Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2

We characterized modulation of the gamma-aminobutyric acid (GABA)-evoked responses of the diazepam-insensitive alpha 4 beta 2 gamma2 and alpha 6 beta 2 gamma 2 recombinant GABAA receptors. The partial agonist bretazenil potentiated the responses of both receptors with similar dose dependence but with a higher maximal enhancement at the alpha 4 beta 2 gamma 2 receptor. The bretazenil-induced potentiation was reduced by the benzodiazepine antagonist flumazenil. At a high concentration (10 microM), flumazenil was a weak potentiator of the GABA response. The partial agonist imidazenil was inactive. The imidazobenzodiazepine inverse agonist Ro 15-4513, which is known to bind with high affinity to the alpha 6 beta 2 gamma 2 receptor, potentiated the GABA responses of the alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor subtypes with similar dose dependence over the concentration range of 0.1-10 microM. Methyl-6, 7-dimethoxy-4-ethyl-beta-carboline, a beta-carboline inverse agonist, had a similar potentiating effect when tested at a concentration of 10 microM. The alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor-mediated currents had equal sensitivities to furosemide and Zn2+ ions, both of which reduced the GABA-evoked responses. The alpha 6 beta 2 gamma 2 receptor but not the alpha 4 beta 2 gamma 2 receptor exhibited a low level of spontaneous activity in the absence of GABA; this resting current could be directly potentiated by Ro 15-4513, methyl-6,7-dimethoxy-4-ethyl-beta-carboline, bretazenil and flumazenil and was blocked by picrotoxin. Thus, although the alpha 4 beta 2 gamma 2 receptors are insensitive to benzodiazepine binding site full agonists, such as diazepam, they can be modulated by certain ligands acting as partial and inverse agonists at diazepam-sensitive receptors and thereby contribute to the respective pharmacological profiles.     

Inhibition of GABAA receptor function by tyrosine kinase inhibitors and their inactive analogues

The effects of tyrosine kinase inhibitors which target the ATP binding site or the substrate binding site of tyrosine kinases were assessed on murine recombinant type A gamma-aminobutyric acid (GABAA) receptors expressed in Xenopus oocytes or HEK cells using two-electrode voltage clamp or patch clamp recording. Genistein inhibited in a noncompetitive manner GABA-activated currents recorded from alpha1beta1gamma2S receptor constructs by reducing the maximum normalized response from 1.83 +/- 0.04 to 0.71 +/- 0.04 and reducing the EC50 from 35.7 +/- 2.1 microM to 15.1 +/- 3.9 microM. After mutating the two "functionally active" substrate tyrosine (Y) residues in gamma2S and expressing the mutant receptor alpha1beta1gamma2S(Y365F, Y367F), genistein still noncompetitively inhibited the responses to GABA reducing the maximum current from 1. 81 +/- 0.03 to 0.26 +/- 0.01 and the EC50 from 33.1 +/- 2.3 microM to 5.8 +/- 2.2 microM. The inactive compound, daidzein, also similarly inhibited responses to GABA on these two receptor constructs. Inhibitors targeting the substrate binding site of tyrosine kinases, the tyrphostins, also inhibited both the wild-type and the tyrosine mutant GABAA receptors. Tyrphostin A25 and the inactive tyrphostin A1 reduced the maximum normalized responses for alpha1beta1gamma2S and alpha1beta1gamma2S(Y365F, Y367F) receptors by 73 and 64%, respectively. The tyrosine kinase inhibitors and their inactive controls did not display any significant voltage sensitivity to the antagonism of GABA-activated responses. Moreover, genistein or tyrphostin A25 did not affect the potentiation of responses to GABA by pentobarbitone or diazepam. Mutating the two "functionally silent" tyrosine residues, Y370 and Y372, known to be substrates for tyrosine kinases in the beta1 subunit and coexpression in the alpha1beta1(Y370F, Y372F)gamma2S(Y365F, Y367F) construct failed to affect the inhibitory action of genistein. The study concludes that tyrosine kinase inhibitors and their inactive controls can directly interact with GABAA receptors completely independent of any effects on tyrosine kinases.     

Modulation of GABAA receptor function by alcohols: effects of subunit composition and differential effects of ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABAA receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABAA receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABAA receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 microM GABA in oocytes expressing the alpha 1 beta 2 gamma 2L and alpha 2 beta 2 gamma 2L receptor isoforms. In the alpha 1 beta 2 gamma 2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABAA receptor function, but 2-butanol had a greater efficacy. In the alpha 2 beta 2 gamma 2L receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the alpha 2 beta 2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 microM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a gamma 2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the alpha 1 beta 2 gamma 2L receptor construct. Ethanol potentiation of GABAA receptor function was completely blocked by the benzodiazepine receptor partial inverse agonist RO15-4513 at a concentration (0.5 microM) that did not alter the control GABA response. In contrast, RO15-4513 did not block potentiation of GABAA receptor activity induced by n-propanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,6-diisopropylphenol). These results suggest that alcohols have specific interactions with GABAA receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit

BACKGROUND: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity. METHODS: Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods. RESULTS: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations. CONCLUSIONS: Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.     

Activation kinetics and single channel properties of recombinant alpha1beta2gamma2L GABA(A) receptor channels

Recombinant alpha1beta2gamma2L GABA(A) receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose-response relationship revealed an EC50 of 11.6 +/- 0.9 microM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 +/- 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 +/- 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.     

Functional characterization of human gamma-aminobutyric acidA receptors containing the alpha 4 subunit

The alpha subunits are an important determinant of the pharmacology of gamma-aminobutyric acidA (GABAA) receptors with respect to agonists, antagonists, and modulatory compounds, particularly the benzodiazepines. The alpha 4 subunit is the least abundant subunit in the brain and the most similar in deduced primary amino acid sequence to the alpha 6 subunit. We demonstrate that the human alpha 4 subunit forms a functional receptor when expressed with beta gamma 2, demonstrating some properties similar to alpha 6 beta gamma 2 and some properties more akin to alpha 1 beta gamma 2. It also exhibited some properties that were unlike any other alpha subunit-containing receptor. GABA affinity seemed to be identical to that of the alpha 1 beta 1 gamma 2 receptor; however, the partial agonists 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid showed lower efficacy than at either alpha 1 beta 1 gamma 2 or alpha 6 beta 1 gamma 2. Benzodiazepine pharmacology of alpha 4-containing receptors was similar to that of alpha 6-containing receptors with the exception of dimethoxy-4-ethyl-beta-carboline-3-carboxylate, which behaved as a partial inverse agonist. Pentobarbital potentiated alpha 4 beta 1 gamma 2 receptor GABA responses to a level comparable with alpha 6 beta 1 gamma 2 (approximately 700% of EC20); however, unlike alpha 6 beta 1 gamma 2 receptors, it did not elicit any direct activation of the receptor. Propofol also potentiated alpha 4 beta 1 gamma 2 GABA responses but to a level more comparable to that of alpha 1 beta 1 gamma 2, suggesting that these compounds act via different sites. Unlike other subunit combinations, propofol did not elicit a direct activation of the receptor. These results suggest that the mechanism for direct activation of the GABAA receptor by pentobarbital and propofol is absent on alpha 4-containing receptors. Furosemide, which non-competitively inhibits the GABAA receptor, showed 700-fold selectivity for alpha 6 beta 3 gamma 2 receptors over alpha 1-, alpha 2-, alpha 3-, and alpha 5-containing receptors and exhibited selectivity for alpha 4 beta 3 gamma 2 receptors (> 50-fold). These experiments reveal a unique pharmacology for alpha 4-containing receptors with some similarities to both alpha 6- and alpha 1-containing receptors.     

U-89843A is a novel allosteric modulator of gamma-aminobutyric acidA receptors

A group of pyrrolopyrimidine derivatives were examined for their interaction with rat recombinant gamma-aminobutyric acid (GABA)A receptors using the whole cell patch clamp and equilibrium binding techniques. In the alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in human embryonic kidney cells, a prototype pyrrolopyrimidine, U-89843A (7H-pyrrol[2,3-d]pyrimidine,6,7-methyl-2,4-di- 1-pyrrolidinyl,hydrochloride), dose-dependently enhanced 5 microM GABA-induced Cl- currents with a maximal enhancement of 362 +/- 91%, a half-maximal concentration of 2 +/- 0.4 microM and a slope factor of 1.1 +/- 0.4. The drug also inhibited [35S]t-butylbicyclophosphorothionate binding in rat cerebrocortical membranes with a similar half-maximal inhibitory concentration. The enhancement of Cl- currents by U-89843A was insensitive to Ro 15-1788 (a benzodiazepine antagonist), was also observed in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes (no selectivity to different alpha-isoforms unlike many benzodiazepines), but was absent in the receptor subtypes consisting of two subunits (alpha 1 beta 2, alpha 1 gamma 2 and beta 2 gamma 2). It has been known that neurosteroids and barbiturates are uniformly active in both the two subunit receptors, substituted pyrazinones are only active in the alpha 1 beta 2 subtype and loreclezole is active in the subtypes containing beta 2. We propose that U-89843A interacts with an allosteric site on GABAA receptors distinct from the sites for benzodiazepines, barbiturates, neurosteroids, substituted pyrazinones or loreclezole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5-HT (300 nM - 10 microM) evoked a transient, concentration-dependent (EC50 = 1.1+/-0.1 microM), inward current. The response reversed in sign at a holding potential of -2.1+/-1.6 mV. The response to 5-HT was mimicked by the 5-HT3 receptor selective agonists 2-methyl-5-HT (EC50= 4.1+/-0.2 microM), 1-phenylbiguanide (EC50=3.0+/-0.1 microM), 3-chlorophenylbiguanide (EC50 = 140+/-10 nM), 3,5-dichlorophenylbiguanide (EC50 = 14.5+/-0.4 nM) and 2,5-dichlorophenylbiguanide (EC50 = 10.2+/-0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 microM) of 5-HT. Responses evoked by 5-HT at EC50 were blocked by the 5-HT3 receptor selective antagonist ondansetron (IC50=231+/-22 pM) and by the less selective agents (+)-tubocurarine (IC50=31.9+/-0.01 nM) and cocaine (IC50 = 2.1+/-0.2 microM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT3 receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT3A subunit orthologues.     

The role of an alpha subtype M2-M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations

Sensitivity of GABAA receptors (GABARs) to inhibition by zinc and other divalent cations is influenced by the alpha subunit subtype composition of the receptor. For example, alpha6beta3gamma2L receptors are more sensitive to inhibition by zinc than alpha1beta3gamma2L receptors. We examined the role of a His residue located in the M2-M3 extracellular domain (rat alpha6 H273) in the enhanced zinc sensitivity conferred by the alpha6 subtype. The alpha1 subtype contains an Asn (N274) residue in the equivalent location. GABA-activated whole-cell currents were obtained from L929 fibroblasts after transient transfection with expression vectors containing GABAA receptor cDNAs. Mutation of alpha1 (alpha1(N274H)) or alpha6 (alpha6(H273N)) subtypes did not alter the GABA EC50 of alphabeta3gamma2L receptors. alpha1(N274H)beta3gamma2L receptor currents were as sensitive to zinc as alpha6beta3gamma2L receptor currents, although alpha6(H273N)beta3gamma2L receptor currents had the reduced zinc sensitivity of alpha1beta3gamma2L receptor currents. We also examined the activity of other inhibitory divalent cations with varying alpha subtype dependence: nickel, cadmium, and copper. alpha6beta3gamma2L receptor currents were more sensitive to nickel, equally sensitive to cadmium, and less sensitive to copper than alpha1beta3gamma2L receptor currents. Studies with alpha1 and alpha6 chimeric subunits indicated that the structural dependencies of the activity of some of these cations were different from zinc. Compared with alpha6beta3gamma2L receptor currents, alpha6(H273N)beta3gamma2L receptor currents had reduced sensitivity to cadmium and nickel, but the sensitivity to copper was unchanged. Compared with alpha1beta3gamma2L receptor currents, alpha1(N274H)beta3gamma2L receptor currents had increased sensitivity to nickel, but the sensitivity to cadmium and copper was unchanged. These findings indicate that H273 of the alpha6 subtype plays an important role in determining the sensitivity of recombinant GABARs to the divalent cations zinc, cadmium, and nickel, but not to copper. Our results also suggest that the extracellular N-terminal domain of the alpha1 subunit contributes to a regulatory site(s) for divalent cations, conferring high sensitivity to inhibition by copper and cadmium.     

Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes

GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.     

Enhancement of recombinant gamma-aminobutyric acid type A receptor currents by chronic activation of cAMP-dependent protein kinase

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.     

Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.     

Protein tyrosine kinase activity as a prognostic parameter in breast cancer, a pilot study

The interaction of omega (benzodiazepine) modulatory drugs with transiently expressed alpha 1 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 forms of the rat GABAA receptor was investigated using [3H]flumazenil as a probe in in vitro radioligand binding assays. The imidazopyridines alpidem and zolpidem exhibited pronounced selectivity for the alpha 1- compared to the alpha 5-containing construct, whereas omega (benzodiazepine) site modulatory compounds from other chemical series including diazepam, tetrazepam, zopiclone, triazolam, bretazenil and midazolam behaved as relatively non-selective drugs. In the presence of 10 microM gamma-aminobutyric acid (GABA) the potencies of diazepam, flunitrazepam and midazolam to inhibit [3H]flumazenil binding to the alpha 1-construct were increased 3 to 5 fold, whereas with 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester a 2.5-fold reduction in potency was observed. Similar modulatory effects of GABA were obtained with these drugs, using the alpha 5-construct. We suggest that these GABA shift determinations of [3H]flumazenil binding can be used as a rapid test to evaluate the intrinsic activities of omega modulatory compounds.     

Direct activation of GABAA receptors by loreclezole, an anticonvulsant drug with selectivity for the beta-subunit

Loreclezole, an anticonvulsant and antiepileptic compound, potentiates gamma-aminobutyric acid (GABA) type A receptor function, by interacting with a specific allosteric modulatory site on receptor beta-subunits. A similar selectivity for GABAA receptor beta-subunits is apparent for the direct activation of receptor-operated Cl- channels, by the general anesthetics propofol and pentobarbital. The ability of loreclezole to activate GABAA receptors directly has now been compared, biochemically and electrophysiologically, with that of propofol. In well-washed rat cortical membranes (devoid of endogenous GABA), loreclezole and propofol increased t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding by up to 28% (at 5 microM) and 80% (at 10 microM), respectively. Higher concentrations (50-100 microM) of both compounds inhibited [35S]TBPS binding with great efficacy, an effect mimicked by GABA. In contrast, the benzodiazepine diazepam increased [35S]TBPS binding, but failed to inhibit this parameter, even at high concentrations. At concentrations of 50-100 microM, loreclezole induced inward Cl- currents in the absence of GABA, in Xenopus oocytes expressing human recombinant GABAA receptors, comprised of alpha 1-, beta 2- and gamma 2S-subunits. At 100 microM, the current evoked by loreclezole was 26% of that induced by 5 microM GABA. The current evoked by 100 microM propofol was 98% of that induced by 5 microM GABA. Currents induced by loreclezole, like those evoked by propofol, were potentiated by diazepam in a flumazenil-sensitive manner and blocked by either bicuculline or picrotoxin. These data suggest that loreclezole shares, with propofol, an agonistic action at GABAA receptors containing the beta 2-subunit and that the different efficacies of the two compounds in this regard, may underlie the difference in their pharmacological profiles. The failure of loreclezole to activate GABAA receptors containing the beta 1-subunit may be responsible for its lack of hypnotic effect.     

Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors expressed in mammalian cells and Xenopus oocytes

GABAA receptors composed of human alpha 1 beta 2 gamma 2L, alpha 1 beta 2 gamma 2S, alpha 1 beta 3 gamma 2S, alpha 6 beta 3 gamma 2S, and alpha 5 beta 3 gamma 3 subunits as well as bovine alpha 1 beta 1 gamma 2L and alpha 1 beta 1 subunits were stably expressed in mammalian L(tk-) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a 36Cl- flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk-) cells than oocytes; this difference ranged from 2.6-to 26-fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk-) cells, low (5-50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the gamma 2L subunit. In oocytes, concentrations of 30-100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except alpha 6 beta 3 gamma 2S and alpha 1 beta 1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk-) cells and oocytes, except for alpha 6 beta 3 gamma 2S where the pentobarbital enhancement was much greater in oocytes than cells. The alpha 6 beta 3 gamma 2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABAA receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.     

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.     

The role of alpha1 and alpha6 subtype amino-terminal domains in allosteric regulation of gamma-aminobutyric acida receptors

The gamma-aminobutyric acidA (GABA) receptor in the mammalian central nervous system is composed of pentameric combinations of alpha1-6, beta1-4, gamma1-3, delta1, and/or epsilon1 subunit subtypes. Although each of the different subunits influences the functional properties of gamma-aminobutyric acidA receptors (GABARs), the alpha subunit subtypes have been shown to be important for activation of the receptor by GABA and pentobarbital and the regulation of GABARs by numerous allosteric regulators, including benzodiazepines, furosemide, zinc, and lanthanum. However, with the exception of the benzodiazepines, the alpha subtype domain that is responsible for the action of these allosteric compounds is unknown. The alpha1 and alpha6 subtypes are among the most diverse of the alpha subunit family and confer a different responsiveness of GABARs to GABA and many of the allosteric modulators. These regulatory compounds act after extracellular application and therefore likely act on extracellular GABAR sites, the largest of which is the amino-terminal extracellular domain. To determine the role of this domain in the action of these allosteric regulatory agents, we constructed chimeras of the rat alpha1 and alpha6 subtypes with a splice site within the first putative transmembrane domain (TM). This separated the large extracellular amino-terminal domain from the transmembrane, intracellular, and TM2-3 and carboxyl-terminal extracellular domains of the subunit. The chimeric subtypes were expressed in L929 fibroblasts along with beta3 and gamma2L subtypes, and their pharmacological properties were determined with whole-cell electrophysiological recording. The alpha subtype amino-terminal extracellular domain was the primary determinant of GABA sensitivity and was responsible for the functional properties of activation by pentobarbital, sensitivity to diazepam, potentiation by lanthanum, and high affinity inhibition by furosemide. The remaining carboxyl-terminal domains influenced the GABA sensitivity and determined zinc sensitivity and low affinity inhibition by furosemide. Both domains were apparently required for inhibition by lanthanum.     

Human brain cholecystokinin: release of cholecystokinin-like immunoreactivity (CCK-LI) from isolated cortical nerve endings and its modulation through GABA(B) receptors

The release of cholecystokinin-like immunoreactivity (CCK-LI) in human brain was investigated using synaptosomes prepared from neocortical specimens removed during neurosurgery. CCK-LI basal release from superfused synaptosomes was increased 3 to 4-fold during depolarization with 15 mM KCI. The K(+)-evoked overflow of CCK-LI was strictly Ca(++)-dependent. The gamma-aminobutyric acidB (GABA(B)) receptor agonist (-)baclofen (0.3-100 microM) inhibited CCK-LI overflow in a concentration-dependent manner (EC50 = 2.20 microM; maximal effect: 45%). The novel GABA(B) receptor ligand CGP 47656 mimicked (-)baclofen (EC50 = 2.45 microM; maximal effect: 50%), whereas the GABA(A) agonist muscimol was ineffective up to 100 microM. The inhibitory effect of 10 microM (-)baclofen on the CCK-LI overflow was concentration-dependently prevented by two selective GABA(B) receptor antagonists, CGP 35348 (IC50 = 13.91 microM) and CGP 52432 (IC50 = 0.08 microM). The effect of 10 microM CGP 47656 was abolished by 1 microM CGP 52432. In experiments on [3H]GABA release, CGP 47656 behaved as an antagonist at the GABA(B) autoreceptors: added at 10 microM, it prevented the inhibitory effect of 10 microM (-)baclofen on the K+ (15 mM)-evoked release of [3H]GABA from human synaptosomes. We conclude that 1) the release of CCK-LI evoked from human brain tissue appears of neuronal origin; 2) the CCK-releasing terminal possess inhibitory presynaptic GABA(B) receptors; 3) these receptors differ pharmacologically from human neocortex GABA(B) autoreceptors, which are CGP 35348-insensitive (Fassio et al., 1994) but can be blocked by CGP 47656; 4) because cholecystokinin has been implicated in anxiety, the GABA(B) receptors here characterized may represent targets for novel anxiolytic agents.     

Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.