 +/- 0.03 mM and had a maximal effect amounting to 3.3 +/- 0.4 fold of the response evoked by the EC10 concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 microM picrotoxin and potentiated by 0.3 microM flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5. 5 alpha-Pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 87 +/- 3 nM and a maximal enhancement of 6.7 +/- 0.8 fold of that produced by the GABA EC10 concentration. By contrast, relatively high concentrations (3-10 microM) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6. A small direct effect of 5 alpha-pregnan-3 alpha-ol-20-one (0.3-10 microM) was detected for the human alpha 3 beta 1 gamma 2L receptor (maximal effect only 0.08 +/- 0.01 times that of the GABA EC10). This response was antagonized by 30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). 5 alpha-Pregnan-3 alpha-ol-20-one did not directly activate the invertebrate GABA receptors. 7. Propofol enhanced GABA-evoked currents mediated by human alpha 3 beta 1 gamma 2L and Rdl splice variant receptors with EC50 values of 3.5 +/- 0.1 microM and 8 +/- 0.3 microM respectively. The maximal enhancement was similar at the two receptor types (human 11 +/- 1.8 fold; invertebrate 8.8 +/- 1.4 fold that of the GABA EC10). 8. Propofol directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 129 +/- 10 microM, and at a maximally effective concentration, evoked a current amounting to 3.5 +/- 0.5 times that elicited by a concentration of GABA producing 10% of the maximal response. The response to propofol was blocked by 10-30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). Propofol did not directly activate the invertebrate Rdl splice variant receptor. 9. GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor were potentiated by etomidate (EC50 = 7.7 +/- 0.2 microM) and maximally enhanced to 8 +/- 0.8 fold of the response to an EC10 concentration of GABA. By contrast, the Rdl, or Rdl splice variant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mammalian nor the invertebrate receptors, were directly activated by etomidate. 10. delta-Hexachlorocyclohexane enhanced GABA-evoked currents with EC50 values of 3.4 +/- 0.1 microM and 3.0 +/- 0.1 microM for the human alpha 3 beta 1 gamma 2L receptor and the Rdl splice variant receptor respectively. The maximal enhancement was 4.5     

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.     

Novel plant substances acting as beta subunit isoform-selective positive allosteric modulators of GABAA receptors

GABAA receptors are modulated by a large variety of compounds. A common chemical characteristic of most of these modulators is that they contain a cyclic entity. Three linear molecules of a polyacetylene structure were isolated from the East African medicinal plant Cussonia zimmermannii Harms and shown to allosterically stimulate GABAA receptors. Stimulation was not abolished by the absence of the gamma2 subunit, the benzodiazepine antagonist Ro15-1788 (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester), or the point mutation beta2N265S that abolishes effects by loreclezole. At a concentration of 30 microM, the substances by themselves elicited only tiny currents. Maximal stimulation at alpha1beta2gamma2 amounted to 110 to 450% for the three substances, and half-maximal stimulation was observed at concentrations of 1 to 2 muM. Stimulation was subunit composition-dependent and was for the substance MS-1, alpha1beta2gamma2 approximately alpha1beta2 approximately alpha3beta2gamma2 > alpha2beta2gamma2 > alpha5beta2gamma2 approximately alpha1beta3gamma2 approximately alpha6beta2gamma2 > alpha1beta1gamma2, for MS-2 alpha1beta2gamma2 approximately alpha3beta2gamma2 approximately alpha1beta2 > alpha2beta2gamma2 approximately alpha6beta2gamma2 approximately alpha5beta2gamma2 > alpha1beta1gamma2, and for MS-4, alpha1beta2gamma2 approximately alpha1beta2 approximately alpha5beta2gamma2 approximately alpha3beta2gamma2 approximately alpha2beta2gamma2 > alpha6beta2gamma2 > alpha1beta1gamma2. Maximal stimulation by MS-1 was 450% at alpha1beta2gamma2, 80% at alpha1beta1gamma2, and 150% at alpha1beta3gamma2. MS-1 was thus specific for receptors containing the beta2 subunit. The reversal potential was unaffected by 10 microM MS-1, whereas apparent picrotoxin affinity for current inhibition was increased approximately 3-fold. In summary, these positive allosteric modulators of GABAA receptors of plant origin have a novel unusual chemical structure and act at a site independent of that of benzodiazepines and loreclezole.     

Cultured Hippocampal Pyramidal Neurons Express Two Kinds of GABAA Receptors

We combined a study of the subcellular distribution of the alpha1, alpha2, alpha4, beta1, beta2/3, gamma2, and delta subunits of the GABAA receptor with an electrophysiological analysis of GABAA receptor currents determine the to types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that alpha1, alpha2, beta2/3, and gamma2 subunits formed distinct clusters of various sizes, which were colocalized with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22 to 58%. In contrast, alpha4, beta1, and delta subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA receptor currents were moderately sensitive to GABAA and were modulated by diazepam. The whole-cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn2+, furosemide, and loreclezole; they were insensitive to diazepam. These studies suggest that two kinds of GABAA receptors are expressed on cultured hippocampal neurons. One kind of receptor formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The alpha1, alpha2, beta2/3, and gamma2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The alpha4, beta1, and delta subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing alpha4, beta1, and delta subunits.     

6-Methylflavanone, a more efficacious positive allosteric modulator of gamma-aminobutyric acid (GABA) action at human recombinant alpha2beta2gamma2L than at alpha1beta2gamma2L and alpha1beta2 GABA(A) receptors expressed in Xenopus oocytes

6-Methylflavanone acted as a positive allosteric modulator of gamma-aminobutyric acid (GABA) responses at human recombinant alpha1beta2gamma2L, alpha2beta2gamma2L and alpha1beta2 GABA(A) receptors expressed in Xenopus laevis oocytes. It was essentially inactive at rho1 GABA(C) receptors. The EC50 values for 6-methylflavanone for the positive modulation of the EC(10-20) GABA responses were 22 microM, 10 microM and 6 microM and the maximum potentiations were 120%, 417% and 130% at alpha1beta2gamma2L, alpha2beta2gamma2L and alpha1beta2 GABA(A) receptors respectively. Thus 6-methylflavanone was much more efficacious as a positive modulator at alpha2beta2gamma2L than at alpha1beta2gamma2L and alpha1beta2 GABA(A) receptors. This may be significant since diazepam-induced anxiolysis is considered to be mediated via alpha2-containing GABA(A) receptors, while sedation is thought to be mediated via alpha1-containing GABA(A) receptors. We have previously reported that 6-methylflavone (1-100 microM) produced positive allosteric modulation at alpha1beta2gamma2L and alpha1beta2 GABA(A) receptors with no significant difference between the enhancement seen at either receptor subtype. In the present study, 6-methylflavone was tested at alpha2beta2gamma2L GABA(A) receptors and found to maximally potentiate the EC(10-20) GABA response by 183+/-39% which is similar to that previously observed for 6-methylflavone at alpha1beta2gamma2L GABA(A) receptors. Thus, 6-methylflavone did not show a preference for alpha2beta2gamma2L over alpha1beta2gamma2L GABA(A) receptors in terms of efficacy. Compared to 6-methylflavone, 6-methylflavanone is more efficacious as a positive allosteric modulator at alpha2beta2gamma2L GABA(A) receptors, and less efficacious at alpha1beta2gamma2L GABA(A) receptors. This may represent a relatively unique type of selectivity for positive modulators of GABA-A receptor subtypes based on efficacy as distinct from potency. As was previously shown for 6-methylflavone at alpha1beta2gamma2L GABA(A) receptors, the positive modulation of GABA responses at alpha1beta2gamma2L and alpha2beta2gamma2L GABA(A) receptors by 6-methylflavanone was insensitive to antagonism by flumazenil, indicating that this action is not mediated via "high-affinity" benzodiazepine sites.     

Immortalized hypothalamic GT1-7 neurons express functional gamma-aminobutyric acid type A receptors.

Neuronal cell lines provide a source of pure populations of neurons and allow the properties of many neurotransmitter receptors to be studied. However, none of these cells have been reported to express functional gamma-aminobutyric acid (GABA)A receptors. Indeed, there have been no reports of cell lines expressing functional amino acid receptors. Using biochemical and electrophysiological techniques, we have identified a neuronal cell line expressing functional GABAA receptors. Membranes from immortalized hypothalamic (GT1-7) neurons bound [3H]muscimol but not [3H]flunitrazepam. GABA-activated chloride currents, recorded from GT1-7 cells, were blocked by bicuculline and Zn2+ but were insensitive to diazepam. These results suggest that GABAA receptors on GT1-7 cells lack gamma subunits. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one and pentobarbital both modulated GABAA receptors in these cells. Polymerase chain reaction analysis of the cells revealed the presence of mRNAs encoding alpha 1, beta 1, and beta 3 polypeptides. GT1-7 cells provide a useful model system for studying the regulation of GABAA receptor polypeptide expression.     

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.     

Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids

Effects of common monoterpenoid alcohols and ketones were investigated on recombinant human gamma-aminobutyric acid A (GABAA; alpha1beta2gamma2s) and glycine (alpha1 homomers) receptors expressed in Xenopus oocytes. GABA currents were enhanced by coapplications of 10-300 microM: (+)-menthol>(-)-menthol>(-)-borneol>(-)-menthone=camphor enantiomers>carvone enantiomers, with menthol acting stereoselectively. By contrast, thujone diastereomers inhibited GABAA receptor currents while glycine currents were only markedly potentiated by menthol. Positive modulation by (+)-menthol was explored given its pronounced effects (e.g., at 100 microM, GABA and glycine EC20 responses increased by 496+/-113% and 135+/-56%, respectively). (+)-Menthol, 100 microM, reduced EC50 values for GABA and glycine from 82.8+/-9.9 to 25.0+/-1.8 microM, and from 98.7+/-8.6 to 75.7+/-9.4 microM respectively, with negligible effects on maximal currents. This study reveals a novel neuroactive role for menthol as a stereoselective modulator of inhibitory ligand-gated channels.     

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.     

Residues in transmembrane domains I and II determine gamma-aminobutyric acid type AA receptor subtype-selective antagonism by furosemide.

GABAA receptors in cerebellar granule cells are unique in expressing a subtype containing the alpha6 subunit. This receptor subtype has high affinity for GABA and produces a degree of tonic inhibition on cerebellar granule cells, modulating the firing of these cells via spillover of GABA from GABAergic synapses. This receptor subtype also has selective affinity for the diuretic furosemide over receptors containing other alpha-subunits. Furosemide exhibits approximately 100-fold selectivity for alpha6-containing receptors over alpha1-containing receptors. By making alpha1/alpha6 chimeras we have identified a transmembrane region (209-279) responsible for the high furosemide sensitivity of alpha6beta3gamma2s receptors. Within the alpha1 transmembrane region, a single amino acid was identified that when mutated from threonine to isoleucine, increased furosemide sensitivity by 20-fold. We demonstrate the beta-subunit selectivity of furosemide to be due to asparagine 265 in the beta2 and beta3 transmembrane-domain II similar to that observed with potentiation by the anticonvulsant loreclezole. We also show that Ile in transmembrane-domain I accounts for the increased GABA sensitivity observed at alpha6beta3gamma2s compared with alpha1beta3gamma2s receptors, but did not affect direct activation by pentobarbital or potentiation by the benzodiazepine flunitrazepam. Location of these residues within transmembrane domains leads to speculation that they may be involved in the channel-gating mechanism conferring increased receptor activation by GABA, in addition to conferring furosemide sensitivity.