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The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.

Tryptophan substitutions were made in the second transmembrane domain of the gamma-aminobutyric acid(A) (GABA(A)) receptor alpha and beta subunits and the resulting mutant receptors, containing alpha(2)(S270W) and/or beta(1)(S265W), were expressed in Xenopus oocytes. Mutation of either or both subunits resulted in receptors that exhibited enhanced sensitivity to agonist and were spontaneously active in the absence of GABA. The spontaneous activity was blocked by picrotoxin or bicuculline. The enhancement of GABA-induced currents by pentobarbital, by the neurosteroid 5alpha-pregnan-3alpha-ol-20-one, and by the benzodiazepine flunitrazepam was dramatically reduced in the mutant receptors. These results are consistent with the idea that a mutation that promotes gating behavior in a ligand-gated ion channel will also show reduced effects of all positive allosteric modulators in a generalized manner, even when these modulators act at distinct sites on the receptor.

1. Each residue in the second transmembrane segment (TM2) of the human GABA(A) receptor alpha(2) subunit was individually mutated to tryptophan. The wild-type or mutant alpha(2) subunits were expressed with the wild-type human GABA(A) receptor beta(2) subunit in Xenopus oocytes, and the effects of these mutations were investigated using two-electrode voltage-clamp recording. 2. Four mutations (V257W, T262W, T265W and S270W) produced receptors which were active in the absence of agonist, and this spontaneous open channel activity was blocked by both picrotoxin and bicuculline, except in the alpha(2)(V257W)beta(2) mutant receptor, which was not sensitive to picrotoxin. 3. Six mutations (V257W, V260W, T262W, T267W, S270W and A273W) enhanced the agonist sensitivity of the receptor, by 10 - 100 times compared with the wild-type alpha(2)beta(2) receptor. Other mutations (T261W, V263W, L269W, I271W and S272W) had little or no effect on the apparent affinity of the receptor to GABA. Eight of the tryptophan mutations (R255, T256, F258, G259, L264, T265, M266 or T268) resulted in undetectable GABA-induced currents. 4. The S270W mutation eliminated potentiation of GABA by ethanol, whereas T261W markedly increased the action of ethanol. The T262W mutation produced direct activation (10% of maximal GABA response) by ethanol in the absence of GABA, while other mutations did not alter the action of ethanol significantly. 5. These results are consistent with a unique role for S270 in the action of ethanol within the TM2 region, and with models of GABA(A) receptor channel function, in which specific residues within TM2 are critical for the regulation of channel gating (S270, L264), while other residues (L269, I271 and S272) have little effect on these functions and may be non-critical structural residues.

Alanine-scanning mutagenesis and the whole cell voltage clamp technique were used to investigate the function of the extracellular loop between the second and third transmembrane domains (TM2-TM3) of the gamma-aminobutyric acid type A receptor (GABA(A)-R). A conserved arginine residue in the TM2-TM3 loop of the GABA(A)-R alpha(2) subunit was mutated to alanine, and the mutant alpha(2)(R274A) was co-expressed with wild-type beta(1) and gamma(2S) subunits in human embryonic kidney (HEK) 293 cells. The GABA EC(50) was increased by about 27-fold in the mutant receptor relative to receptors containing a wild-type alpha(2) subunit. Similarly, the GABA EC(50) at alpha(2)(L277A)beta(1)gamma(2S) and alpha(2)(K279A)beta(1)gamma(2S) GABA(A)-R combinations was increased by 51- and 4-fold, respectively. The alpha(2)(R274A) or alpha(2)(L277A) mutations also reduced the maximal response of piperidine-4-sulfonic acid relative to GABA by converting piperidine-4-sulfonic acid into a weak partial agonist at the GABA(A)-R. Based on these results, we propose that alpha(2)(Arg-274) and alpha(2)(Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA(A)-R.

The actions of 13 general anaesthetics (diethyl ether, enflurane, isoflurane, methoxyflurane, sevoflurane, chloral hydrate, trifluoroethanol, tribromoethanol, tert-butanol, chloretone, brometone, trichloroethylene, and alpha-chloralose) were studied on agonist-activated Cl(-) currents at human GABA(A) alpha(2)beta(1), glycine alpha(1), and GABA(C) rho(1) receptors expressed in human embryonic kidney 293 cells. All 13 anaesthetics enhanced responses to submaximal (EC(20)) concentrations of agonist at GABA(A) and glycine receptors, except alpha-chloralose, which did not enhance responses at the glycine alpha(1) receptor. None of the anaesthetics studied potentiated GABA responses at the GABA(C) rho(1) receptor. Potentiation of submaximal agonist currents by the anaesthetics was studied at GABA(A) and glycine receptors harbouring mutations in putative transmembrane domains 2 and 3 within GABA(A) alpha(2), beta(1), or glycine alpha(1) receptor subunits: GABA(A) alpha(2)(S270I)beta(1), alpha(2)(A291W)beta(1), alpha(2)beta(1)(S265I), and alpha(2)beta(1)(M286W); glycine alpha(1)(S267I) and alpha(1)(A288W). For all anaesthetics studied except alpha-chloralose, at least one of the mutations above abolished drug potentiation of agonist responses at GABA(A) and glycine receptors. alpha-Chloralose produced efficacious direct activation of the GABA(A) alpha(2)beta(1) receptor (a 'GABA-mimetic' effect). The other 12 anaesthetics produced minimal or no direct activation of GABA(A) and glycine receptors. A non-anaesthetic isomer of alpha-chloralose, beta-chloralose, was inactive at GABA(A) and glycine receptors and did not antagonize the actions of alpha-chloralose at GABA(A) receptors. The implications of these findings for the molecular mechanisms of action of general anaesthetics at GABA(A) and glycine receptors are discussed.

Using the whole-cell patch-clamp technique, we have determined that propofol, but not midazolam, increases the efficacy of piperidine-4-sulphonic acid (P4S), a partial agonist at alpha1beta1gamma2s, GABA(A) receptors expressed in HEK 293 cells. These findings are consistent with the idea that propofol facilitates receptor gating, while midazolam increases receptor occupancy by the agonist.

Gamma-aminobutyric acid type A (GABA(A) )receptors are targets for allosteric modulation by general anesthetics. Mutation of Ser270 within the second transmembrane domain of the GABA(A) receptor alpha subunit can ablate the modulation of the receptor by the anesthetic ether isoflurane. To investigate further the function of this critical amino acid residue, we made multiple amino acid substitutions at Ser270 and analyzed the concentration-dependent gating by GABA and regulation by isoflurane in each mutant receptor. There is a strong negative correlation between the EC(50) for GABA and the molecular volume of the amino acid residue at position 270. Replacement of Ser by large residues such as His and Trp produced a shift of the GABA concentration-response curve to the left, whereas replacement of Ser with Gly had the opposite effect. There also was a strong negative association between the molecular volume of the amino acid residue at 270 and the degree of enhancement of submaximal GABA responses by isoflurane. These results indicate the significance of the amino acid at position alpha270 in gating of the GABA(A) receptor. In addition, the data on isoflurane are consistent with the existence of a cavity of finite size in the region of alpha270 that may be filled by the anesthetic molecule or by the side chain of a larger residue at alpha270. The introduction of isoflurane, or of a large residue, into this cavity may stabilize the open state of the GABA(A) receptor relative to the closed state.

The molecular mechanisms of general anaesthetics have remained largely obscure since their introduction into clinical practice just over 150 years ago. This review describes the actions of general anaesthetics on mammalian neurotransmitter-gated ion channels. As a result of research during the last several decades, ligand-gated ion channels have emerged as promising molecular targets for the central nervous system effects of general anaesthetics. The last 10 years have witnessed an explosion of studies of anaesthetic modulation of recombinant ligand-gated ion channels, including recent studies which utilize chimeric and mutated receptors to identify regions of ligand-gated ion channels important for the actions of general anaesthetics. Exciting future directions include structural biology and gene-targeting approaches to further the understanding of general anaesthetic molecular mechanisms.

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.

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a "cutoff" is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the alpha subunit. We now demonstrate that these residues in the glycine alpha1 and the gamma-aminobutyric acid rho1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine alpha1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the gamma-aminobutyric acid rho1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

The actions of 2,2,2,-trichloroethanol were studied on agonist-activated Cl- currents in gamma-aminobutyric acid type A (GABAA), glycine and GABA rho 1 receptors by use of the whole-cell patch-clamp technique. Recombinant wild-type and mutant receptor subunits were transiently expressed in human embryonic kidney (HEK) 293 cells. Trichloroethanol enhanced currents elicited by submaximal (EC20) agonist concentrations at GABAA alpha 2 beta 1 receptors and glycine alpha 1 homomeric receptors in a reversible, concentration-dependent manner. Trichloroethanol, at concentrations of < or = 2 mM, did not significantly alter the magnitude of submaximal GABA currents at GABA rho 1 receptors, whereas higher concentrations inhibited submaximal GABA currents. Recent work has identified residues within putative transmembrane domains 2 and 3 as critical for positive modulation of GABAA and glycine receptors by n-alkanols and volatile ether anesthetics. Submaximal glycine currents at receptors containing either of two specific mutations within the glycine receptor alpha 1 subunit (S267I and A288W) were not enhanced by low concentrations of trichloroethanol and were inhibited by higher concentrations of trichloroethanol. In the GABAA alpha 2 beta 1 receptor, a specific mutation within transmembrane domain 3 of the beta 1 subunit (M286W) also abolished positive modulation by trichloroethanol. Mutations within the GABAA alpha 2 receptor subunit did not alter positive modulation by TCEt, whereas such mutations ablate positive modulation by n-alkanols and volatile anesthetics. In summary, trichloroethanol modulation of GABAA, glycine and GABA rho 1 receptors shares some, but not all, features in common with the requirements for modulation by n-alkanols and volatile anesthetics.

Two sets of chimeras between alphaxalone-sensitive GABA(A) receptor alpha2 or beta1 subunits and the alphaxalone-insensitive glycine receptor alpha1 subunit were constructed to determine the structural domains important for the modulatory actions of neuroactive steroids. These data suggest that the site of action for neurosteroids on GABA(A) receptors is not the same as that for volatile anesthetics and ethanol, but is on the N-terminal side of the middle of TM2.

Glycine and gamma-aminobutyric acid (GABA)A receptors are members of the "superfamily" of ion channels, and are sensitive to allosteric modulation by n-alcohols such as ethanol and butanol. We recently demonstrated that the mutation of Ser-267 to Ile in the alpha1 subunit abolished ethanol regulation of glycine receptors (Gly-R). In the present study, a pair of chimeric receptors was studied, in which a 45-amino acid domain comprising transmembrane domains 2 and 3 was exchanged between the Gly-Ralpha1 and gamma-aminobutyric acid rho1 subunits. Detailed pharmacologic analysis of these chimeras confirmed that this domain of the Gly-R confers enhancement of receptor function by ethanol and butanol. An extensive series of mutations at Ser-267 in the Gly-Ralpha1 subunit was also prepared, and the resulting homomeric receptors were expressed and tested for sensitivity to glycine, and allosteric modulation by alcohols. All of the mutant receptors expressed successfully in Xenopus oocytes. Mutation of Ser-267 to small amino acid residues such as Gly or Ala produced receptors in which glycine responses were potentiated by ethanol. As we have reported previously, the mutant Gly-Ralpha1 (Ser-267 --> Ile) was completely insensitive to ethanol; mutation of Ser-267 to Val had a similar effect. Mutation of Ser-267 to large residues such as His, Cys, or Tyr resulted in inhibition of Gly-R function by ethanol. These results demonstrate that the size of the amino acid residue at position alpha267 plays a crucial role in determining the functional consequences of allosteric modulation of the Gly-R by alcohols.

Mice whose gamma-aminobutyric acid type A (GABA(A)) beta3 subunit gene is inactivated ('beta3 knockout mice') have been previously shown to have epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal mortality. In this study, we analyze whole-cell responses to GABA in neurons from beta3+/+, beta3+/- and beta3-/- mice. We demonstrate markedly decreased responses to GABA in both hippocampal and dorsal root ganglion neurons isolated from beta3-/- mice without major differences in the GABA concentration-response curves. We also utilize the subunit selective pharmacology of Zn2+ and the anticonvulsant drug loreclezole to help infer the presence of beta2 and gamma subunits in the GABA(A) receptors remaining in neurons from beta3-/- mice.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The beta3 subunit is an essential component of the GABA(A)-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a beta3 gene inactivated by gene targeting. GABA(A)-R density is approximately halved in brain of beta3-deficient mice, and GABA(A)-R function is severely impaired. Most beta3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. beta3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the beta3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. beta3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.

Whole cell voltage clamp recordings were used to study the effects of two barbiturates, methohexital and pentobarbital, on inwardly rectifying K+ currents in the plasma membrane of a rat basophilic granulocyte cell line (RBL-1). Inwardly rectifying K+ currents are responsible for maintaining the resting membrane potential in a variety of cell types including skeletal and cardiac muscle, neurons, glia, blood cells, and endothelial cells. RBL-1 cells are unusual because the inward rectifier is the only apparent voltage-dependent current in these cells. Steps to command potentials between + 80 and -120 mV evoked only this strongly rectifying, rapidly developing current at membrane potentials more hyperpolarized than the reversal potential for K' ions. Extracellular Cs+ (10 mM) and Ba2+ (100 microM and 1 mM) blocked this current in a reversible and voltage-dependent manner. The voltage threshold for activation of the inwardly rectifying K+ current is dependent on the extracellular K+ concentration as predicted by the Nernst equation. Methohexital and pentobarbital reversibly inhibited the current in a concentration-dependent fashion with 50% inhibitory concentration (IC50) values of 145 microM and 218 microM respectively. The Hill slopes for both of these effects were approximately 1. The inhibition was not voltage dependent. These results indicate that fast inwardly rectifying K+ channels are potential molecular targets for barbiturates and could explain some of the diverse clinical effects of these drugs.

GABAA and glycine receptors are close relatives in the "gene superfamily" of ligand-gated ion channels, but have distinctly different pharmacology. For example, barbiturates have two effects on GABAA receptors (GABAA-R): at low micromolar concentrations (2-5 microM), the anesthetic barbiturate methohexital potentiates submaximal chloride current responses to GABA; at higher concentrations (20-50 microM), the barbiturate causes direct gating of the channel in the absence of agonist. Neither of these barbiturate effects is seen on the glycine receptor (Gly-R). In order to study the structural parts of the GABAA-R involved in this barbiturate pharmacology, two unique restriction sites were introduced into the cDNAs encoding the alpha 2 and beta 1 subunits of the human GABAA-R and the alpha 1 subunit of the human gly-R. The first site ('X') corresponded to the C-terminal end of the third transmembrane domain (M3) in each subunit and enabled exchange of C-terminal fragment of approximately 100 amino acids (which includes the large 'cytoplasmic loop' and M4 segment) between GABAA-R and Gly-R subunits. The second site ('S') was approximately 30 amino acids 3'- from the N-terminal end of each subunit and enabled exchange of a small N-terminal fragment between GABAA-R and Gly-R subunits. Several chimeric receptor subunit cDNAs were constructed and the resulting receptors tested for their ability to respond to GABA and glycine and for sensitivity to the barbiturate methohextial (MTX). The results show that neither the large C-terminal fragment nor the smaller N-terminal fragment is associated with the enhancement or direct activation of the GABAA-R by MTX. These results demonstrate the viability of chimeric GABAA/Gly-R and suggest that the method will be suitable for further investigation of the molecular basis of the barbiturate pharmacology of the GABA-R.

Many anxiolytics, anticonvulsants and general anesthetics modulate gamma-aminobutyric acid type A (GABAA) receptors. The anxiolytic benzodiazepines potentiate the actions of GABA, and this only at GABAA receptors with gamma subunits. The general anesthetics both potentiate GABA and activate GABAA receptors directly, but their binding sites on the receptor are poorly defined. We examined whether the gamma 2 subunit was required for the modulation of GABAA receptors by the general anesthetic 2,6-diisopropylphenol (propofol). Using the patch-clamp technique, we recorded membrane currents from HEK293 cells transfected with human alpha 2, beta 1 and gamma 2 cDNAs and with alpha 2 and beta 1 cDNAs alone. Both forms of the receptor were activated by GABA and by propofol at low concentrations. At maximal doses, propofol was considerably less effective than GABA as an activator of alpha 2 beta 1 GABAA receptors, but it had an efficacy similar to that of GABA as an activator of alpha 2 beta 1 gamma 2s receptors. In addition to activating currents directly, propofol potentiated currents elicited by GABA recorded from cells expressing either subunit combination. We conclude that the gamma 2 subunit is not a prerequisite for activation of GABAA receptors by propofol or for its potentiation of GABA-activated currents. However, the subunit may contribute to the efficacy of propofol as a GABAA receptor activator.

It has been suggested that general anesthesia might arise as a consequence of increased cytoplasmic free ionized calcium concentration ([Ca2+]i). The effect of increased [Ca2+]i might be to activate K+ channels or to modulate other ion channels important for the control of excitability, such as the GABAA receptor. A direct test of this hypothesis has not been reported. Microfluorimetry with the calcium-sensitive dye fura-2 was used to study the effects of four anesthetic agents on the regulation of intracellular free Ca2+ in hippocampal neurons cultured from the embryonic rat hippocampus. Basal intracellular free ionized calcium concentration [Ca2+]i in the neurons was 50-100 nM. Depolarization of the neurons with 50 mM K+ resulted in the elevation of [Ca2+]i to 200-800 nM, with subsequent recovery of [Ca2+]i over several minutes. The volatile anesthetics halothane, enflurane and isoflurane did not alter basal [Ca2+]i, even above clinically relevant concentrations; however, they did inhibit elevation of [Ca2+]i by high K+ stimulation. The intravenous anesthetic methohexital caused small increases in basal [Ca2+]i at concentrations > or = 50 microM; methohexital (5-50 microM) also inhibited elevations of [Ca2+]i induced by high K+. The evidence presented here suggests that the anesthetics studied do not produce their actions via sustained or transient increases in [Ca2+]i. However, all of the anesthetics studied appear to possess inhibitory effects on hippocampal voltage-dependent Ca2+ channels, in addition to their previously described effects at GABAA receptors.

1. The mechanisms of Zn2+ modulation of transient outward current (TOC) were studied in cultured rat hippocampal neurons, using the voltage-clamp technique. In the presence of micromolar concentrations of external Zn2+, the voltage dependence of activation and inactivation was shifted to more positive membrane potentials. The gating of TOC was unaltered by internal application of Zn2+. The effect of Zn2+ were not mimicked by external Ca2+, except at very high concentrations (> 10 mM). 2. The modulatory effects of external Zn2+ on TOC gating were not reproduced, antagonized, nor enhanced by lowering external ionic strength, indicating that modulation by Zn2+ does not occur via screening of bulk surface negative charge. 3. A range of other divalent and trivalent metal ions also was studied, and several were found to modulate the transient outward current when added to the extracellular medium. In particular, Pb2+, La3+, and Gd3+ were potent modulators, showing activity in the low micromolar range. Other metal ions were weaker modulators (e.g., Cd2+) or were without activity at the concentrations tested (Fe3+, Cu2+, Ni2+). 4. The same range of ions also was tested on the delayed rectifier K+ current in cultured rat hippocampal neurons. None of the ions studied had significant effects on delayed rectifier gating, although high (> or = 100 microM) concentrations of Pb2+ and La3+ reduced maximal current amplitude, suggesting the possibility of channel block.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of extracellular applications of Zn2+ ions on the strychnine-sensitive glycine receptor were studied in cultured rat spinal cord neurons and with recombinant glycine receptors expressed in human embryonic kidney 293 cells. Nanomolar concentrations of Zn2+ enhanced the chloride ion current in response to brief applications of 100 microM glycine. The enhancement of glycine responses increased from 20 nM to 1 microM Zn2+. Higher concentrations of Zn2+ caused a reversal of the potentiation, followed by progressive inhibition of the glycine response up to approximately 20-50 microM Zn2+. The biphasic modulation by Zn2+ appeared essentially identical in native and recombinant glycine receptors. Biphasic Zn2+ modulation was observed both with picrotoxin-insensitive heteromeric (alpha 2/beta) receptors and with picrotoxin-sensitive homomeric receptors consisting only of alpha 2 subunits. This suggests that the alpha subunit alone is sufficient for formation of two distinct Zn2+ binding sites on the glycine receptor. The demonstration of Zn2+ modulation of the strychnine-sensitive glycine receptor is of potential physiological importance, in view of the likely range of subsynaptic Zn2+ concentrations to which the receptor is exposed.

Ten general anesthetics of varying structure and potency were investigated for possible modulatory effects on gamma-aminobutyric acid, (GABAA) receptors, using the voltage clamp technique. All 10 anesthetics studied were observed to prolong the duration of responses to exogenously applied GABA recorded in cultured rat hippocampal neurons. These modulatory effects of the anesthetics occurred at pharmacologically relevant concentrations. An excellent correlation exists between drug potency as modulators of the GABAA receptor and anesthetic potency in vivo. These data suggest an alternative interpretation of the historical association between anesthetic potency and lipophilicity. It is proposed that hydrophobic binding sites on ligand-gated ion channel proteins, such as the GABAA receptors, constitute a molecular target site for many general anesthetics.

1. The effects of the volatile anesthetics enflurane, halothane, and isoflurane on gamma-aminobutyric acid (GABA) receptor-mediated inhibitory postsynaptic currents (IPSCs) were studied in cultured rat hippocampal neurons. The experimental concentrations of anesthetics were measured directly using gas chromatography. All three anesthetics increased the overall duration of IPSCs, measured as the time to half-decay (T1/2). Clinically effective concentrations of anesthetics [between 0.5 and 1.5 times MAC (minimum alveolar concentration)] produced between 100 and 400% increases in T1/2. These effects were fully reversible, and did not involve alterations in the reversal potential for the IPSC (EIPSC). 2. The decay of the IPSC was fitted as a sum of two exponential functions, yielding a fast component (tau fast = 20 ms), and a slow component (tau slow = 77 ms), such that the fast component accounted for 79% of the IPSC amplitude and 52% of the total charge transfer. All three anesthetics produced concentration-related increases in the amplitude and charge transfer of the slow component, while simultaneously decreasing the amplitude and charge transfer of the fast component. Thus T1/2 approximated tau fast under control conditions, but approximated tau slow in the presence of the anesthetics. 3. Varying the calcium chelating agents in the recording pipettes had no effect on the quality or magnitude of alterations in IPSC kinetics produced by halothane, suggesting that variations in intracellular calcium levels are not required for the effect of halothane on the time course of the IPSC. 4. The (+)-stereoisomer of isoflurane produced greater increases in the duration of the IPSC than the (-)-isomer when applied at approximately equal concentrations, suggesting that there is a structurally selective site of interaction for isoflurane that modulates the GABAA receptor. 5. These results suggest that the previously shown abilities of volatile anesthetics to potentiate responses to exogenously applied GABA and to prolong the duration of GABA-mediated synaptic inhibition may be due to an alteration in the gating kinetics of the GABAA receptor/channel complex. Prolongation of synaptic inhibition in the CNS is consistent with the physiological effects that accompany anesthesia and may contribute to the mechanism of anesthetic action.

The interactions of the inhalation anesthetic agent isoflurane with ligand-gated chloride channels were studied using transient expression of recombinant human receptors in a mammalian cell line. Isoflurane enhanced gamma-aminobutyric acid (GABA)-activated chloride currents in cells that expressed heteromeric GABAA receptors consisting of combinations of alpha 1 or alpha 2, beta 1, and gamma 2 subunits and in cells that expressed receptors consisting of combinations of only alpha and beta subunits. Receptors consisting of alpha 2 and gamma 2 subunits were poorly expressed but were sensitive to isoflurane. Receptors consisting of beta 1 and gamma 2 subunits were not expressed. Isoflurane also enhanced glycine-activated chloride currents through homomeric alpha glycine receptors but did not enhance GABA currents in cells expressing homomeric rho 1 receptors. These results show that not all ligand-gated chloride channel receptors are sensitive to isoflurane and, therefore, that the anesthetic interacts with specific structural determinants of these ion channel proteins.

The actions of zinc ions on three species of K+ channels were studied using mouse fibroblasts stably transfected with a plasmid containing both the appropriate K+ channel gene and a steroid-inducible promotor. The channels studied were rKv1.1 and hKv1.5, delayed rectifiers cloned from rat and human tissue, respectively, and hKv1.4, an inactivating human K+ channel. Zn2+ shifted the activation curves for all three K+ currents in the depolarizing direction and also shifted the steady state inactivation curve for hKv1.4 in the depolarizing direction. The effect of Zn2+ was concentration dependent between 2 and 1000 microM. As a consequence of the modulation of gating, the activation kinetics of the K+ currents were slowed by Zn2+, an effect likely to delay repolarization of the neuronal action potential. The action of Zn2+ on these diverse K+ channels suggests the existence of a common Zn2+ binding domain, the occupation of which influences the voltage sensor. The resulting modulation of gating of hKv1.4 by Zn2+ may well be of physiological significance, in view of the localization of this channel in mossy fiber nerve terminals in the hippocampus, where Zn2+ is found in abundance.

We have examined the actions of trichloroethanol (TCEt), the active metabolite of the general anesthetic chloral hydrate, on responses mediated by gamma-aminobutyric acid (GABA)A receptors in response to application of exogenous GABA and activation of endogenous GABAergic transmission, by using hippocampal neurons in cell culture and in brain slices. In the presence of TCEt, Cl- current activated by exogenous GABA was both enhanced in amplitude and prolonged, leading to a net increase in total charge passing through GABAA receptor channels. Prolongation of GABA-activated current increased in magnitude in a concentration-dependent manner from 0.2 to 10 mM TCEt. Inhibitory postsynaptic currents produced at synapses between pairs of cultured GABAergic neurons or by activation of interneurons in hippocampal slices were also prolonged by TCEt, at concentrations from 0.5 to 10 mM. Application of TCEt at concentrations of 1 mM and above produced a small amplitude current which was directed outwardly at -40 mV in neurons in which methylsulfate or gluconate was the major intracellular anion and directed inwardly in neurons filled with Cl-. Our observations indicate that TCEt potentiates GABAergic transmission; presumably by potentiating the function of GABAA receptors in a manner similar to barbiturate or steroid anesthetics. This action is likely to contribute to the general anesthetic effect of TCEt which occurs after chloral hydrate administration.

Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).

The actions of zinc ions (Zn2+) on transient outward current (TOC) were studied in cultured embryonic rat hippocampal neurons and adult guinea pig CA1 neurons acutely isolated from hippocampal slices. Zn2+ (1-1000 microM) shifted both the activation and inactivation curves for the TOC in the depolarizing direction. These effects of Zn2+ were modeled, assuming a single class of binding sites for Zn2+, yielding apparent dissociation constants for Zn2+ between 10 and 35 microM. In addition, the activation kinetics of the TOC were significantly slowed by Zn2+; deactivation was slowed at the highest concentrations of Zn2. These complex actions of Zn2+ on the TOC are likely to delay repolarization and promote spontaneous activity, resulting in action potential prolongation and hyperexcitability of hippocampal neurons.

1. We have examined the effects of neuropeptide Y (NPY) on synaptic transmission and [Ca2+]i signals in rat hippocampal neurones grown in culture. [Ca2+]i in individual neurones displayed frequent spontaneous fluctuations often resulting in an elevated plateau [Ca2+]i. These fluctuations were reduced by tetrodotoxin (1 microM) or combinations of the excitatory amino acid antagonists 6-cyano-7-dinitro-quinoxaline (CNQX) (10 microM) and aminophosphonovalerate (APV) (50 microM), indicating that they were the result of glutamatergic transmission occurring between hippocampal neurones. 2. [Ca2+]i fluctuations were also prevented by Ni2+ (200 microM), by the GABAB receptor agonist, baclofen (10 microM) and by NPY (100 nM) or Y2 receptor-selective NPY agonists. Following treatment of cells with pertussis toxin, NPY produced only a brief decrease in [Ca2+]i fluctuations which rapidly recovered. 3. Perfusion of hippocampal neurones with 50 mM K+ produced a large rapid increase in [Ca2+]i. This increase was slightly reduced by NPY or by a combination of CNQX and APV. The effects of CNQX/APV occluded those of NPY. NPY had no effect on Ba2+ currents measured in hippocampal neurones under whole cell voltage-clamp even in the presence of intracellular GTP-gamma-S. On the other hand, Ba2+ currents were reduced by both Cd2+ (200 microM) and baclofen (10 microM). 4. Current clamp recordings from hippocampal neurones demonstrated the occurrence of spontaneous e.p.s.ps and action potential firing which were accompanied by increases in [Ca2+]i. This spontaneous activity and the accompanying [Ca2+]i signals were prevented by application of NPY (100 nM). When hippocampal neurones were induced to fire trains of action potentials in the absence of synaptic transmission, these were accompanied by an increase in cell soma [Ca2+]j. NPY (100 nM) had no effect on these cell soma [Ca2+], signals. NPY (100 nM) also had no effect on inward currents generated in hippocampal neurones by micropipette application of glutamate (50 microM).5. Thus, NPY is able to abolish excitatory neurotransmission in hippocampal cultures through a pertussis toxin-sensitive mechanism. However, no effect of NPY on Ca2+ influx into the cell soma of these hippocampal neurones could be discerned. These results are consistent with a localized presynaptic inhibitory effect of NPY on glutamate release in hippocampal neurones in culture.

1. The effects of the volatile anaesthetics enflurane, halothane and isoflurane on gamma-aminobutyric acid (GABA)A receptor-mediated chloride currents were studied in cultured rat hippocampal neurones. Transient current responses were obtained by brief pressure application of GABA to the cell body of neurones under voltage clamp. 2. All three anaesthetics increased the peak amplitude and duration of current 2. All three anaesthetics increased the peak amplitude and duration of current responses to brief applications of GABA. These effects were fully reversible, and did not involve alterations in the reversal potential for GABA responses. 3. The experimental concentrations of anaesthetics were measured directly using gas chromatography. The enhancement of GABA currents increased with increasing anaesthetic concentration. Clinically effective concentrations of anaesthetics (between 1 and 1.5 times MAC (minimum alveolar concentration) produced significant enhancement of GABA currents. 4. These results demonstrate that the changes in the time course of synaptic inhibition reported in the presence of the volatile anaesthetics are likely to result from modification of the function of postsynaptic GABAA receptor-channel complexes. These findings also support the hypothesis that GABAA receptor complexes serve as common molecular target sites for a variety of structurally diverse anaesthetic molecules.

The actions of 3-aminopropanephosphinic acid (APPA) were examined using whole-cell patch-clamp recording in rat hippocampal slice. In recordings from neurons in subfield CA1 of slices from young (2-4 weeks) and adult (greater than 2 month) rats, APPA (0.5-50 microM) produced membrane hyperpolarization and outward current under voltage-clamp. APPA also inhibited excitatory postsynaptic potentials with an IC50 of 2.3 microM, and reduced inhibitory postsynaptic potentials at concentrations from 0.1 to 1 microM. The hyperpolarizing and synaptic depressant effects of APPA were reduced by 2-OH-saclofen an antagonist at the B-type receptor for the neurotransmitter gamma-aminobutyric acid (GABA). In this preparation APPA exhibited potencies similar to those previously reported for the GABAB receptor agonist baclofen. APPA was much less effective in inhibiting synaptic transmission measured using field potential recordings. The observations made with whole-cell patch-clamp recording indicate that in hippocampus APPA acts as a potent agonist at presynaptic GABAB receptors associated with both excitatory and inhibitory synapses, and also activates postsynaptic GABAB receptors.

The possibility that zinc (Zn2+) induces giant depolarizing potentials (GDPs) by blocking pre- and postsynaptic gamma-aminobutyric acidB (GABAB) receptors in area CA1 of rat hippocampal slices was investigated. Monosynaptic GABAA receptor-mediated fast and GABAB receptor-mediated late inhibitory postsynaptic potentials (IPSPs) were evoked in the presence of the excitatory amino acid (EAA) receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-amino-5-phosphonovalerate (APV). Addition of Zn2+ (0.3 mM) resulted in the appearance of long-lasting GDPs which obscured monosynaptic late IPSPs. The GABAA receptor antagonist bicuculline methiodide (BMI; 30 microM) blocked fast monosynaptic IPSPs and GDPs, revealing a monosynaptic late IPSP that was prolonged in the presence of Zn2+ and blocked by the GABAB receptor antagonist CGP 35,348 (100 microM). The selective GABAB receptor agonist baclofen (10 microM) depressed monosynaptic IPSPs and population excitatory postsynaptic potentials (pEPSPs) by acting at presynaptic GABAB receptors. Depression of synaptic potentials by baclofen was unaffected by Zn2+. These results suggest that induction of GDPs in area CA1 does not result from an action of Zn2+ at GABAB receptors. We suggest instead that Zn2+ induces GDPs by inducing synchronized discharge of GABAergic interneurons.

The mechanism of disinhibition produced by (+/-)-baclofen was studied using intracellular recording in area CA1 of rat hippocampal slices. Baclofen reversibly depressed monosynaptic IPSPs evoked by direct activation of interneurons in the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV). Ba2+ prevented baclofen-induced hyperpolarization of pyramidal neurons but not depression of monosynaptic IPSPs by baclofen. Baclofen reversibly depressed monosynaptic IPSPs when applied close to the recording site, but was ineffective when applied close to the stimulating site in stratum radiatum. These results suggest that baclofen disinhibits pyramidal neurons in area CA1 of the rat hippocampus by activating receptors on the terminals of inhibitory neurons that are coupled to a Ba(2+)-insensitive effector mechanism.

The mechanism of disinhibition produced by opioid peptides was studied using intracellular recording in area CA1 of rat hippocampal slices. The mu-selective opioid peptide [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAGO) reversibly depressed directly-activated, monosynaptic inhibitory postsynaptic potentials (IPSPs) evoked in the presence of the excitatory amino acid receptor antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovalerate (APV) in a naloxone-sensitive manner. Depression of monosynaptic inhibitory postsynaptic potentials (IPSPs) by DAGO was not prevented by 1-2 mM Ba2+. DAGO reversibly depressed monosynaptic IPSPs when applied locally close to the recording site, but was ineffective when applied close to the stimulating site in stratum radiatum. These results suggest that DAGO disinhibits pyramidal neurons in area CA1 of the rat hippocampus by activating mu opiate receptors located on the terminals of inhibitory neurons, and by a Ba(2+)-insensitive mechanism.

The actions of 2-hydroxy-saclofen (2-OH-S), a recently developed analog of baclofen, were studied at presynaptic GABAB receptors in the rat hippocampal slice. Baclofen (0.5-20 microM) reduces the amplitude of excitatory postsynaptic potentials (EPSPs) recorded from hippocampal CA1 pyramidal neurons. In the presence of 200-500 microM 2-OH-S, the synaptic depressant action of baclofen is significantly reduced. These data show that 2-OH-S is an effective antagonist at presynaptic GABAB receptors on excitatory terminals in the hippocampus.

We have studied the effects of the membrane-permeant cyclic AMP analogs 8-bromo-cyclic AMP and 8-(4-chlorophenylthio)-cyclic AMP (CPT-cAMP) on the gamma-aminobutyric acidA (GABAA) receptor-mediated chloride current in cultured rat hippocampal neurons. External perfusion with 8-bromo-cyclic AMP or CPT-cAMP caused a reversible, concentration-dependent decrease in the response to GABA. Adding the protein kinase inhibitor H-8 to the perfusing medium or the intracellular recording solution did not affect the response to GABA, which was decreased by CPT-cAMP as before. L858051, a water-soluble derivative of the adenylate cyclase activator forskolin, did not decrease the response to GABA even in the presence of the phosphodiesterase inhibitor 3-isobutylmethylxanthine. External cyclic AMP also caused a reversible, concentration-dependent decrease in the response to GABA with a potency similar to that of 8-Br-cAMP. When cAMP was present in the intracellular recording solution cAMP and CPT-cAMP decreased the response to GABA as before. These experiments suggest that analogs of cAMP decrease GABAA receptor-activated chloride current by acting at an extracellular site.

The actions of the GABA analog 3-aminopropanephosphinic acid (3-APA) were studied in the guinea-pig isolated ileal preparation and at synapses between cultured rat hippocampal neurons. Like the GABAB receptor agonist, baclofen, 3-APA inhibited the electrically evoked ileal twitch. The EC50 for 3-APA was 0.8 microM; the EC50 for baclofen was 9 microM. In addition, the depressant responses to 3-APA and baclofen were blocked by the GABAB receptor antagonists phaclofen, saclofen, 2-hydroxy-saclofen and delta-aminovaleric acid. 3-APA also mimicked the presynaptic action of baclofen at GABAergic synapses between embryonic rat hippocampal neurons in culture. 3-APA reduced the amplitude of inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) by greater than 50% at a concentration of 1 microM, while baclofen reduced synaptic transmission to a similar degree at 10 microM. 3-APA did not alter membrane conductance, nor did the drug alter postsynaptic responses to GABA. These data show that 3-APA is a potent agonist at presynaptic GABAB receptors in the periphery and on GABAergic neurons from the central nervous system. The activity of 3-APA at central postsynaptic GABAB receptors remains to be studied.

1. (-)Baclofen reduces inhibitory postsynaptic potentials (IPSPs) and the associated synaptic currents (IPSCs) at inhibitory GABAergic synapses between cultured rat hippocampal neurones. The reversal potential for the IPSC is unaltered. 2. The effect of (-)baclofen is concentration dependent; the EC50 for (-)baclofen is approximately 5 microM. 3. Statistical analyses of the amplitude fluctuations of the IPSC in the presence of (-)baclofen suggested a presynaptic location for the depression of synaptic transmission by (-)baclofen. In control experiments, lowering extracellular Ca2+ produced similar effects. (-)Baclofen has no detectable postsynaptic actions in these cultured neurones. 4. Phaclofen (0.2-0.5 mM) increases IPSC amplitude but does not significantly block the depressant effect of (-)baclofen on synaptic transmission. 5. The effect of (-)baclofen is not blocked by pertussis toxin pre-treatment. 6. It is concluded that (-)baclofen acts presynaptically to reduce the release of GABA. The mechanism by which release is reduced may involve a phaclofen-insensitive GABAB receptor.

GABA-mediated monosynaptic inhibitory postsynaptic currents were recorded at synapses between embryonic rat hippocampal neurons in culture. The GABAB receptor agonist L(-)-baclofen reduced inhibitory postsynaptic current amplitude substantially at 10 microM, while the D(+)-isomer of baclofen was without effect. L(-)-Baclofen did not increase postsynaptic membrane conductance or block postsynaptic membrane responses to GABA. It is concluded that L(-)-baclofen depresses GABA-mediated inhibitory postsynaptic potentials by a presynaptic action at GABAB receptors on or near the terminals of the inhibitory neuron. The possible physiological importance of these presynaptic 'autoreceptors' is discussed.

Certain steroids are potent barbiturate-like modulators of the gamma-aminobutyric acidA (GABA) receptor-chloride ionophore complex in rat brain membranes. At nanomolar to low micromolar concentrations, these steroids stimulate [3H]flunitrazepam and [3H] muscimol binding and displace the convulsant [35S]t-butylbicyclophosphorothionate from its binding site in an allosteric manner, in addition to enhancing Cl- conductance responses to GABA recorded in cultured rat hippocampal and spinal neurons. A stringent structure-activity relationship exists for these interactions of steroids with the GABAA receptor complex. Comparison of the structure-activity relationship data obtained in this study with those for steroid-induced general anesthesia strongly suggests that steroidal anesthesia may result from the interaction between steroids and the GABAA receptor. The essential features of the active structures are a 5 alpha or 5 beta-reduced pregnane skeleton with a hydroxyl at C3 in the alpha-position and a ketone group at C20. These features are all present in some naturally occurring steroids, including metabolites of deoxycorticosterone and progesterone, that show potent activity at the GABAA receptor complex. Two of the compounds investigated are known to be formed in vivo as reduced metabolites of endogenous steroid hormones: 5 alpha-pregnane-3 alpha -ol-20-one and 5 alpha-pregnane-3 alpha,21-diol-20-one, which are derived from progesterone and deoxycorticosterone, respectively. These two steroids produce a striking prolongation of GABA-mediated inhibitory postsynaptic currents recorded at synapses between rat hippocampal neurons in culture and could conceivably regulate GABA-mediated inhibition under some physiologic and pathologic conditions.