GABA Receptors and the Pharmacology of Sleep.

Current GABAergic sleep-promoting medications were developed pragmatically, without making use of the immense diversity of GABAA receptors. Pharmacogenetic experiments are leading to an understanding of the circuit mechanisms in the hypothalamus by which zolpidem and similar compounds induce sleep at α2ßγ2-type GABAA receptors. Drugs acting at more selective receptor types, for example, at receptors containing the α2 and/or α3 subunits expressed in hypothalamic and brain stem areas, could in principle be useful as hypnotics/anxiolytics. A highly promising sleep-promoting drug, gaboxadol, which activates αßδ-type receptors failed in clinical trials. Thus, for the time being, drugs such as zolpidem, which work as positive allosteric modulators at GABAA receptors, continue to be some of the most effective compounds to treat primary insomnia.

The role of K2p channels in anaesthesia and sleep.

Tandem two-pore potassium channels (K2Ps) have widespread expression in the central nervous system and periphery where they contribute to background membrane conductance. Some general anaesthetics promote the opening of some of these channels, enhancing potassium currents and thus producing a reduction in neuronal excitability that contributes to the transition to unconsciousness. Similarly, these channels may be recruited during the normal sleep-wake cycle as downstream effectors of wake-promoting neurotransmitters such as noradrenaline, histamine and acetylcholine. These transmitters promote K2P channel closure and thus an increase in neuronal excitability. Our understanding of the roles of these channels in sleep and anaesthesia has been largely informed by the study of mouse K2P knockout lines and what is currently predicted by in vitro electrophysiology and channel structure and gating.

Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels.

Although it is generally agreed that general anesthetics ultimately act on neuronal ion channels, there is considerable controversy over whether this occurs by direct binding to protein or secondarily by nonspecific perturbation of lipids. Very pure optical isomers of the inhalational general anesthetic isoflurane exhibited clear stereoselectivity in their effects on particularly sensitive ion channels in identified molluscan central nervous system neurons. At the human median effect dose (ED50) for general anesthesia, the (+)-isomer was about twofold more effective than the (-)-isomer both in eliciting the anesthetic-activated potassium current IK(An) and in inhibiting a current mediated by neuronal nicotinic acetylcholine receptors. For inhibiting the much less sensitive transient potassium current IA, the (-)-isomer was marginally more potent than the (+)-isomer. Both isomers were equally effective at disrupting lipid bilayers.

The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway.

We investigated the role of regionally discrete GABA (gamma-aminobutyric acid) receptors in the sedative response to pharmacological agents that act on GABA(A) receptors (muscimol, propofol and pentobarbital; 'GABAergic agents') and to ketamine, a general anesthetic that does not affect GABA(A) receptors. Behavioral studies in rats showed that the sedative response to centrally administered GABAergic agents was attenuated by the GABA(A) receptor antagonist gabazine (systemically administered). The sedative response to ketamine, by contrast, was unaffected by gabazine. Using c-Fos as a marker of neuronal activation, we identified a possible role for the tuberomammillary nucleus (TMN): when gabazine was microinjected directly into the TMN, it attenuated the sedative response to GABAergic agents. Furthermore, the GABA(A) receptor agonist muscimol produced a dose-dependent sedation when it was administered into the TMN. We conclude that the TMN is a discrete neural locus that has a key role in the sedative response to GABAergic anesthetics.

Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes.

BACKGROUND: Firefly luciferase is a 62 kDa protein that catalyzes the production of light. In the presence of MgATP and molecular oxygen, the enzyme oxidizes its substrate, firefly luciferin, emitting yellow-green light. The reaction proceeds through activation of the substrate to form an adenylate intermediate. Firefly luciferase shows extensive sequence homology with a number of enzymes that utilize ATP in adenylation reactions. RESULTS: We have determined the crystal structure of firefly luciferase at 2.0 A resolution. The protein is folded into two compact domains. The large N-terminal domain consists of a beta-barrel and two beta-sheets. The sheets are flanked by alpha-helices to form an alphabetaalphabetaalpha five-layered structure. The C-terminal portion of the molecule forms a distinct domain, which is separated from the N-terminal domain by a wide cleft. CONCLUSIONS: Firefly luciferase is the first member of a superfamily of homologous enzymes, which includes acyl-coenzyme A ligases and peptide synthetases, to have its structure characterized. The residues conserved within the superfamily are located on the surfaces of the two domains on either side of the cleft, but are too far apart to interact simultaneously with the substrates. This suggests that the two domains will close in the course of the reaction. Firefly luciferase has a novel structural framework for catalyzing adenylate-forming reactions.

Fatty acid binding to human serum albumin: new insights from crystallographic studies.

Human serum albumin possesses multiple fatty acid binding sites of varying affinities, but the precise locations of these sites have remained elusive. The determination of the crystal structure of human serum albumin complexed with myristic acid recently revealed the positions and architecture of six binding sites on the protein. While the structure of the complex is consistent with a great deal of the biochemical and biophysical data on fatty acid binding, it is not yet possible to provide a completely rigorous correlation between the structural and binding data. The challenge now is to use the new structural information to design experiments that will identify the physiologically important binding sites on HSA and provide a much richer description of fatty acid interactions with the protein.

An X-ray diffraction analysis of oriented lipid multilayers containing basic proteins.

X-ray diffraction techniques have been used to study the structures of lipid bilayers containing basic proteins. Highly ordered multilayer specimens have been formed by using the Langmuir-Blodgett method in which a solid support is passed through a lipid monolayer held at constant surface pressure at an air/water interface. If the lipid monolayer contains acidic lipids then basic proteins in the aqueous subphase are transferred with the monolayer and incorporated into the multi-membrane stack. X-ray diffraction patterns have been recorded from multilayers of cerebroside sulphate and 40% (molar) cholesterol both with and without polylysine, cytochrome c and the basic protein from central nervous system myelin. Electron density profiles across the membranes have been derived at between 6 A and 12 A resolution. All of the membrane profiles have been placed on an absolute scale of electron density by the isomorphous exchange of cholesterol with a brominated cholesterol analog. The distributions and conformations of the various basic proteins incorporated within the cerebroside sulphate/cholesterol bilayer are very different. Polylysine attaches to the surface of the lipid bilayer as a fully extended chain while cytochrome c maintains its native structure and attaches to the bilayer surface with its short axis approximately perpendicular to the membrane plane. The myelin basic protein associates intimately with the lipid headgroups in the form of an extended molecule, yet its dimension perpendicular to the plane of the membrane of approx. 15 A is consistent with the considerable degree of secondary structure found in solution. In the membrane plane, the myelin basic protein extends to cover an area of about 2500 A2. There is no significant penetration of the protein into the hydrocarbon region of the bilayer or, indeed, beyond the position of the sulphate group of the cerebroside sulphate molecule.

The effects of general anaesthetics on carbachol-evoked gamma oscillations in the rat hippocampus in vitro.

The effects of general anaesthetics and temperature on carbachol-evoked gamma oscillations in the rat hippocampal brain slice preparation were investigated. The frequency of the oscillations was found to be dependent on temperature in the range 32-25 degrees C, with a linear reduction in frequency from 40-17 Hz over this temperature range. The volatile anaesthetics isoflurane and halothane, and the intravenous anaesthetics thiopental, propofol and R(+)-etomidate caused a reduction in the frequency of the oscillations, in a concentration-dependent manner, over a range of clinically relevant concentrations. On the other hand, the intravenous agent ketamine and the "inactive" S(-)-isomer of etomidate had no significant effect on the oscillation frequency. The oscillations were markedly asymmetric over one cycle with a relatively rapid "rising" phase followed by a slower "decaying" phase. The decrease in oscillation frequency was due to an increase in the time-course of the "decaying phase" of the oscillation with little effect on the "rising" phase, consistent with the idea that carbachol-evoked gamma oscillations are trains of GABAergic inhibitory postsynaptic potentials and that the anaesthetics are acting postsynaptically at the GABA(A) receptor.

Synthesis and use of the n-bromododecane-1,12-diols as conformational probes for general anesthetic target sites.

The n-bromododecane-1,12-diols with bromine on carbons 2, 3, 5, and 6, respectively, were synthesized and found to be potent general anesthetics. They were also found to be potent inhibitors of firefly luciferase, a protein model for the primary target sites underlying general anesthesia. However, their effects on lipid bilayers were small, lowering the chain-melting phase transition temperature by less than 1 degree C at their EC50 concentrations for general anesthesia. A large dependence upon the position of the bromine atom was found for both n-hexadecane/water partition coefficients and inhibition constants for firefly luciferase; a much smaller positional dependence was found for induction of general anesthesia and for disrupting lipids. These results are consistent with the bulky bromine atom inhibiting the conformational flexibility of the diol hydrocarbon chain, making these bromo diols useful probes for ascertaining the shapes of apolar binding sites. In particular, our measurements suggest that these novel anesthetics produce general anesthesia by binding to long and relatively narrow apolar target sites in the central nervous system.

Actions of general anaesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurones of Lymnaea stagnalis.

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were studied under two-electrode voltage-clamp. Neuronal nicotinic acetylcholine receptor currents were studied at low acetylcholine (ACh) concentrations (< or = 200 nM). At these levels, control currents were non-desensitizing and proportional to the square of the ACh concentration. 2. IC50 concentrations were determined for the steady-state inhibition of the ACh-activated current by 31 general anaesthetics plus the non-anaesthetic alcohol n-tridecanol. The general anaesthetics included inhalational agents, n-alcohols, n-alkane-(alpha,omega)-diols, cycloalcohols and an n-alkane. 3. Anaesthetic inhibition was independent of voltage and consistent with two anaesthetic-binding sites on the receptor. 4. IC50 concentrations for inhibiting the neuronal nicotinic ACh receptor correlated well (r = 0.97) with EC50 concentrations for general anaesthesia. The maximum deviation from the line of identity was less than fourfold. The inhalational agents tended to be more potent as inhibitors of the ACh receptor than as general anaesthetics, while the alcohols and diols were less potent. 5. The inhibition of the ACh-induced current by the homologous series of n-alcohols exhibited a cutoff at the same position (just after dodecanol) as found for the induction of general anaesthesia in tadpoles. 6. Polarity profile maps of the anaesthetic-binding sites on the neuronal nicotinic ACh receptor were calculated from IC50 concentrations for the homologous series of n-alcohols and n-alkane-(alpha,omega)-diols. They reveal amphiphilic sites with apolar regions capable of accommodating the hydrocarbon chains of n-alcohols as large as decanol. A striking resemblance was found to profiles previously calculated from data for tadpole general anaesthesia.

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor.

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.

Actions of general anaesthetics on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. NIE-115 mouse neuroblastoma cells were studied under voltage clamp in the whole-cell patch-clamp configuration. Peak currents induced by bath application of 5-hydroxytryptamine (5-HT) were inwardly rectifying, reversed at 0.4 +/- 0.2 mV (mean +/- s.e.mean), and were approximately half-inhibited (at 1 microM 5-HT) by 2 nM of the 5-HT3 selective antagonist MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). 2. Peak inward currents activated by a low concentration of 5-HT at a holding potential of -50 mV were potentiated by volatile general anaesthetics. At their human minimum alveolar concentrations (MACs), the degree of potentiation increased in the order isoflurane < halothane < enflurane < methoxyflurane. Potentiation by methoxyflurane was independent of membrane potential in the range -70 mV to +40 mV. The reversal potential was the same in the presence and absence of methoxyflurane. 3. Methoxyflurane shifted the 5-HT dose-response curve to lower 5-HT concentrations, without significantly changing the Hill coefficient or maximum response. The EC50 concentration for 5-HT decreased from 1.86 +/- 0.02 microM to 1.07 +/- 0.11 microM (means +/- s.e.mean) due to the presence of 1 MAC (270 microM) methoxyflurane. 4. In contrast to the volatile anaesthetics, the barbiturate anaesthetic, thiopentone, inhibited the 5-HT3 receptor. Hill analysis of thiopentone dose-response data gave an average IC50 = 117 +/- 8 microM thiopentone and Hill coefficient = 1.6 +/- 0.2 (means +/- s.e.mean). These parameters were not significantly different for data obtained at 5-HT concentrations above and below the control EC50 concentration for 5-HT, consistent with non-competitive inhibition. 5. The n-alcohols occupied an intermediate position between the volatile and barbiturate anaesthetics. The lower alcohols (butanol and hexanol) potentiated 5-HT responses at low alcohol concentrations but inhibited them at high concentrations. In contrast, the higher alcohols (octanol, decanol, dodecanol, tridecanol, tetradecanol and pentadecanol) produced no potentiation, but only inhibition, at all alcohol concentrations. 6. Inhibition of the 5-HT3 receptor by the n-alcohols exhibited a cutoff in potency similar to those previously found for tadpoles, luciferase enzymes and a neuronal nicotinic acetylcholine receptor channel.

Stereoselective and non-stereoselective actions of isoflurane on the GABAA receptor.

1. Acutely dissociated cerebellar Purkinje neurones from 8-14 day old rats were studied under voltage clamp in the whole-cell patch-clamp configuration. Cl- currents induced by bath application of gamma-aminobutyric acid (GABA) were measured (using symmetrical Cl- solutions) at both low (2 microM) non-desensitizing and high (300 microM) desensitizing concentrations of GABA. 2. At 2 microM GABA, the bicuculline-sensitive Cl- currents were potentiated by racemic isoflurane and both of its optical isomers. Isoflurane had no effect on membrane current in the absence of GABA. The dose-response data for potentiation by racemic isoflurane could be fitted with a Hill equation with an EC50 = 320 +/- 20 microM isoflurane and a Hill coefficient of h = 2.7 +/- 0.4 (means +/- s.e.mean). 3. The potentiations produced by the optical isomers of isoflurane at 2 microM GABA were stereoselective at moderate and high anaesthetic concentrations. The maximum stereoselectivity, about two fold, occurred at the EC50 concentration for general anaesthesia (310 microM isoflurane), with S(+)-isoflurane being more effective than R(-)-isoflurane. At sub-anaesthetic concentrations, the stereoselectivity was less marked and vanished at the lowest concentration used (77 microM isoflurane). 4. The sustained residual current remaining after exposure of neurons to a desensitizing concentration of GABA (300 microM) was inhibited non-stereoselectively, but only at high concentrations of isoflurane. The ratio of inhibitions by S(+)- and R(-)-isoflurane (mean +/- s.e.mean) was 1.14 +/- 0.21 at 770 microM isoflurane. At the EC50 concentration for general anaesthesia, however, the inhibition was barely significant. 5. The above results are discussed in relation to the possible role of the GABAA receptor channel in general anaesthesia.

Actions of general anaesthetics and arachidonic pathway inhibitors on K+ currents activated by volatile anaesthetics and FMRFamide in molluscan neurones.

1. K+ currents activated by volatile general anaesthetics (IK(An)) and by the neuropeptide FMRFamide (IK(FMRFa)) were studied under voltage clamp in isolated identified neurones from the pond snail Lymnaea stagnalis. 2. IK(An) was activated by all the volatile anaesthetics studied. The maximal responses varied from agent to agent, with halothane sevoflurane > isoflurane > enflurane approximately chloroform. 3. IK(An) was inhibited rather than activated by the n-alcohols from hexanol to dodecanol and by the 6- and 8-carbon cycloalcohols. The n-alcohols exhibited a cutoff effect, with dodecanol being unable to half-inhibit IK(An). 4. Unlike IK(An) which did not desensitize at reasonable halothane concentrations, IK(FMRFa) desensitized at most FMRFamide concentrations studied. This desensitization could be substantially removed by halothane. Nonetheless, both IK(An) and IK(FMRFa) had similar sensitivities to the potassium channel blockers tetraethylammonium and 4-aminopyridine, consistent with both currents flowing through the same channels. Responses to low concentrations of halothane and FMRFamide showed synergy. 5. The phospholipase A2 inhibitor aristolochic acid inhibited IK(An), consistent with a role for arachidonic acid (AA). The lipoxygenase and cyclooxygenase inhibitor nordihydroguaiaretic acid blocked IK(FMRFa) but did not affect IK(An). IK(An) and IK(FMRFa) were little affected by the cyclooxygenase inhibitor indomethacin. These findings suggest that neither lipoxygenase nor cyclooxygenase pathways of AA metabolism are involved in the anaesthetic activation of IK(An. 6. Inhibitors of a third, cytochrome P450-mediated, pathway of AA metabolism (clotrimazole and econazole) potently blocked IK(An), suggesting possible roles for certain cytochrome P450 isoforms in the activation of IK(An).

Probing the molecular dimensions of general anaesthetic target sites in tadpoles (Xenopus laevis) and model systems using cycloalcohols.

1. The series of cycloalcohols C6, C7, C8 and C10 have been used to probe the molecular dimensions of a variety of general anaesthetic target sites. 2. The general anaesthetic EC50 concentrations of the cycloalcohols were determined for tadpoles (Xenopus laevis). All of the cycloalcohols tested were found to be potent general anaesthetics (on average EC50/Csat = 0.03). 3. The effects of the cycloalcohols on highly purified luciferase enzymes from fireflies (Photinus pyralis) and bacteria (Vibrio harveyi) were also investigated. Both enzymes were inhibited competitively, with the cycloalcohols competing with firefly luciferin for binding to the firefly enzyme and with n-decanal for binding to the bacterial enzyme. 4. The binding site on the firefly enzyme could accommodate two molecules of cycloalcohols C6 and C7 but only a single molecule of the larger cycloalcohols (C8 and C10), implying a volume of the binding site of about 250 cm3 mol-1. In contrast, the binding site on the bacterial luciferase could bind only a single cycloalcohol molecule between C6 and C10. 5. While all of the cycloalcohols were potent inhibitors of the firefly luciferase enzyme (on average EC50/Csat = 0.015), they were very weak inhibitors of the bacterial luciferase enzyme (on average EC50/Csat = 0.12). Since both enzymes bind long-chain aliphatic n-alcohols tightly, the differing affinities of the cycloalcohols for the two enzymes is probably a consequence of geometrical factors. 6. The cycloalcohols produced very small effects on lipid bilayers. At EC50 concentrations which produce general anaesthesia, lipid bilayer phase transitions were shifted, on average, by only 0.43 degrees C. 7. We conclude that the general anaesthetic effects of the cycloalcohols can most economically be explained by assuming that the cycloalcohols act at protein binding sites in the central nervous system. These target sites would have binding properties similar to those of the anaesthetic-binding site on firefly luciferase, but their average volume would be somewhat smaller than 250 cm3 mol -1.

Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes.

1. Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine-sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 +/- 5 microM and Hill coefficient nH = 1.70 +/- 0.05 (means +/- s.e. means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 +/- 1 microM and Hill coefficient nH = 1.76 +/- 0.08. The current-voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = -21 +/- 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl- was linear (with Vrev = 3.2 +/- 0.6 mV). 2. Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60-22%) was approximately twice that found with the medullary neurones (between 40-80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane approximately sevoflurane < halothane approximately isoflurane approximately enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane. 3. For both the recombinant and native receptors, isoflurane potentiated the currents in a dose-dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine-activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration-response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 +/- 5 microM to 84 +/- 7 microM glycine at 610 microM isoflurane, while that for the medullary neurones decreased from a control value of 30 +/- 1 microM to 18 +/- 2 microM glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential. 4. Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 +/- 0.2 to 0.9 +/- 0.1 mM taurine in the presence of 305 microM isoflurane, while the maximum response to taurine increased from 47 +/- 2 to 59 +/- 2% of the maximum response to glycine. 5. Glycine receptors, like other members of the fast ligand-gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.

Mechanisms of general anesthesia.

Although general anesthetics are often said to be nonspecific agents, it is likely that they act at a much more restricted set of target sites than commonly believed. The traditional view has been that the primary targets are lipid portions of nerve membranes, but recent evidence shows that the effects on lipid bilayers of clinically relevant levels of anesthetics are very small. Effects on most proteins are also small, but there are notable examples of proteins that are extremely sensitive to anesthetics and mimic the pharmacological profile of anesthetic target sites in animals. Such target sites are amphiphilic in nature, having both hydrophobic and polar components. The polar components appear to behave as good hydrogen-bond acceptors but poor hydrogen-bond donors. Although the targets can accept molecules with a wide variety of shapes and chemical groupings, they are unaffected by molecules exceeding a certain size. Overall, the data can be explained by supposing that the primary target sites underlying general anesthesia are amphiphilic pockets of circumscribed dimensions on particularly sensitive proteins in the central nervous system.

A simple method for recording single-channel activity from synaptic plasma membranes.

Due to the small size of most nerve terminals, the ion channels which underlie presynaptic currents are usually inaccessible to investigation by conventional electrophysiological techniques. Here we describe a simple method for obtaining single-channel recordings from synaptic plasma membranes that does not require exposure of the native membranes to exogenous lipids or fusogens. To illustrate the method, we have recorded single-channel activity from rat cerebrocortical synaptosomal membranes. Under conditions designed to isolate calcium-independent currents, we describe three channel types that are most commonly observed.