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.     

The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors

Murine gamma-aminobutyric acid type A (GABAA) receptor beta 1, beta 2, and beta 3 subunits were expressed in Xenopus oocytes and studied using the two electrode voltage clamp technique. Although all three beta-subunits were unresponsive to GABA when expressed as homomers, the intravenous general anaesthetics pentobarbital, etomidate and propofol induced currents in beta 2 and beta 3 homomers. The pentobarbital-induced currents in beta 3 homomers showed a dose dependence with an ED50 of 89 +/- 8.9 microM and a Hill coefficient of 0.94 +/- 0.08. Zinc (50 microM) blocked (61.1 +/- 5.6% of control) and 200 microM lanthanum potentiated (139 +/- 8.6% of control) the pentobarbital-induced current. This current was also blocked by picrotoxin but was insensitive to the GABAA receptor antagonist bicuculline. These observations indicate that the full expression of the agonistic action of GABA requires the presence of an alpha-subunit, in contrast to the agonistic action of intravenous general anesthetics, where the presence of a beta2 or beta 3-subunit is sufficient. The difference in the agonistic action of intravenous anaesthetics among these highly homologous beta-subunits suggests that the beta-subunit homomeric receptors may be useful to further define the molecular sites of action of intravenous general anaesthetics and other functional domains on GABAA receptors.     

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.     

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.     

Ethanol increases GABAA responses in cells stably transfected with receptor subunits

Ethanol enhancement of GABAA receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., 36Cl- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk-) cells stably transfected with alpha 1 + beta 1 or alpha 1 + beta 1 + gamma 2L GABAA receptor subunit DNAs and compared 36Cl- flux with whole-cell, patch-clamp measurements of GABAA receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the gamma-subunit and was maximal at a concentration of 10 mM. Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing alpha 1 beta 1-gamma 2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 mM. Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABAA receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a gamma-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.     

Development of a high-performance liquid chromatographic-electrospray mass spectrometric assay for the specific and sensitive quantification of the novel immunosuppressive macrolide 40-O-(2-hydroxyethyl)rapamycin

Propofol (2,6-diisopropylphenol) is an intravenous general anaesthetic which can directly activate and positively modulate the GABAA receptor. The effects of propofol on human recombinant GABAA receptors were studied in Xenopus oocytes expressing either alpha1beta2, alpha1beta2gamma2L, or alpha2beta2gamma2L receptor isoforms. In all receptor isoforms tested, propofol was able to potentiate the GABA-activated currents in a concentration-dependent manner. Although propofol potentiated both alpha1beta2 and alpha1beta2gamma2L receptor isoforms with equal affinity, the efficacy of propofol potentiation was markedly greater in the alpha1beta2 receptor isoform. In contrast, potentiation of the alpha2beta2gamma2L receptor isoform by propofol occurred with higher affinity and lower efficacy than in the alpha1beta2gamma2L receptor isoform. Propofol directly activated all three receptor isoforms in a concentration dependent manner. Addition of the gamma2L subunit subtype to the alpha1beta2 receptor isoform decreased receptor sensitivity to direct activation by propofol. Replacement of the alpha1-subunit subtype with the alpha2-subunit subtype increased receptor sensitivity to propofol's direct effects. These results suggest that the alpha-and gamma2L-subunit subtypes each have the ability to influence both the direct and modulatory actions of propofol on GABAA receptor function.     

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.     

Creating an education model for cardiac patients

gamma-Aminobutyric acidA (GABA(A)) gated chloride ion channels were expressed from human recombinant cDNA using the baculovirus/Sf-9 insect cell expression system. The electrophysiological effects in whole-cell currents of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, were investigated on GABA(A) receptor complexes of alpha1beta2gamma2S subunits as well as a slightly modified construct of alpha1(valine 121)beta2gamma2S subunits. Here we report that (1)4-PIOL induces an inward whole-cell current in a concentration-dependent manner in both alpha1(val 121)beta2gamma2S and alpha1(ile 121)beta2gamma2S receptor subunit combinations. (2) The 4-PIOL induced whole-cell currents were more pronounced in alpha1(val 121)beta2gamma2S than in alpha1(ile 121)beta2gamma2S receptor subunit combinations. (3) 4-PIOL inhibited GABA-induced responses on alpha1(ile 121)beta2gamma2S and alpha1(val 121)beta2gamma2S receptor combinations with similar potency.     

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.     

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.     

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.     

Distinct deactivation and desensitization kinetics of recombinant GABAA receptors

The functional role of the large heterogeneity in GABAA receptor subunit genes and its role in setting the properties of inhibitory synapses in the CNS is poorly understood. A kinetic comparison between currents elicited by ultra-rapid application with a piezoelectric translator of 1 mM GABA to mammalian cells transfected with cDNAs encoding distinct GABAA receptor subunits revealed that the intrinsic deactivation and desensitization properties depend on subunit combination. In particular, receptors containing alpha 6 with beta 2 gamma 2 subunits were endowed with a significantly slower deactivation as compared to those receptors containing alpha 1 with beta 2 gamma 2 subunits. While desensitization produced by prolonged GABA applications on alpha 1 beta 2 gamma 2 receptors was characterized by a rapid exponential decay followed by a slower decay and a steady state response, alpha 6 beta 2 gamma 2 receptors lacked desensitization. Furthermore, GABAA receptors lacking the gamma 2 subunit were characterized by a much larger non-desensitization component and a very rapid deactivation. Lastly, analysis of GABA-activated currents in cells cotransfected with alpha 1 and alpha 6 together with beta 2 gamma 2 subunit revealed unique kinetic properties. Our results suggest that distinct subunit composition confers specific deactivation and desensitization properties that may profoundly affect synaptic decay kinetics and the capability to sustain high frequency synaptic inputs.     

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.     

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.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

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.     

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.     

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