Comparison of the effects of zaleplon,zolpidem, and triazolam at various GABA(A) receptor subtypes

The pyrazolopyrimidine zaleplon is a hypnotic agent that acts at the benzodiazepine recognition site of GABA(A) receptors. Zaleplon, like the hypnotic agent zolpidem but unlike classical benzodiazepines, exhibits preferential affinity for type I benzodiazepine (BZ(1)/omega(1)) receptors in binding assays. The modulatory action of zaleplon at GABA(A) receptors has now been compared with those of zolpidem and the triazolobenzodiazepine triazolam. Zaleplon potentiated GABA-evoked Cl(-) currents in Xenopus oocytes expressing human GABA(A) receptor subunits with a potency that was higher at alpha1beta2gamma2 receptors than at alpha2- or alpha3-containing receptors. Zolpidem, but not triazolam, also exhibited selectivity for alpha1-containing receptors. However, the potency of zaleplon at these various receptors was one-third to one-half that of zolpidem. Zaleplon and zolpidem also differed in their actions at receptors containing the alpha5 or gamma3 subunit. Zaleplon, zolpidem, and triazolam exhibited similar patterns of efficacy among the different receptor subtypes. The affinities of zaleplon for [(3)H]flunitrazepam or t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding sites in rat brain membranes were lower than those of zolpidem or triazolam. Furthermore, zaleplon, unlike zolpidem, exhibited virtually no affinity for the peripheral type of benzodiazepine receptor.     

Subunit selectivity of topiramate modulation of heteromeric GABA(A) receptors

Topiramate (TPM) is an anticonvulsant of novel chemical structure whose mechanism of action remains elusive. Reports of TPM modulation of ligand- and voltage-gated ion channel functions are variable and often inconsistent. In fact, TPM has been found to produce enhancement, inhibition, and no effect on GABA-currents of cultured neurons and GABA(A) receptors expressed in Xenopus laevis oocytes. To identify possible causes for the variable effects of TPM on GABA(A) receptors, multiple combinations of recombinant GABA(A) receptor subunits were expressed in Xenopus oocytes. TPM modulation of GABA-currents was sensitive to GABA concentrations and the beta subunit isoform co-expressed in heteromeric GABA(A) receptors. TPM potentiated and directly activated heteromeric receptors containing either beta(2) or beta(3) subunit. TPM's direct activation was most effective on receptors comprised of alpha(4)beta(3)gamma(2S) subunits and activated approximately 74% of the peak GABA-current. TPM modulation of beta(1)-containing heteromeric receptors depended on the co-expressed alpha subunit isoform (i.e., either TPM enhancement or inhibition). Depolarized potentials decreased TPM enhancement and increased TPM inhibition depending on the beta subunit present. These results suggest that the effects of TPM on GABA(A) receptor function will depend on the expression of specific subunits that can be regionally and temporally distributed, and altered by neurological disorders.     

Subunit mutations affect ethanol actions on GABA(A) receptors expressed in Xenopus oocytes.

1. Mutations of specific amino acids were introduced in transmembrane domains (TM) of GABA(A) receptor alpha2, beta1 and gamma2L subunits. The effects of these mutations on the action of ethanol were studied using the Xenopus oocyte expression system and two-electrode voltage-clamp recording techniques. 2. Mutant alpha2 subunits containing S270I (TM2) or A291W (TM3) made the receptor more sensitive to GABA, as compared to wild-type alpha2beta1gamma2L receptor. The mutation S265I (TM2) of beta1 and S280I (TM2) or S30IW (TM3) in gamma2L subunits did not alter apparent affinity of the receptor for GABA. M286W (TM3) in the beta1 subunit resulted in a receptor that was tonically open. 3. Using an EC5 concentration of GABA, the function of the wild-type receptor with alpha2beta1gamma2L subunits was potentiated by ethanol (50-200 mM). The mutations in TM2 or TM3 of the alpha2 subunit diminished the potentiation by ethanol. The action of ethanol was also eliminated with a mutation in the TM2 site of the beta1 subunit. Ethanol produced significant inhibition of GABA responses in receptors containing the combination of alpha2 and beta1 TM2 mutants with a wild-type gamma2L subunit. A small but significant reduction in the potentiation by ethanol was observed with gamma2L TM2 and/or TM3 mutants. 4. From these results, we suggest that in heteromeric GABA(A) receptors composed of the alpha, beta and gamma subunits, ethanol may bind in a cavity formed by TM2 and TM3, and that binding to the alpha or beta subunit may be more critical than the gamma subunit.     

Effects of gamma-HCH and delta-HCH on human recombinant GABA(A) receptors: dependence on GABA(A) receptor subunit combination

1. Human GABA(A) receptors containing different alpha and beta subunits with or without the gamma 2S or gamma 2L subunits were expressed in XENOPUS: oocytes and the effects of the insecticides gamma- and delta-hexachlorocyclohexane (gamma-HCH and delta-HCH, respectively) on these receptor subunit combinations were examined using two electrode voltage-clamp procedures. 2. gamma-HCH produced incomplete inhibition of GABA responses on all receptor combinations examined with affinities in the range of 1.1--1.9 microM. Affinity was not dependent on subunit composition but the maximum percentage of inhibition was significantly reduced in beta 1-containing receptors. delta-HCH both potentiated GABA(A) receptors and activated them in the absence of GABA at concentrations higher than those producing potentiation. Allosteric enhancement of GABA(A) receptor function by delta-HCH was not affected by the subunit composition of the receptor, By contrast the GABA mimetic actions of delta-HCH were abolished in receptors containing either alpha 4, beta 1 or gamma 2L subunits. 4. Sensitivity to the direct actions were not restored in receptors containing the mutant beta 1(S290N) subunit, but alpha 1 beta 2 gamma 2L receptors became sensitive to the direct actions of delta-HCH when oocytes were treated for 24 h with the protein kinase inhibitor isoquinolinesulphonyl-2-methyl piperazine dihydrochloride (H-7). 5. We have shown the influence of various alpha, beta and gamma subunits on the inhibitory, GABA mimetic and allosteric effects of HCH isomers. The data reveal that neither the inhibitory actions of gamma-HCH nor the allosteric effects delta-HCH has a strict subunit dependency. By contrast, sensitivity to the direct actions of delta-HCH are abolished in receptors containing alpha 4, beta 1 or gamma 2L subunits.     

The novel anxiolytic ELB139 displays selectivity to recombinant GABA(A) receptors different from diazepam

A chemically heterogeneous group of compounds acts at the benzodiazepine (BZ) recognition site of the diverse gamma-aminobutyric acid type A (GABA(A)) receptor complexes which can assemble from more than 16 known subunits. Most 1,4-BZs like diazepam recognize all GABA(A)/BZ receptors containing the alpha1-3 or alpha5 together with any beta and the gamma2 subunit. Other compounds differentiate less, e.g. Ro15-4513, that additionally recognizes alpha4- and a6-containing receptors, or differentiate more, e.g. zolpidem, that recognizes preferentially alpha1-containing receptors. Here we describe the functional properties of 1-(4-chloro-phenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on (ELB139) in the presence and absence of the BZ receptor antagonist flumazenil (Ro15-1788) on recombinant alphaibeta2gamma2 (i=1-5) receptor subtypes expressed in HEK 293 cells. The properties were measured with the whole-cell variation of the patch-clamp technique and compared to those of diazepam. Like the latter, ELB139 did not potentiate GABA-induced currents in alpha4-containing receptors, but it displays functional subtype specificity between alpha1, alpha2, alpha3, and alpha5beta2gamma2 receptors with highest potency in alpha3-containing receptors but highest efficacy in alpha1- or alpha2-containing receptors, respectively. ELB139 acted as a partial agonist on these receptor subtypes reaching 40-50% of the efficacy of diazepam.     

Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions

Based on an unexpected high maximum response to piperidine-4-sulphonic acid (P4S) at human alpha1alpha6beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABA(A) receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine-4-sulphonic acid (P4S) as partial agonists, respectively, at alpha1beta2gamma2, alpha6beta2gamma2 and alpha1alpha6beta2gamma2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole-cell patch-clamp recordings were used to investigate the pharmacological profile of the partial GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP), P4S, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), and 3-(4-piperidyl)isoxazol-5-ol (iso-4-PIOL), and the competitive GABA(A) receptor antagonists Bicuculline Methbromide (BMB) and 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly alpha1, alpha2, alpha3, and alpha5 containing receptors and alpha1, alpha6 and alpha1alpha6 containing receptors, respectively. 4-PIOL and iso-4-PIOL, which at GABA(A) receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4-PIOL was slightly more potent at cortical neurones than at granule neurones and iso-4-PIOL was more potent in inhibiting isoguvacine-evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4-PIOL corresponded to that of a partial agonist, whereas that of iso-4-PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.     

Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement

All GABA(A) receptor (GABAR) subunits include an invariant proline in a consensus motif in the first transmembrane segment (M1). In receptors containing bovine alpha1, beta1 and gamma2 subunits, we analyzed the effect of mutating this M1 proline to alanine in the alpha1 or beta1 subunit using 3 different expression systems. The beta1 subunit mutant, beta1(P228A), reduced the EC(50) for GABA about 10-fold in whole cell recordings in HEK293 cells and L929 fibroblasts. The corresponding alpha1 subunit mutant (alpha1(P233A)) also reduced the GABA EC(50) when expressed in Xenopus oocytes; alpha1(P233A)beta1gamma2S receptors failed to assemble in HEK293 cells. Binding of [(3)H]flumazenil and [(3)H]muscimol to transfected HEK293 cell membranes showed similar levels of receptor expression with GABARs containing beta1 or beta1(P228A) subunits and no change in the affinity for [(3)H]flumazenil; however, the affinity for [(3)H]muscimol was increased 6-fold in GABARs containing beta1(P228A) subunits. In L929 cells, presence of the beta1(P228A) subunit reduced enhancement by barbiturates without affecting enhancement by diazepam or alfaxalone. Single channel recordings from alpha1beta1gamma2S and alpha1beta1(P228A)gamma2L GABARs showed similar channel kinetics, but beta-mutant containing receptors opened at lower GABA concentrations. We conclude that the beta1 subunit M1 segment proline affects the linkage between GABA binding and channel gating and is critical for barbiturate enhancement. Mutation of the M1 proline in the alpha1 subunit also inhibited receptor assembly.     

6,3'-Dinitroflavone is a low efficacy modulator of GABA(A) receptors

6,3'-Dinitroflavone (6,3'-DNF) is a synthetic flavone derivative that exerts anxiolytic effects in the elevated plus maze. Based on the finding that this effect is blocked by Ro15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) which is a specific antagonist at the benzodiazepine binding site of GABA(A) receptors we investigated the interaction of 6,3'-DNF with several recombinant GABA(A) receptor subtypes. Inhibition of [(3)H]flunitrazepam binding to recombinant GABA(A) receptors in transiently transfected HEK293 cells indicated that 6,3'-DNF exhibited the highest affinity for GABA(A) receptors composed of alpha1beta2gamma2 subunits and a 2-20 fold lower affinity for homologous receptors containing alpha2, alpha3, or alpha5 subunits. Two-electrode voltage-clamp experiments in Xenopus oocytes indicated that 6,3'-DNF does not induce chloride flux in the absence of GABA, but exerts low efficacy inverse agonistic modulatory effects on GABA-elicited currents in the GABA(A) receptor subtypes alpha1beta2gamma2 and alpha5beta2gamma2. In the subtypes alpha2beta2gamma2, alpha3beta2gamma2, alpha4beta2gamma2, alpha6beta2gamma2 or alpha4beta2delta and alpha4beta3delta, 6,3'-DNF exerts either none or very low efficacy positive modulatory effects. In contrast, 100 nM Ro15-1788 exhibited weak to moderate partial agonistic effects on each receptor investigated. These data indicate that Ro15-1788 only can antagonize the weak inverse agonist effects of 6,3'-DNF on alpha1beta2gamma2 and alpha5beta2gamma2 receptors, but will enhance the weak agonistic effects on the other receptor subtypes investigated. The possible mechanism of the Ro15-1788 sensitive anxiolytic effect of 6,3'-DNF is discussed.     

Determinants of amentoflavone interaction at the GABA(A) receptor

We investigated the recognition properties of different GABA(A) receptor subtypes and mutant receptors for the biflavonoid amentoflavone, a constituent of St. John's Wort. Radioligand binding studies showed that amentoflavone recognition paralleled that of the classical benzodiazepine diazepam in that it had little or no affinity for alpha4- or alpha6-containing receptors. Lysine and alanine substitutions at position 101 of the rat alpha1 subunit resulted in a complete loss of competitive amentoflavone binding, but functional analysis of the alanine mutant expressed with beta2 and gamma2 subunits in Xenopus oocytes revealed no significant difference in the negative modulation of GABA-induced currents brought about by amentoflavone. Furthermore, elimination of the gamma subunit had no effect on the negative modulation of these currents. This negative modulation was also observed at alpha1beta1gamma2 GABA(A) receptors and is therefore not likely mediated by the loreclezole site. These results suggest a complex mechanism of amentoflavone interaction at GABA(A) receptors.     

(3-Aminocyclopentyl)methylphosphinic acids: novel GABA(C) receptor antagonists

Our understanding of the role GABA(C) receptors play in the central nervous system is limited due to a lack of specific ligands. Here we describe the pharmacological effects of (+/-)-cis-3- and (+/-)-trans-3-(aminocyclopentyl)methylphosphinic acids ((+/-)-cis- and (+/-)-trans-3-ACPMPA) as novel ligands for the GABA(C) receptor showing little activity at GABA(A) or GABA(B) receptors. (+/-)-cis-3-ACPMPA has similar potency to (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) at human recombinant rho1 (K(B)=1.0+/-0.2microM) and rat rho3 (K(B)=5.4+/-0.8microM) but is 15 times more potent than TPMPA on human recombinant rho2 (K(B)=1.0+/-0.3microM) GABA(C) receptors expressed in Xenopus oocytes. (+/-)-cis- and (+/-)-trans-3-ACPMPA are novel lead compounds for developing into more potent and selective GABA(C) receptor antagonists with increased lipophilicity for in vivo studies.     

Valerenic acid potentiates and inhibits GABA(A) receptors: molecular mechanism and subunit specificity

Valerian is a commonly used herbal medicinal product for the treatment of anxiety and insomnia. Here we report the stimulation of chloride currents through GABA(A) receptors (I(GABA)) by valerenic acid (VA), a constituent of Valerian. To analyse the molecular basis of VA action, we expressed GABA(A) receptors with 13 different subunit compositions in Xenopus oocytes and measured I(GABA) using the two-microelectrode voltage-clamp technique. We report a subtype-dependent stimulation of I(GABA) by VA. Only channels incorporating beta(2) or beta(3) subunits were stimulated by VA. Replacing beta(2/3) by beta(1) drastically reduced the sensitivity of the resulting GABA(A) channels. The stimulatory effect of VA on alpha(1)beta(2) receptors was substantially reduced by the point mutation beta(2N265S) (known to inhibit loreclezole action). Mutating the corresponding residue of beta(1) (beta(1S290N)) induced VA sensitivity in alpha(1)beta(1S290N) comparable to alpha(1)beta(2) receptors. Modulation of I(GABA) was not significantly dependent on incorporation of alpha(1), alpha(2), alpha(3) or alpha(5) subunits. VA displayed a significantly lower efficiency on channels incorporating alpha(4) subunits. I(GABA) modulation by VA was not gamma subunit dependent and not inhibited by flumazenil (1 microM). VA shifted the GABA concentration-effect curve towards lower GABA concentrations and elicited substantial currents through GABA(A) channels at > or = 30 microM. At higher concentrations (> or = 100 microM), VA and acetoxy-VA inhibit I(GABA). A possible open channel block mechanism is discussed. In summary, VA was identified as a subunit specific allosteric modulator of GABA(A) receptors that is likely to interact with the loreclezole binding pocket.     

The alpha and gamma subunit-dependent effects of local anesthetics on recombinant GABA(A) receptors

Although convulsions due to local anesthetic systemic toxicity are thought to be due to inhibition of GABA(A) receptor-linked currents in the central nervous system, the mechanism of action remains unclear. We therefore examined the effects of local anesthetics on gamma-aminobutyric acid (GABA)-induced currents using recombinant GABA(A) receptors with specific combinations of subunits. Murine GABA(A) receptors were expressed by injection of cRNAs encoding each subunit into Xenopus oocytes. The effects of local anesthetics (lidocaine, bupivacaine, procaine and tetracaine) on GABA-induced currents of receptors expressing different subunit combinations (alpha1beta2, alpha1beta2gamma2s, alpha4beta2gamma2s and beta2) were examined via the two electrode voltage clamp method. At alpha1beta2, alpha1beta2gamma2s and alpha4beta2gamma2s GABA(A) receptors, all local anesthetics inhibited GABA-induced currents in a dose-dependent manner. The presence of the gamma2s subunit resulted in a greater inhibition by all local anesthetics, but the presence of the alpha4 subunit resulted in less inhibition. At beta2 homomeric receptors, local anesthetics directly induced an outward current similar to that of picrotoxin. These data indicated that (1) the alpha and gamma subunits of GABA(A) receptors modulated the inhibitory effects of local anesthetics on GABA(A) function, and (2) local anesthetics can activate the beta2 subunit and may block the GABA(A) receptor channel pore.     

Effect of alpha subunit on allosteric modulation of ion channel function in stably expressed human recombinant gamma-aminobutyric acid(A) receptors determined using (36)Cl ion flux

Inhibitory gamma-aminobutyric acid (GABA)(A) receptors are subject to modulation at a variety of allosteric sites, with pharmacology dependent on receptor subunit combination. The influence of different alpha subunits in combination with beta3gamma2s was examined in stably expressed human recombinant GABA(A) receptors by measuring (36)Cl influx through the ion channel pore. Muscimol and GABA exhibited similar maximal efficacy at each receptor subtype, although muscimol was more potent, with responses blocked by picrotoxin and bicuculline. Receptors containing the alpha3 subunit exhibited slightly lower potency. The comparative pharmacology of a range of benzodiazepine site ligands was examined, revealing a range of intrinsic efficacies at different receptor subtypes. Of the diazepam-sensitive GABA(A) receptors (alpha1, alpha2, alpha3, alpha5), alpha5 showed the most divergence, being discriminated by zolpidem in terms of very low affinity, and CL218,872 and CGS9895 with different efficacies. Benzodiazepine potentiation at alpha3beta3gamma2s with nonselective agonist chlordiazepoxide was greater than at alpha1, alpha2, or alpha5 (P < 0.001). The presence of an alpha4 subunit conferred a unique pharmacological profile. The partial agonist bretazenil was the most efficacious benzodiazepine, despite lower alpha4 affinity, and FG8205 displayed similar efficacy. Most striking were the lack of affinity/efficacy for classical benzodiazepines and the relatively high efficacy of Ro15-1788 (53 +/- 12%), CGS8216 (56 +/- 6%), CGS9895 (65 +/- 6%), and the weak partial inverse agonist Ro15-4513 (87 +/- 5%). Each receptor subtype was modulated by pentobarbital, loreclezole, and 5alpha-pregnan-3alpha-ol-20-one, but the type of alpha subunit influenced the level of potentiation. The maximal pentobarbital response was significantly greater at alpha4beta3gamma2s (226 +/- 10% increase in the EC(20) response to GABA) than any other modulator. The rank order of potentiation for pregnanolone was alpha5 > alpha2 > alpha3 = alpha4 > alpha1, for loreclezole alpha1 = alpha2 = alpha3 > alpha5 > alpha4, and for pentobarbital alpha4 = alpha5 = alpha2 > alpha1 = alpha3.     

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.     

Tracazolate reveals a novel type of allosteric interaction with recombinant gamma-aminobutyric acid(A) receptors

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with gamma-aminobutyric acid (GABA)(A) receptors, adenosine receptors, and phosphodiesterases. Its anxiolytic effect is thought to be via its interaction with GABA(A) receptors. We now report the first detailed pharmacological study examining the effects of tracazolate on a range of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes. Replacement of the gamma2s subunit within the alpha1beta3gamma2s receptor with the epsilon subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The gamma2s subunit was not critical for potentiation because alpha1beta3 receptors were also potentiated by tracazolate. gamma2/epsilon chimeras revealed a critical N-terminal domain between amino acids 206 and 230 of gamma2, governing the nature of this response. Replacement of the beta3 subunit with the beta1 subunit within alpha1beta3gamma2s and alpha1beta3epsilon receptors also revealed selectivity of tracazolate for beta3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of gamma2s with gamma1 or gamma3 revealed a profile intermediate to that of alpha1beta1epsilon and alpha1beta1gamma2s. alpha1beta1delta receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC(20) was much greater than on alpha1beta1gamma2. Concentration-response curves to GABA in the presence of tracazolate for alpha1beta1epsilon and alpha1beta1gamma2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC(50) only on alpha1beta1gamma2. Like alpha1beta1gamma2s, GABA concentration-response curves on alpha1beta1delta receptors were shifted to the left with increased maximum responses. Tracazolate has a unique pharmacological profile on recombinant GABA(A) receptors: its potency (EC(50)) is influenced by the nature of the beta subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (gamma1-3, delta, or epsilon) within the receptor complex.     

Activity of novel 4-PIOL analogues at human alpha 1 beta 2 gamma 2S GABA(A) receptors--correlation with hydrophobicity

A series of novel 5-(4-piperidyl)-3-isoxazolol (4-PIOL) analogues where the 4-position of the 3-isoxazolol ring was substituted with groups of different size, flexibility, and lipophilicity have been characterised. Their activity as agonists and/or antagonists on human alpha(1)beta(2)gamma(2S) GABA(A) receptors expressed in Xenopus oocytes was studied using two-electrode voltage clamp electrophysiology. Methyl- and ethyl-substituted 4-PIOL analogues were characterised as partial agonists since weak agonist responses could be potentiated with lorazepam and inhibited by the competitive antagonist 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyradizinum bromide (SR95531). All larger substituents in the 4-position of the 3-isoxazolol ring of 4-PIOL converted the compounds into pure competitive antagonists. Additionally, for GABA, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), piperidine-4-sulphonic acid (P4S), and 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL), a negative linear correlation was found between the agonist efficacy of the compound and the ability of lorazepam to potentiate EC(95) responses. Furthermore, a positive linear correlation between the lipophilicity of the substituents in the 4-position of the 3-isoxazolol ring of 4-PIOL and the antagonist affinity was found. These data suggest that the GABA(A) receptor contains a hydrophobic binding pocket at the GABA recognition site and that the binding of the 4-PIOL analogues is largely determined by the transfer from the aqueous phase to the hydrophobic pocket.     

Thiocolchicoside inhibits the activity of various subtypes of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes

Thiocolchicoside is a myorelaxant drug with anti-inflammatory and analgesic properties as well as pronounced convulsant activity. To characterize the mechanisms of action of this drug at the molecular level, we examined its effects on the function of various recombinant neurotransmitter receptors expressed in Xenopus oocytes. Electrophysiological recordings from recombinant human gamma-aminobutyric acid type A (GABA(A)) receptors consisting of alpha1beta1gamma2L, alpha1beta2gamma2L, or alpha2beta2gamma2L subunit combinations revealed that thiocolchicoside inhibited GABA-evoked Cl(-) currents with similar potencies (median inhibitory concentrations of 0.13 to 0.2 microM) and in a competitive manner. Consistent with previous observations, thiocolchicoside also inhibited the binding of GABA to rat cerebral cortical membranes. Thiocolchicoside inhibited the function of recombinant human strychnine-sensitive glycine receptors composed of the alpha1 subunit with a potency (median inhibitory concentration of 47 microM) lower than that apparent with recombinant GABA(A) receptors. It also inhibited the function of human nicotinic acetylcholine receptors composed of the alpha4 and beta2 subunits, but this effect was only partial and apparent at high concentrations. In contrast, thiocolchicoside had no effect on the function of 5-HT(3A) serotonin receptors. Our results thus provide molecular evidence that the epileptogenic activity of thiocolchicoside might be due to inhibition of the function of inhibitory receptors in the central nervous system, especially that of GABA(A) receptors.     

Homologous sites of GABA(A) receptor alpha(1), beta(3) and gamma(2) subunits are important for assembly

GABA(A) receptors are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two alpha, two beta and one gamma subunit that assemble around an aqueous pore and form an intrinsic chloride ion channel. Using full-length or truncated chimeric subunits it was demonstrated that homologous sequences from different subunit classes, alpha(1)(54-68), beta(3)(52-66), and gamma(2)(67-81), are important for assembly of GABA(A) receptors composed of alpha(1), beta(3), and gamma(2) subunits. In addition, evidence was provided that these sequences all are located in topologically homologous regions of the different subunits. Finally, it was demonstrated that the sequences investigated cause a selective assembly with certain subunits only and thus influence subunit arrangement within GABA(A) receptors.     

The beta-lactam antibiotics, penicillin-G and cefoselis have different mechanisms and sites of action at GABA(A) receptors

The action of the beta-lactam antibiotics, penicillin-G (PCG) and cefoselis (CFSL) on GABA(A) receptors (GABA(A)-R) was investigated using the two-electrode voltage clamp technique and Xenopus oocyte expressed murine GABA(A)-R. Murine GABA(A)-Rs were expressed in Xenopus oocytes by injecting cRNA that encoded for each subunit (alpha1, beta2, and gamma2) and the effects of PCG and CFSL on the alpha1beta2gamma2s subunit receptors were examined using two-electrode voltage clamp. Using the alpha1beta2gamma2s GABA(A)-R, PCG and CFSL inhibited GABA-induced currents in a concentration-dependent manner, with IC(50)s of 557.1+/-125.4 and 185.0+/-26.6 microM, respectively. The inhibitory action of PCG on GABA-induced currents was non-competitive whereas that of CFSL was competitive. Mutation of tyrosine to phenylalanine at position 256 in the beta2 subunit (beta2(Y256F)), which is reported to abolish the inhibitory effect of picrotoxin, drastically reduced the potency of PCG (IC(50)=28.4+/-1.42 mM) for the alpha1beta2(Y256F)gamma2s receptor without changing the IC(50) of CFSL (189+/-26.6 microM). These electrophysiological data indicate that PCG and CFSL inhibit GABA(A)-R in a different manner, with PCG acting non-competitively and CFSL competitively. The mutational study indicates that PCG might act on an identical or nearby site to that of picrotoxin in the channel pore of the GABA(A)-R.     

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.