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EFFECTS OF GENERAL ANAESTHETICS ON LIGAND-GATED ION CHANNELS
British Journal of Anaesthesia 1993; 7 1 : 59-64
EFFECTS OF GENERAL ANAESTHETICS ON LIGAND-GATED ION CHANNELS S. DANIELS AND E. B. SMITH
(Br. J. Anaesth. 1993; 71: 59-64) KEY WORDS Anaesthetic action. Electrophysiology: ligand-gated channels. General anaesthesia: enflurane, halothane, isoflurane, propofol.
ACETYLCHOLINE-ACTIVATED ION CHANNELS
The effects of anaesthetics on the acetylcholine receptor have been studied extensively [13,18,23,24,44,48]. The reason for the widespread use of this receptor is that the electric tissue of Torpedo provides a source of receptor-rich membranes and, furthermore, the receptor derived from this tissue is homologous with that at the mammalian neuromuscular junction [2] and with that from the human brain [74]. The large density of acetylcholine receptors in the electric tissue has made it possible to use ion flux techniques to investigate the effect of anaesthetics on the acetylcholine receptor [44,48]. Rapid kinetic studies, in which the flux of a radioactive cation (typically 86Rb+) is measured, have been performed and the results, in the presence and absence of anaesthetics, have been interpreted on the basis of a model of the acetylcholine receptor in which there are two resting states for the receptor, normal and desensitized, and that when two molecules of acetylcholine bind there is rapid transition to an open channel state. Normally, acetylcholinesterase rapidly removes acetylcholine and the channel returns to the resting state. However, there is the possibility that an anaesthetic molecule could block the open channel, preventing return to the resting state. It is assumed that the channel must be unblocked before it can return to the resting state (sequential block model) [48]. All general anaesthetics have been shown to increase the proportion of receptors in the desensitized state. Furthermore, it appears that this action may be mediated by some non-specific membrane perturbation [48]. In addition, most general anaesthetics block the ion channel also, although it is not clear by what mechanism [23]. However, it has been established recently that the site at which general anaesthetics block the channel is different from that at which local anaesthetics bind to block the channel [48]. Electrophysiological measurements of single channel activity, under conditions of patch-clamp, have shown that general anaesthetics cause the channel to flicker, that is openings become more frequent, briefer and appear to be grouped into bursts [18, 24, 50]. If a simple, sequential channel block model of anaesthetic action is assumed, in which the S. DANIELS, M.A., D.PHIL., E. B. SMITH, M.A., PH.D., D.SC, Oxford Hyperbaric Group, Physical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ.
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The site at which anaesthetics act within the central nervous system (CNS) has been the subject of research for almost 100 years. Most success has been obtained in defining the physical nature of the site [70]. For example, the well established correlation of anaesthetic potency to fat solubility indicates that the site of action is hydrophobic. Research using anaesthetics with unusual solubility properties (sulphur hexafluoride and carbon tetrafluoride) failed to provide support for the alternative hypothesis that interaction within the aqueous phase of the CNS was responsible for anaesthesia [45,46]. It is now generally agreed that interaction at a hydrophobic site is involved, but debate continues as to whether or not this is within the membrane lipids or at a hydrophobic region within specific proteins. Evidence for the latter view has been provided by the fact that bacterial and firefly luciferases are sensitive to anaesthetics with potency ratios comparable to those of mammalian anaesthesia [21, 22, 32, 42, 43]. However, there is no evidence for a substance related to the luciferases within the mammalian CNS and for the protein model of general anaesthesia to be advanced such a target site must be identified. General anaesthetics may affect signal transmission by altering action potential propagation or synaptic transmission or, indeed, both. At clinically relevant concentrations, general anaesthetics appear primarily to affect synaptic processes [5, 53, 58-61], although impulse conduction in small unmyelinated fibres is reduced [4]. There is some evidence that anaesthetics may inhibit neurotransmitter release also [56], which if it were to occur at an excitatory synapse could produce the necessary depression of synaptic transmission. Anaesthetics also have effects on the postsynaptic neurotransmitter receptor/ion channel complex. Much of the early work concentrated on the nicotinic acetylcholine receptor as a model for ligand-gated ion channels in general. However, attention is now being devoted to the receptors activated by excitatory amino acids, which probably form the majority of the fast excitatory synapses in the CNS, and to the principal inhibitory receptors, activated by yaminobutyric acid (GABA) and glycine.
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GLUTAMATE-ACTIVATED ION CHANNELS
Glutamate-activated ion channels are obvious targets for general anaesthetics because they are probably the principal neurotransmitter receptors that mediate synaptic excitation in the vertebrate CNS. Glutamate receptors have been categorized, on the basis of their preferred agonist, into a group of three ionotropic (i.e. gated by the neurotransmitter) receptors (NMDA (N-methyl-D-aspartate), KA (kainate) and AMPA (quisqualate)) and two metabotropic (i.e. accessing the metabolic machinery of the cell) G protein coupled subtypes (L-AP4 (2-amino-4phosphonobutyrate) linked to the hydrolysis of cGMP and ACPD (trans-1 -amino-cyclopentane1,3-dicarboxylate) linked to inositol phosphorate diacylglycerol formation). The three ionotropic receptors operate on a fast (ms) time scale, whilst the two G protein coupled receptors operate on a time scale of hundreds of milliseconds to seconds [26]. NMD A receptors show a voltage-dependent Ca2+ permeability with a relatively slow rise time and a decay time of several hundred milliseconds [20,63]. AMPA receptors conduct mainly Na+ currents [14]. The distinction between AMPA and KA receptors is at present unclear. It was made on the basis of discrete high affinity binding sites for these two ligands [15]. However, high affinity binding sites need not necessarily imply a functional receptor/channel complex. Recent DNA cloning experiments have revealed a large number of receptor subtypes [26,73]. At present there are approximately 10 ionotropic receptor subunits known, although not any subunit may assemble with any other to form a functional glutamate-activated channel with distinct properties.
It appears that there may be four distinct AMPA receptors, three KA receptors and one NMDA receptor, together with three high affinity KA subunits which do not form a functional receptor. Further experimental progress is required before the complete categorization of this receptor family may be achieved. In mice, the non-competitive NMDA antagonists MK-801 ((+ )-5-methyl-10,l l-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine), phencyclidine and ketamine increase the potency of the general anaesthetics ethanol, pentobarbitone,halothane and diethyl ether, in parallel with their potency as NMDA antagonists [16]. Ethanol 30 mmol litre"1, a concentration which produces intoxication in man, induces 50% inhibition in NMDA-induced currents in cultured hippocampal neurones but less than 10% inhibition in AMPA- or KA-induced currents [77]. The IC50 values for currents elicited by NMDA for a range of alcohols (methanol, ethanol, 1 -butanol, isopentanol) correlated with both the membrane/buffer partition coefficient and the ED3 for intoxication for these alcohols [77]. Similarly, the volatile anaesthetic diethyl ether, inhibits currents elicited by NMDA but not AMPA or KA [77]. In contrast, pentobarbitone and phenobarbitone do not affect currents elicited by NMDA. However, pentobarbitone inhibits KA and AMPA currents elicited in cultured hippocampal cells (IC50 approximately 50 umol litre"1) [77]. The inhibition was antagonized rapidly and not voltage dependent, suggesting that the block does not occur deep within the channel [77]. Phenobarbitone inhibits similarly KA and AMPA currents elicited in cultured hippocampal cells [77]. The current elicited by KA in Xenopus oocytes, in which glutamate receptors are induced by microinjection with mRNA from rat brain, is also inhibited by phenobarbitone [17]. The inhibition by phenobarbitone of currents elicited in oocytes was not sensitive to pressures up to 135 bar [17]. The steroid anaesthetic alphaxalone had no effect on NMDA-, AMPA- or KA-induced currents [77]. Experiments using cultured hippocampal cells have shown that isoflurane, at concentrations greater than 2 mmol litre"1, induces channel flickering in NMDA receptors, with a decrease in channel open time that is dependent on the dose of anaesthetic [79]. Burst durations also showed a dose-dependent decrease with no decrease in apparent single channel conductance. These actions of isoflurane are different to its actions on the acetylcholine receptor. For the acetylcholine receptor channel, small concentrations of isoflurane produced a dose-dependent decrease in channel open times and the frequency of channel openings increased [12]. In another study using cultured rat hippocampal CA1 neurones, the intermediate chain length nalkanols decreased both the mean channel open time and the probability of opening of single NMDA channels [41]. There was no significant effect on the amplitude of single channel currents or channel conductance. No evidence for channel block (flickering) was observed.
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anaesthetic may only bind to the open channel and the flickering is caused by the open channel being repeatedly blocked, it follows that the mean burst duration should increase linearly with increasing concentration of anaesthetic. Experimental evidence indicates that this does not occur, rather the bursts get shorter with increasing concentration [18]. To account for this it has been proposed that the open blocked state can be terminated by the closing of the blocked channel. This extension to the simple sequential block model of the mode of action does predict correctly the concentration-dependent behaviour of the burst duration and the number of openings/burst [18, 50]. However, even this extended model for the interaction of anaesthetics and the nicotinic acetylcholine receptor cannot be complete. For example, the frequency of bursts increases in the presence of anaesthetics [18]. It is not clear at present if this occurs as a result of changes in acetylcholine binding or as a result of changes in the rate of induced conformational change. Finally, anaesthetics accelerate the fast desensitization process that occurs in the presence of high concentrations of acetylcholine [18]. This implies that the model will have to incorporate modified desensitization rates or, alternatively, additional desensitized states.
EFFECTS OF ANAESTHETICS ON LIGAND-GATED ION CHANNELS GABA-ACTIVATED ION CHANNELS (see also Goodchild
[27])
tization processes or to differential effects on different receptor subtypes [80] awaits clarification. GLYCINE-ACTIVATED ION CHANNELS
Glycine is the postsynaptic inhibitory neurotransmitter in the brain stem and spinal cord. The chloride channel activated by glycine has similar conductance properties to that activated by GABA [6, 7]. The glycine receptor has been isolated from mammalian CNS tissue and shown to comprise two integral membrane proteins (a and P) and a peripheral membrane protein [3, 28, 55]. It has been suggested that the channel core is a pentameric arrangement of three a and two P subunits [37]. This subunit arrangement closely resembles that of die nicotinic acetylcholine receptor [75]. As with other ligandgated receptors, recent evidence from DNA sequencing experiments have indicated considerable heterogeneity in die glycine receptor subunits. To date, four variants of the a subunit have been identified but no variants of the P subunit have been identified [29, 35, 36]. In contrast to acetylcholine, glutamate and GABA receptors, relatively little work has been performed on the effect of anaesdietics on glycine receptors. It has been shown, using whole cell patch recording from cultured embryonic spinal neurones, that glycine-evoked currents were potentiated by propofol and chlormethiazole [30,31], but not by pentobarbitone [30, 31] or alphaxalone [33]. Preliminary experiments recording whole cell currents from voltage clamped oocytes microinjected with mRNA derived from rat spinal cord have indicated that the strychnine-sensitive glycine-evoked current is inhibited by pentobarbitone [unpublished observations] . CONCLUSIONS
It is becoming increasingly apparent that the nicotinic acetylcholine, glutamate, GABAA and glycine receptors are members of a ligand-gated receptor super family that evolved from a common ancestor [35, 66]. The transmembrane segments in particular appear to show significant amino acid sequence homology and topology, of four hydrophobic a helices. However, it is also clear diat anaesthetics affect members of this receptor super family in different ways and that to use the nicotinic acetylcholine receptor as a model for which the interaction of anaesthetics with ligand-gated receptors in general can be elucidated is quite wrong. Anaesthetics appear to act at the nicotinic acetylcholine receptor to block the channel, whereas not all anaesthetics block glutamate channels and the action at the GABAA receptor appears to be an allosteric potentiation of GABA binding and, for some anaesthetics, an effect on die gating properties. That large pressures (of the order of 10 MPa) will antagonize general anaesthesia [38] led to a theory of general anaesthesia that assumed that anaesthetics and pressure acted at a common site and via a common mechanism [47]. More recently it was observed that species diat do not use glycine as a neurotransmitter do not exhibit pressure reversal of
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GABA receptors are distributed widely diroughout the CNS and are responsible for both pre- and postsynaptic inhibition. GABA receptors have traditionally been divided into ionotropic GABAA and metabotropic GABAB subtypes. GABAA receptors are activated by muscimol, inactivated by bicuculline and sensitive to benzodiazepines, whereas GABAB receptors are activated by baclofen, inactivated by phacolfen and unaffected by benzodiazepines. GABAA receptors conduct Cl~ ions [1,6, 62] whereas the GABAB receptor is coupled to Ca2+ channels, K+ channels and G proteins [9, 19, 34, 51, 78]. Recently, the GABAA receptor has been separated into a number of subtypes, based on the multiple
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anaesthesia [72] and that pressure, far from acting in a non-specific manner, appears to act in a highly selective way [10,11,71]. At the level of the neurotransmitter receptor-ion channel complex, it has been shown that the kainate channel is unaffected by pressure but that the glycine channel is highly pressure sensitive [16, 69]. However, this does not allow postulation that pressure and anaesthetics act via the same mechanism and the observed interaction could arise through a balance between inhibition and excitation in the CNS. Given that the mechanisms that induce anaesthesia are not understood, it is difficult to establish if the effects observed in in vitro systems are related to general anaesthesia in animals. One important requirement is that a wide range of molecular structures should produce similar effects with potency ratios comparable to those observed in vivo. For glutamate, GABAA and glycine receptors, such behaviour is not observed. The simple anaesthetics (alcohols and volatile agents) affect NMDA receptors, whereas barbiturates affect AMPA and KA receptors and steroid anaesthetics do not appear to have any effect on glutamate receptors. Barbiturates have a discrete binding site at GABAA receptors and affect allosterically GABA binding. Steroid anaesthetics appear to act similarly but utilize a different binding site. Volatile anaesthetics not only affect GABA binding but also gating properties. At the glycine receptor, some anaesthetics appear to potentiate glycine binding whilst others are either ineffective or even act to inhibit, either by channel blocking or by enhancing desensitization. Furthermore, it is possible that when it becomes feasible to experiment with specific receptor subtypes, differences in the action of anaesthetic on the different subtypes may be observed. It appears that anaesthetics act differently on ligand-gated ion channels and that therefore the interaction between general anaesthetics and the ligand-gated ion channels studied so far cannot provide a basis for a general mechanism of anaesthesia. However, if we assume that different anaesthetics, or classes of anaesthetics, act via different mechanisms, the difficulty arises of identifying which of the different effects are responsible for general anaesthesia. Thus barbiturates block nicotinic acetylcholine channels, potentiate GABA binding, block KA and AMPA receptors and inhibit glycine receptors. All of these effects, apart from the effect on the glycine receptor, could be responsible for the induced loss of consciousness.
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EFFECTS OF GENERAL ANAESTHETICS ON LIGAND-GATED ION CHANNELS S. DANIELS AND E. B. SMITH
(Br. J. Anaesth. 1993; 71: 59-64) KEY WORDS Anaesthetic action. Electrophysiology: ligand-gated channels. General anaesthesia: enflurane, halothane, isoflurane, propofol.
ACETYLCHOLINE-ACTIVATED ION CHANNELS
The effects of anaesthetics on the acetylcholine receptor have been studied extensively [13,18,23,24,44,48]. The reason for the widespread use of this receptor is that the electric tissue of Torpedo provides a source of receptor-rich membranes and, furthermore, the receptor derived from this tissue is homologous with that at the mammalian neuromuscular junction [2] and with that from the human brain [74]. The large density of acetylcholine receptors in the electric tissue has made it possible to use ion flux techniques to investigate the effect of anaesthetics on the acetylcholine receptor [44,48]. Rapid kinetic studies, in which the flux of a radioactive cation (typically 86Rb+) is measured, have been performed and the results, in the presence and absence of anaesthetics, have been interpreted on the basis of a model of the acetylcholine receptor in which there are two resting states for the receptor, normal and desensitized, and that when two molecules of acetylcholine bind there is rapid transition to an open channel state. Normally, acetylcholinesterase rapidly removes acetylcholine and the channel returns to the resting state. However, there is the possibility that an anaesthetic molecule could block the open channel, preventing return to the resting state. It is assumed that the channel must be unblocked before it can return to the resting state (sequential block model) [48]. All general anaesthetics have been shown to increase the proportion of receptors in the desensitized state. Furthermore, it appears that this action may be mediated by some non-specific membrane perturbation [48]. In addition, most general anaesthetics block the ion channel also, although it is not clear by what mechanism [23]. However, it has been established recently that the site at which general anaesthetics block the channel is different from that at which local anaesthetics bind to block the channel [48]. Electrophysiological measurements of single channel activity, under conditions of patch-clamp, have shown that general anaesthetics cause the channel to flicker, that is openings become more frequent, briefer and appear to be grouped into bursts [18, 24, 50]. If a simple, sequential channel block model of anaesthetic action is assumed, in which the S. DANIELS, M.A., D.PHIL., E. B. SMITH, M.A., PH.D., D.SC, Oxford Hyperbaric Group, Physical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ.
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The site at which anaesthetics act within the central nervous system (CNS) has been the subject of research for almost 100 years. Most success has been obtained in defining the physical nature of the site [70]. For example, the well established correlation of anaesthetic potency to fat solubility indicates that the site of action is hydrophobic. Research using anaesthetics with unusual solubility properties (sulphur hexafluoride and carbon tetrafluoride) failed to provide support for the alternative hypothesis that interaction within the aqueous phase of the CNS was responsible for anaesthesia [45,46]. It is now generally agreed that interaction at a hydrophobic site is involved, but debate continues as to whether or not this is within the membrane lipids or at a hydrophobic region within specific proteins. Evidence for the latter view has been provided by the fact that bacterial and firefly luciferases are sensitive to anaesthetics with potency ratios comparable to those of mammalian anaesthesia [21, 22, 32, 42, 43]. However, there is no evidence for a substance related to the luciferases within the mammalian CNS and for the protein model of general anaesthesia to be advanced such a target site must be identified. General anaesthetics may affect signal transmission by altering action potential propagation or synaptic transmission or, indeed, both. At clinically relevant concentrations, general anaesthetics appear primarily to affect synaptic processes [5, 53, 58-61], although impulse conduction in small unmyelinated fibres is reduced [4]. There is some evidence that anaesthetics may inhibit neurotransmitter release also [56], which if it were to occur at an excitatory synapse could produce the necessary depression of synaptic transmission. Anaesthetics also have effects on the postsynaptic neurotransmitter receptor/ion channel complex. Much of the early work concentrated on the nicotinic acetylcholine receptor as a model for ligand-gated ion channels in general. However, attention is now being devoted to the receptors activated by excitatory amino acids, which probably form the majority of the fast excitatory synapses in the CNS, and to the principal inhibitory receptors, activated by yaminobutyric acid (GABA) and glycine.
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GLUTAMATE-ACTIVATED ION CHANNELS
Glutamate-activated ion channels are obvious targets for general anaesthetics because they are probably the principal neurotransmitter receptors that mediate synaptic excitation in the vertebrate CNS. Glutamate receptors have been categorized, on the basis of their preferred agonist, into a group of three ionotropic (i.e. gated by the neurotransmitter) receptors (NMDA (N-methyl-D-aspartate), KA (kainate) and AMPA (quisqualate)) and two metabotropic (i.e. accessing the metabolic machinery of the cell) G protein coupled subtypes (L-AP4 (2-amino-4phosphonobutyrate) linked to the hydrolysis of cGMP and ACPD (trans-1 -amino-cyclopentane1,3-dicarboxylate) linked to inositol phosphorate diacylglycerol formation). The three ionotropic receptors operate on a fast (ms) time scale, whilst the two G protein coupled receptors operate on a time scale of hundreds of milliseconds to seconds [26]. NMD A receptors show a voltage-dependent Ca2+ permeability with a relatively slow rise time and a decay time of several hundred milliseconds [20,63]. AMPA receptors conduct mainly Na+ currents [14]. The distinction between AMPA and KA receptors is at present unclear. It was made on the basis of discrete high affinity binding sites for these two ligands [15]. However, high affinity binding sites need not necessarily imply a functional receptor/channel complex. Recent DNA cloning experiments have revealed a large number of receptor subtypes [26,73]. At present there are approximately 10 ionotropic receptor subunits known, although not any subunit may assemble with any other to form a functional glutamate-activated channel with distinct properties.
It appears that there may be four distinct AMPA receptors, three KA receptors and one NMDA receptor, together with three high affinity KA subunits which do not form a functional receptor. Further experimental progress is required before the complete categorization of this receptor family may be achieved. In mice, the non-competitive NMDA antagonists MK-801 ((+ )-5-methyl-10,l l-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine), phencyclidine and ketamine increase the potency of the general anaesthetics ethanol, pentobarbitone,halothane and diethyl ether, in parallel with their potency as NMDA antagonists [16]. Ethanol 30 mmol litre"1, a concentration which produces intoxication in man, induces 50% inhibition in NMDA-induced currents in cultured hippocampal neurones but less than 10% inhibition in AMPA- or KA-induced currents [77]. The IC50 values for currents elicited by NMDA for a range of alcohols (methanol, ethanol, 1 -butanol, isopentanol) correlated with both the membrane/buffer partition coefficient and the ED3 for intoxication for these alcohols [77]. Similarly, the volatile anaesthetic diethyl ether, inhibits currents elicited by NMDA but not AMPA or KA [77]. In contrast, pentobarbitone and phenobarbitone do not affect currents elicited by NMDA. However, pentobarbitone inhibits KA and AMPA currents elicited in cultured hippocampal cells (IC50 approximately 50 umol litre"1) [77]. The inhibition was antagonized rapidly and not voltage dependent, suggesting that the block does not occur deep within the channel [77]. Phenobarbitone inhibits similarly KA and AMPA currents elicited in cultured hippocampal cells [77]. The current elicited by KA in Xenopus oocytes, in which glutamate receptors are induced by microinjection with mRNA from rat brain, is also inhibited by phenobarbitone [17]. The inhibition by phenobarbitone of currents elicited in oocytes was not sensitive to pressures up to 135 bar [17]. The steroid anaesthetic alphaxalone had no effect on NMDA-, AMPA- or KA-induced currents [77]. Experiments using cultured hippocampal cells have shown that isoflurane, at concentrations greater than 2 mmol litre"1, induces channel flickering in NMDA receptors, with a decrease in channel open time that is dependent on the dose of anaesthetic [79]. Burst durations also showed a dose-dependent decrease with no decrease in apparent single channel conductance. These actions of isoflurane are different to its actions on the acetylcholine receptor. For the acetylcholine receptor channel, small concentrations of isoflurane produced a dose-dependent decrease in channel open times and the frequency of channel openings increased [12]. In another study using cultured rat hippocampal CA1 neurones, the intermediate chain length nalkanols decreased both the mean channel open time and the probability of opening of single NMDA channels [41]. There was no significant effect on the amplitude of single channel currents or channel conductance. No evidence for channel block (flickering) was observed.
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anaesthetic may only bind to the open channel and the flickering is caused by the open channel being repeatedly blocked, it follows that the mean burst duration should increase linearly with increasing concentration of anaesthetic. Experimental evidence indicates that this does not occur, rather the bursts get shorter with increasing concentration [18]. To account for this it has been proposed that the open blocked state can be terminated by the closing of the blocked channel. This extension to the simple sequential block model of the mode of action does predict correctly the concentration-dependent behaviour of the burst duration and the number of openings/burst [18, 50]. However, even this extended model for the interaction of anaesthetics and the nicotinic acetylcholine receptor cannot be complete. For example, the frequency of bursts increases in the presence of anaesthetics [18]. It is not clear at present if this occurs as a result of changes in acetylcholine binding or as a result of changes in the rate of induced conformational change. Finally, anaesthetics accelerate the fast desensitization process that occurs in the presence of high concentrations of acetylcholine [18]. This implies that the model will have to incorporate modified desensitization rates or, alternatively, additional desensitized states.
EFFECTS OF ANAESTHETICS ON LIGAND-GATED ION CHANNELS GABA-ACTIVATED ION CHANNELS (see also Goodchild
[27])
tization processes or to differential effects on different receptor subtypes [80] awaits clarification. GLYCINE-ACTIVATED ION CHANNELS
Glycine is the postsynaptic inhibitory neurotransmitter in the brain stem and spinal cord. The chloride channel activated by glycine has similar conductance properties to that activated by GABA [6, 7]. The glycine receptor has been isolated from mammalian CNS tissue and shown to comprise two integral membrane proteins (a and P) and a peripheral membrane protein [3, 28, 55]. It has been suggested that the channel core is a pentameric arrangement of three a and two P subunits [37]. This subunit arrangement closely resembles that of die nicotinic acetylcholine receptor [75]. As with other ligandgated receptors, recent evidence from DNA sequencing experiments have indicated considerable heterogeneity in die glycine receptor subunits. To date, four variants of the a subunit have been identified but no variants of the P subunit have been identified [29, 35, 36]. In contrast to acetylcholine, glutamate and GABA receptors, relatively little work has been performed on the effect of anaesdietics on glycine receptors. It has been shown, using whole cell patch recording from cultured embryonic spinal neurones, that glycine-evoked currents were potentiated by propofol and chlormethiazole [30,31], but not by pentobarbitone [30, 31] or alphaxalone [33]. Preliminary experiments recording whole cell currents from voltage clamped oocytes microinjected with mRNA derived from rat spinal cord have indicated that the strychnine-sensitive glycine-evoked current is inhibited by pentobarbitone [unpublished observations] . CONCLUSIONS
It is becoming increasingly apparent that the nicotinic acetylcholine, glutamate, GABAA and glycine receptors are members of a ligand-gated receptor super family that evolved from a common ancestor [35, 66]. The transmembrane segments in particular appear to show significant amino acid sequence homology and topology, of four hydrophobic a helices. However, it is also clear diat anaesthetics affect members of this receptor super family in different ways and that to use the nicotinic acetylcholine receptor as a model for which the interaction of anaesthetics with ligand-gated receptors in general can be elucidated is quite wrong. Anaesthetics appear to act at the nicotinic acetylcholine receptor to block the channel, whereas not all anaesthetics block glutamate channels and the action at the GABAA receptor appears to be an allosteric potentiation of GABA binding and, for some anaesthetics, an effect on die gating properties. That large pressures (of the order of 10 MPa) will antagonize general anaesthesia [38] led to a theory of general anaesthesia that assumed that anaesthetics and pressure acted at a common site and via a common mechanism [47]. More recently it was observed that species diat do not use glycine as a neurotransmitter do not exhibit pressure reversal of
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GABA receptors are distributed widely diroughout the CNS and are responsible for both pre- and postsynaptic inhibition. GABA receptors have traditionally been divided into ionotropic GABAA and metabotropic GABAB subtypes. GABAA receptors are activated by muscimol, inactivated by bicuculline and sensitive to benzodiazepines, whereas GABAB receptors are activated by baclofen, inactivated by phacolfen and unaffected by benzodiazepines. GABAA receptors conduct Cl~ ions [1,6, 62] whereas the GABAB receptor is coupled to Ca2+ channels, K+ channels and G proteins [9, 19, 34, 51, 78]. Recently, the GABAA receptor has been separated into a number of subtypes, based on the multiple
61
62
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