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The GABAA receptor: An important target for the general anaesthetic etomidate
International Congress Series 1283 (2005) 67 – 72
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The GABAA receptor: An important target for the general anaesthetic etomidate J.J. Lambert a,*, A.R. Haythornthwaite a, D.R. Peden a, M.B. Herd a, K.A. Wafford b, T.W. Rosahl b, D. Belelli a a
b
Neuroscience Institute, Division of Pathology and Neuroscience, Dundee University, Ninewells Hospital and Medical School, Dundee DD19SY, Scotland, UK Merck Sharp and Dohme Research Laboratories, The Neuroscience Research Centre, Harlow, Essex CM20 2QR, UK
Abstract. The general anaesthetic etomidate potently and selectively enhances the function of the GABAA receptor. Furthermore, etomidate is selective for GABAA receptors that incorporate a h2 or a h3 subunit, but is relatively ineffective at equivalent receptors containing the h1 subunit. This selectivity is governed by the nature of a single amino acid residue (asparagine, N for h2 and h3; serine, S for h1). Utilising this knowledge gene targeting was used to create a mouse in which the h2 subunit was engineered to be relatively insensitive to etomidate (h2N265S). In Purkinje neurones derived from such mice, the effects of etomidate on inhibitory synaptic transmission are greatly reduced. In complementary behavioural experiments, the mutation influences both the sedative and hypnotic actions of etomidate. D 2005 Published by Elsevier B.V. Keywords: GABA; GABAA receptor; Etomidate; General anaesthetic; Sedation
1. Introduction The molecular target(s) that mediate the various behaviours induced by general anaesthetics have proved elusive. General anaesthetics are structurally diverse and can influence the function of numerous proteins that mediate or influence neuronal signalling within the central nervous system (CNS). However, actions at behaviourally relevant anaesthetic concentrations involving physiologically and anatomically plausible targets are more restricted. Applying these criteria, receptors of the transmitter gated ion channel * Corresponding author. Tel.: +44 1382 632161; fax: +44 1382 667120. E-mail address: [email protected] (J.J. Lambert). 0531-5131/ D 2005 Published by Elsevier B.V. doi:10.1016/j.ics.2005.07.071
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family emerge as probable targets for certain intravenous (i.v.) general anaesthetics. Of particular interest in this regard is the i.v. anaesthetic etomidate, which at behaviourally relevant concentrations enhances the interaction of GABA acting at the GABAA receptor but has little or no effect on a variety of other transmitter-gated ion channels including the strychnine-sensitive glycine, neuronal nicotinic, 5-HT3, AMPA and NMDA receptor [1]. The GABA-enhancing and the anaesthetic effects of etomidate exhibit the same enantioselectivity, further implicating the GABAA receptor, and suggests the presence of a specific etomidate binding site on this protein [1,2]. GABAA receptors are a viable target as they mediate the majority of brapidQ neuronal inhibition in the mammalian brain. GABAA receptors are composed of five membrane crossing subunits drawn from a repertoire (a1–6, h1–3, g1–3, y, q, k, u, U1–3) that underpins the expression in the mammalian CNS of ~ 20–30 GABAA receptor isoforms. Such receptor subtypes are heterogeneously expressed in the CNS and exhibit distinct physiological and pharmacological properties [3]. Therefore, if GABAA receptors mediate the behavioural actions of the general anaesthetic etomidate, the question arises as to which receptor isoforms are important and whether the repertoire of behaviours induced by this anaesthetic are mediated by distinct receptor subtypes. 2. Materials and methods 2.1. Xenopus laevis oocytes Oocytes were isolated and prepared as previously described [1]. Such oocytes were injected with cRNA encoding for human GABAA receptor subunits and used for electrophysiological experiments 2–12 days post-injection. The preparation of the human wild type (WT) and mutant GABAA subunit cDNAs and the synthesis of the corresponding cRNAs has been previously described [4,5]. GABA-evoked currents were recorded from RNA-injected oocytes with a two-electrode voltage-clamp at a holding potential of 60 mV [1]. During recording oocytes were continuously perfused with a standard buffered salt solution and both etomidate and GABA were introduced via the perfusate [1]. The GABA-modulatory activity of etomidate was examined against a concentration of GABA that induces a response 10% of the maximal (EC10). Etomidate concentration response relationships were determined, subsequently fitted with a logistical equation and the EC50 (the concentration of etomidate required to produce a GABA-modulatory response 50% of the anaesthetic maximal response) and the E MAX (the maximal GABA-modulatory response induced by etomidate, expressed as a percentage of the response produced by a maximal concentration of GABA) were derived [1]. All experiments reported here are the mean F S.E.M. of 3–5 experiments. 2.1.1. Cerebellar slice recordings Cerebellar slices were prepared from WT or h2N265S mice of either sex (postnatal days 16–25) according to standard protocols [6]. Whole-cell patch-clamp recordings were made at 35 8C from submerged Purkinje neurons in a standard extracellular recording solution containing 2 mM kynurenic acid and 0.5 AM tetrodotoxin to block ionotropic glutamate receptors and action potentials, respectively. For further details of the recording conditions,
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see [6]. The GABA-mediated mIPSC frequency, amplitude, rise time and decay time (the weighted time constant of decay, s W) were determined before and after 10 AM etomidate [6]. All results reported here are from a minimum of 5 neurons.
3. Results Utilising the Xenopus laevis oocyte expression system etomidate produced a concentration dependent (30 nM–10 AM) enhancement of the current mediated by a non-saturating concentration of GABA (EC10) acting at a6h3g2 receptors [5]. Analysis of the etomidate concentration response relationship revealed the GABA-modulatory action of etomidate to be both potent (EC50 = 0.7 F 0.06 AM) and efficacious (E MAX = 135 F 7%; i.e. a ~ 12.5-fold increase) (Fig. 1). Similar results were obtained for a6h2g2 receptors [4]—see Fig. 1. By contrast, etomidate was much less effective at a6h1g2 receptors (EC50 = 7.4 F 0.6 AM; E MAX = 28 F 2%)—see [4,5] and Fig. 1. Therefore, the coexpression of either the h3 or the h2 subunit, with the a6 and g2 subunit greatly favours the GABAmodulatory actions of etomidate in comparison to the equivalent receptors expressing the h1 subunit. Etomidate is structurally related to loreclezole and the GABA-modulatory actions of this anticonvulsant, in common with etomidate, are h subunit selective, a preference governed by the nature of a single amino acid residue (asparagine, N for h2 and h3 subunits and serine, S for the h1 subunit) located towards the extracellular portion of the second transmembrane (TM2) domain of the protein [7]. In agreement, the GABA-modulatory actions of etomidate were greatly reduced by mutation of this key asparagine residue to serine (etomidate EC50 = 5.7 F 2.6 AM; E MAX = 36 F 1% for 1)
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Fig. 1. The GABA-enhancing actions of etomidate are influenced by the nature of a single amino acid residue on the GABAA h subunit. (1) The histogram gives the maximal enhancement (E MAX expressed as a percentage of the maximal response to a saturating concentration of GABA) by etomidate of the GABA (EC10)-evoked response mediated by a6hXg2 GABAA receptors (where X, the nature of the h subunit is specified in the figure) expressed in oocytes. Data obtained are the mean F S.E.M. of 3–5 experiments. (2) Representative traces illustrating the effect of 10 AM etomidate on averaged mIPSCs recorded from wild type and h2N265S Purkinje neurones. (3) The effect of etomidate to prolong the decay (s W) of mIPSCs recorded from wild type and h2N265S Purkinje neurones. In each case the data represent the mean F S.E.M. obtained from 5 neurones.
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a6h3N265Sg2 receptors)—see [5] and Fig. 1. Correspondingly, the GABA enhancing actions of this anaesthetic were facilitated by the mutation of the homologous serine residue of the h1 subunit to asparagine (etomidate EC50 = 1.6 F .3 AM; E MAX = 150 F 13%; for a6h1S265Ng2 receptors)—see [5] and Fig. 1. Based on these findings, gene targeting was used to generate a mouse containing the equivalent asparagine to serine mutation in the h2 subunit (h2N265S)—see [6]. Such mice were used to investigate the role of GABAA receptors in mediating the behavioural actions of etomidate (i.e. in these mice etomidate is presumed to selectively interact with GABAA receptors containing the h3 subunit). However, the properties of recombinant receptors expressed in a foreign host cell (i.e. oocytes) may not faithfully reproduce those of native receptors expressed in neurones. Therefore, we used the whole-cell clamp technique and cerebellar brain slices to compare the actions of etomidate on miniature inhibitory postsynaptic currents (mIPSCs) recorded from Purkinje neurones of wild type (WT) and mutant (h2N265S) mice. Such mIPSCs result from the activation of synaptic GABAA receptors by quantally released GABA. By perturbing the channel kinetics of the GABA-gated ion channels, positive allosteric modulators such as etomidate act primarily to prolong the mIPSC decay. Purkinje neurons were chosen as they are reported to express the h2 subunit together with only a limited repertoire of other GABAA receptor subunits [8]. Importantly, a comparison of the properties (frequency, amplitude, rise and decay times) of mIPSCs recorded from Purkinje neurons derived from WT and mutant mice revealed the mutation to have no effect on any of these parameters (i.e. the mutation was functionally silent). However, the effects of etomidate were greatly suppressed. Application of 10 AM etomidate to brain slices of WT mice caused a marked prolongation of the mIPSC decay (s W of 2.8 F 0.2 ms compared with 29.2 F 5.8 ms) of Purkinje neurones. By contrast, application of this concentration of anaesthetic to brain slices derived from mutant mice produced only a modest prolongation of the mIPSC (s W = 2.4 F 0.1 ms compared with 7.3 F 0.3 ms)—see [6] and Fig. 1.
4. Discussion Many general anaesthetics are relatively low in potency and at similar concentrations influence the function of a variety of receptors and ion channels. These features have confounded attempts to define the important molecular target(s) that mediate the behavioural repertoire induced by general anaesthetics. In this regard, the i.v. general anaesthetic etomidate is unusual and of value. Etomidate at relatively low concentrations enhances the function of the major inhibitory receptor in the mammalian CNS, the GABAA receptor. Furthermore, this anaesthetic appears to be relatively selective, having little or no activity across other representative members of the transmitter-gated ion channel family [1]. The case for an involvement of GABAA receptors in the behavioural actions of etomidate is strengthened by studies utilising the enantiomers of this anaesthetic. Behaviourally, the R-(+) enantiomer of etomidate is ~ 10-fold more potent than the S-( ) form in producing a loss of the righting reflex (LORR) in mice and tadpoles [1,2]. In correspondence this behavioural selectivity is mirrored by the GABA-modulatory properties of the anaesthetic [1,2]. Of particular importance is the observation that the GABA-modulatory actions of etomidate are dependent upon the subunit composition of the receptor [4,5]. In particular, receptors incorporating the h2 or the h3 subunit are highly sensitive to etomidate, whereas this anaesthetic has a much more limited action at equivalent receptors containing the h1
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subunit—see Fig. 1 [4,5]. Furthermore, this subunit selectivity resides exclusively with the nature of a single amino acid (S for h1; N for h2 and h3 subunits), see [5]. This observation provided the impetus to use gene targeting to generate mice carrying a point mutation of the h2 asparagine to serine residue [6]. Purkinje neurones are known to express the h2 subunit [8]. In agreement, etomidate greatly prolonged the decay of GABA-mediated mIPSCs recorded from these neurons and this effect was greatly reduced by the h2 subunit mutation—see Fig. 1 [6]. The use of such mice, together with subtype selective drugs such as etomidate are invaluable in deciphering the identity of GABAA receptor isoforms expressed in neuronal circuits implicated in the effects of general anaesthetics. In this regard we are currently employing this approach to characterise inhibitory transmission in the thalamocortical circuitry [9]. In addition, this genetic approach permits the role of GABAA receptor subtypes in the repertoire of behaviours induced by general anaesthetics to be explored. Given the limited sensitivity of h1 and in the mutant mice h2 subunit containing receptors to etomidate, in the bknock inQ mouse this anaesthetic will mainly act at receptors incorporating the h3 subunit. Importantly, our experiments on Purkinje neurones demonstrate the h2 subunit mutation to be silent (being revealed only in the altered response to etomidate), and in agreement these mice do not have a distinctive phenotype, observations that collectively legitimise this model [6]. However, the behavioural effects of etomidate are considerably altered. In particular, the sedative effects of low (non-anaesthetic) doses of etomidate were absent in the h2N265S mice, and the duration of the loss of the righting reflex induced by this anaesthetic was reduced [6]. We had previously demonstrated that the Drosophila GABA receptor RDL is insensitive to etomidate but that mutation of the homologous TM2 residue (M: methionine) to asparagine uncovered a GABA-modulatory effect of this anaesthetic [10]. Reciprocally, we found that exchange of the N to M residue of the h3 subunit completely suppressed the GABA-modulatory actions of etomidate and additionally those of propofol [10,11]. These findings were developed by a second group to generate a mouse with a h3 subunit that carried this mutation (h3N265M)—see [12]. In these mice, the duration of the LORR induced by either propofol or etomidate, was greatly reduced and the suppression of the hind limb withdrawal reflex (immobilizing action) induced by these anaesthetics was almost abolished [12]. Collectively, the results obtained with the h2 and h3 mutant mice suggest the immobilizing action of etomidate and propofol are mediated by h3 subunit containing receptors, the sedative action of etomidate involves GABAA receptors incorporating the h2 subunit, whereas the hypnotic actions of these anaesthetics require the participation of both h2 and h3 subunit containing receptors. For etomidate and propofol it is now evident that the GABAA receptor is an important target. Furthermore, the results obtained from bknock inQ mice reveal that different components of the repertoire of behaviours induced by general anaesthetics are mediated by distinct GABAA receptor isoforms. These findings should encourage the future development of GABAA receptor subtype selective anaesthetics, offering hopefully an improved side effect profile. More fundamentally, the future development of mice carrying the h subunit mutations described here, but expressed only in specific neuronal populations, should permit the anatomical locus of the dramatic behavioural effects induced by general anaesthetics to finally be revealed.
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Acknowledgements Part of this work was supported by a BBSRC CASE award with Merck Sharp and Dohme and a BBSRC Project Grant. References [1] D. Belelli, et al., The in vitro and in vivo enantioselectivity of etomidate implicates the GABAA receptor in general anaesthesia, Neuropharmacology 45 (2003) 57 – 71. [2] S.L. Tomlin, et al., Stereoselective effects of etomidate optical isomers on gamma-aminobutyric acid type A receptors and animals, Anesthesiology 88 (1998) 708 – 717. [3] H. Mohler, et al., Specific GABAA circuits in brain development and therapy, Biochem. Pharmacol. 68 (2004) 1685 – 1690. [4] C. Hill-Venning, et al., Subunit-dependent interaction of the general anaesthetic etomidate with the g-aminobutyric acid type A receptor, Br. J. Pharmacol. 120 (1997) 749 – 756. [5] D. Belelli, et al., The interaction of the general anesthetic etomidate with the g-aminobutyric acid type A receptor is influenced by a single amino acid, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 11031 – 11036. [6] D.S. Reynolds, et al., Sedation and anesthesia mediated by distinct GABAA receptor isoforms, J. Neurosci. 23 (2003) 8608 – 8617. [7] P.B. Wingrove, et al., The modulatory action of loreclezole at the g-aminobutyric acid type A receptor is determined by a single amino acid in the h2 and h3 subunit, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4569 – 4573. [8] D.J. Laurie, et al., The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain: III. Embryonic and postnatal development, J. Neurosci. 12 (1992) 4151 – 4172. [9] D. Belelli, et al., Etomidate modulation of GABAA receptor mediated synaptic transmission in the thalamus of h2N265S knock-in mice, Abstr.-Soc. Neurosci. (2003) 571.7. [10] K.A. McGurk, et al., The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor, Br. J. Pharmacol. 124 (1998) 13 – 20. [11] M. Pistis, et al., Complementary regulation of anaesthetic activation of human (a6h3g2L) and Drosophila (RDL) GABA receptors by a single amino acid residue, J. Physiol. (Lond.) 515 (Pt. 1) (1999) 3 – 18. [12] U. Rudolph, B. Antkowiak, Molecular and neuronal substrates for general anaesthetics, Nat. Rev., Neurosci. 5 (2004) 709 – 720.
www.ics-elsevier.com
The GABAA receptor: An important target for the general anaesthetic etomidate J.J. Lambert a,*, A.R. Haythornthwaite a, D.R. Peden a, M.B. Herd a, K.A. Wafford b, T.W. Rosahl b, D. Belelli a a
b
Neuroscience Institute, Division of Pathology and Neuroscience, Dundee University, Ninewells Hospital and Medical School, Dundee DD19SY, Scotland, UK Merck Sharp and Dohme Research Laboratories, The Neuroscience Research Centre, Harlow, Essex CM20 2QR, UK
Abstract. The general anaesthetic etomidate potently and selectively enhances the function of the GABAA receptor. Furthermore, etomidate is selective for GABAA receptors that incorporate a h2 or a h3 subunit, but is relatively ineffective at equivalent receptors containing the h1 subunit. This selectivity is governed by the nature of a single amino acid residue (asparagine, N for h2 and h3; serine, S for h1). Utilising this knowledge gene targeting was used to create a mouse in which the h2 subunit was engineered to be relatively insensitive to etomidate (h2N265S). In Purkinje neurones derived from such mice, the effects of etomidate on inhibitory synaptic transmission are greatly reduced. In complementary behavioural experiments, the mutation influences both the sedative and hypnotic actions of etomidate. D 2005 Published by Elsevier B.V. Keywords: GABA; GABAA receptor; Etomidate; General anaesthetic; Sedation
1. Introduction The molecular target(s) that mediate the various behaviours induced by general anaesthetics have proved elusive. General anaesthetics are structurally diverse and can influence the function of numerous proteins that mediate or influence neuronal signalling within the central nervous system (CNS). However, actions at behaviourally relevant anaesthetic concentrations involving physiologically and anatomically plausible targets are more restricted. Applying these criteria, receptors of the transmitter gated ion channel * Corresponding author. Tel.: +44 1382 632161; fax: +44 1382 667120. E-mail address: [email protected] (J.J. Lambert). 0531-5131/ D 2005 Published by Elsevier B.V. doi:10.1016/j.ics.2005.07.071
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family emerge as probable targets for certain intravenous (i.v.) general anaesthetics. Of particular interest in this regard is the i.v. anaesthetic etomidate, which at behaviourally relevant concentrations enhances the interaction of GABA acting at the GABAA receptor but has little or no effect on a variety of other transmitter-gated ion channels including the strychnine-sensitive glycine, neuronal nicotinic, 5-HT3, AMPA and NMDA receptor [1]. The GABA-enhancing and the anaesthetic effects of etomidate exhibit the same enantioselectivity, further implicating the GABAA receptor, and suggests the presence of a specific etomidate binding site on this protein [1,2]. GABAA receptors are a viable target as they mediate the majority of brapidQ neuronal inhibition in the mammalian brain. GABAA receptors are composed of five membrane crossing subunits drawn from a repertoire (a1–6, h1–3, g1–3, y, q, k, u, U1–3) that underpins the expression in the mammalian CNS of ~ 20–30 GABAA receptor isoforms. Such receptor subtypes are heterogeneously expressed in the CNS and exhibit distinct physiological and pharmacological properties [3]. Therefore, if GABAA receptors mediate the behavioural actions of the general anaesthetic etomidate, the question arises as to which receptor isoforms are important and whether the repertoire of behaviours induced by this anaesthetic are mediated by distinct receptor subtypes. 2. Materials and methods 2.1. Xenopus laevis oocytes Oocytes were isolated and prepared as previously described [1]. Such oocytes were injected with cRNA encoding for human GABAA receptor subunits and used for electrophysiological experiments 2–12 days post-injection. The preparation of the human wild type (WT) and mutant GABAA subunit cDNAs and the synthesis of the corresponding cRNAs has been previously described [4,5]. GABA-evoked currents were recorded from RNA-injected oocytes with a two-electrode voltage-clamp at a holding potential of 60 mV [1]. During recording oocytes were continuously perfused with a standard buffered salt solution and both etomidate and GABA were introduced via the perfusate [1]. The GABA-modulatory activity of etomidate was examined against a concentration of GABA that induces a response 10% of the maximal (EC10). Etomidate concentration response relationships were determined, subsequently fitted with a logistical equation and the EC50 (the concentration of etomidate required to produce a GABA-modulatory response 50% of the anaesthetic maximal response) and the E MAX (the maximal GABA-modulatory response induced by etomidate, expressed as a percentage of the response produced by a maximal concentration of GABA) were derived [1]. All experiments reported here are the mean F S.E.M. of 3–5 experiments. 2.1.1. Cerebellar slice recordings Cerebellar slices were prepared from WT or h2N265S mice of either sex (postnatal days 16–25) according to standard protocols [6]. Whole-cell patch-clamp recordings were made at 35 8C from submerged Purkinje neurons in a standard extracellular recording solution containing 2 mM kynurenic acid and 0.5 AM tetrodotoxin to block ionotropic glutamate receptors and action potentials, respectively. For further details of the recording conditions,
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see [6]. The GABA-mediated mIPSC frequency, amplitude, rise time and decay time (the weighted time constant of decay, s W) were determined before and after 10 AM etomidate [6]. All results reported here are from a minimum of 5 neurons.
3. Results Utilising the Xenopus laevis oocyte expression system etomidate produced a concentration dependent (30 nM–10 AM) enhancement of the current mediated by a non-saturating concentration of GABA (EC10) acting at a6h3g2 receptors [5]. Analysis of the etomidate concentration response relationship revealed the GABA-modulatory action of etomidate to be both potent (EC50 = 0.7 F 0.06 AM) and efficacious (E MAX = 135 F 7%; i.e. a ~ 12.5-fold increase) (Fig. 1). Similar results were obtained for a6h2g2 receptors [4]—see Fig. 1. By contrast, etomidate was much less effective at a6h1g2 receptors (EC50 = 7.4 F 0.6 AM; E MAX = 28 F 2%)—see [4,5] and Fig. 1. Therefore, the coexpression of either the h3 or the h2 subunit, with the a6 and g2 subunit greatly favours the GABAmodulatory actions of etomidate in comparison to the equivalent receptors expressing the h1 subunit. Etomidate is structurally related to loreclezole and the GABA-modulatory actions of this anticonvulsant, in common with etomidate, are h subunit selective, a preference governed by the nature of a single amino acid residue (asparagine, N for h2 and h3 subunits and serine, S for the h1 subunit) located towards the extracellular portion of the second transmembrane (TM2) domain of the protein [7]. In agreement, the GABA-modulatory actions of etomidate were greatly reduced by mutation of this key asparagine residue to serine (etomidate EC50 = 5.7 F 2.6 AM; E MAX = 36 F 1% for 1)
3) 200 180
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Fig. 1. The GABA-enhancing actions of etomidate are influenced by the nature of a single amino acid residue on the GABAA h subunit. (1) The histogram gives the maximal enhancement (E MAX expressed as a percentage of the maximal response to a saturating concentration of GABA) by etomidate of the GABA (EC10)-evoked response mediated by a6hXg2 GABAA receptors (where X, the nature of the h subunit is specified in the figure) expressed in oocytes. Data obtained are the mean F S.E.M. of 3–5 experiments. (2) Representative traces illustrating the effect of 10 AM etomidate on averaged mIPSCs recorded from wild type and h2N265S Purkinje neurones. (3) The effect of etomidate to prolong the decay (s W) of mIPSCs recorded from wild type and h2N265S Purkinje neurones. In each case the data represent the mean F S.E.M. obtained from 5 neurones.
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a6h3N265Sg2 receptors)—see [5] and Fig. 1. Correspondingly, the GABA enhancing actions of this anaesthetic were facilitated by the mutation of the homologous serine residue of the h1 subunit to asparagine (etomidate EC50 = 1.6 F .3 AM; E MAX = 150 F 13%; for a6h1S265Ng2 receptors)—see [5] and Fig. 1. Based on these findings, gene targeting was used to generate a mouse containing the equivalent asparagine to serine mutation in the h2 subunit (h2N265S)—see [6]. Such mice were used to investigate the role of GABAA receptors in mediating the behavioural actions of etomidate (i.e. in these mice etomidate is presumed to selectively interact with GABAA receptors containing the h3 subunit). However, the properties of recombinant receptors expressed in a foreign host cell (i.e. oocytes) may not faithfully reproduce those of native receptors expressed in neurones. Therefore, we used the whole-cell clamp technique and cerebellar brain slices to compare the actions of etomidate on miniature inhibitory postsynaptic currents (mIPSCs) recorded from Purkinje neurones of wild type (WT) and mutant (h2N265S) mice. Such mIPSCs result from the activation of synaptic GABAA receptors by quantally released GABA. By perturbing the channel kinetics of the GABA-gated ion channels, positive allosteric modulators such as etomidate act primarily to prolong the mIPSC decay. Purkinje neurons were chosen as they are reported to express the h2 subunit together with only a limited repertoire of other GABAA receptor subunits [8]. Importantly, a comparison of the properties (frequency, amplitude, rise and decay times) of mIPSCs recorded from Purkinje neurons derived from WT and mutant mice revealed the mutation to have no effect on any of these parameters (i.e. the mutation was functionally silent). However, the effects of etomidate were greatly suppressed. Application of 10 AM etomidate to brain slices of WT mice caused a marked prolongation of the mIPSC decay (s W of 2.8 F 0.2 ms compared with 29.2 F 5.8 ms) of Purkinje neurones. By contrast, application of this concentration of anaesthetic to brain slices derived from mutant mice produced only a modest prolongation of the mIPSC (s W = 2.4 F 0.1 ms compared with 7.3 F 0.3 ms)—see [6] and Fig. 1.
4. Discussion Many general anaesthetics are relatively low in potency and at similar concentrations influence the function of a variety of receptors and ion channels. These features have confounded attempts to define the important molecular target(s) that mediate the behavioural repertoire induced by general anaesthetics. In this regard, the i.v. general anaesthetic etomidate is unusual and of value. Etomidate at relatively low concentrations enhances the function of the major inhibitory receptor in the mammalian CNS, the GABAA receptor. Furthermore, this anaesthetic appears to be relatively selective, having little or no activity across other representative members of the transmitter-gated ion channel family [1]. The case for an involvement of GABAA receptors in the behavioural actions of etomidate is strengthened by studies utilising the enantiomers of this anaesthetic. Behaviourally, the R-(+) enantiomer of etomidate is ~ 10-fold more potent than the S-( ) form in producing a loss of the righting reflex (LORR) in mice and tadpoles [1,2]. In correspondence this behavioural selectivity is mirrored by the GABA-modulatory properties of the anaesthetic [1,2]. Of particular importance is the observation that the GABA-modulatory actions of etomidate are dependent upon the subunit composition of the receptor [4,5]. In particular, receptors incorporating the h2 or the h3 subunit are highly sensitive to etomidate, whereas this anaesthetic has a much more limited action at equivalent receptors containing the h1
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subunit—see Fig. 1 [4,5]. Furthermore, this subunit selectivity resides exclusively with the nature of a single amino acid (S for h1; N for h2 and h3 subunits), see [5]. This observation provided the impetus to use gene targeting to generate mice carrying a point mutation of the h2 asparagine to serine residue [6]. Purkinje neurones are known to express the h2 subunit [8]. In agreement, etomidate greatly prolonged the decay of GABA-mediated mIPSCs recorded from these neurons and this effect was greatly reduced by the h2 subunit mutation—see Fig. 1 [6]. The use of such mice, together with subtype selective drugs such as etomidate are invaluable in deciphering the identity of GABAA receptor isoforms expressed in neuronal circuits implicated in the effects of general anaesthetics. In this regard we are currently employing this approach to characterise inhibitory transmission in the thalamocortical circuitry [9]. In addition, this genetic approach permits the role of GABAA receptor subtypes in the repertoire of behaviours induced by general anaesthetics to be explored. Given the limited sensitivity of h1 and in the mutant mice h2 subunit containing receptors to etomidate, in the bknock inQ mouse this anaesthetic will mainly act at receptors incorporating the h3 subunit. Importantly, our experiments on Purkinje neurones demonstrate the h2 subunit mutation to be silent (being revealed only in the altered response to etomidate), and in agreement these mice do not have a distinctive phenotype, observations that collectively legitimise this model [6]. However, the behavioural effects of etomidate are considerably altered. In particular, the sedative effects of low (non-anaesthetic) doses of etomidate were absent in the h2N265S mice, and the duration of the loss of the righting reflex induced by this anaesthetic was reduced [6]. We had previously demonstrated that the Drosophila GABA receptor RDL is insensitive to etomidate but that mutation of the homologous TM2 residue (M: methionine) to asparagine uncovered a GABA-modulatory effect of this anaesthetic [10]. Reciprocally, we found that exchange of the N to M residue of the h3 subunit completely suppressed the GABA-modulatory actions of etomidate and additionally those of propofol [10,11]. These findings were developed by a second group to generate a mouse with a h3 subunit that carried this mutation (h3N265M)—see [12]. In these mice, the duration of the LORR induced by either propofol or etomidate, was greatly reduced and the suppression of the hind limb withdrawal reflex (immobilizing action) induced by these anaesthetics was almost abolished [12]. Collectively, the results obtained with the h2 and h3 mutant mice suggest the immobilizing action of etomidate and propofol are mediated by h3 subunit containing receptors, the sedative action of etomidate involves GABAA receptors incorporating the h2 subunit, whereas the hypnotic actions of these anaesthetics require the participation of both h2 and h3 subunit containing receptors. For etomidate and propofol it is now evident that the GABAA receptor is an important target. Furthermore, the results obtained from bknock inQ mice reveal that different components of the repertoire of behaviours induced by general anaesthetics are mediated by distinct GABAA receptor isoforms. These findings should encourage the future development of GABAA receptor subtype selective anaesthetics, offering hopefully an improved side effect profile. More fundamentally, the future development of mice carrying the h subunit mutations described here, but expressed only in specific neuronal populations, should permit the anatomical locus of the dramatic behavioural effects induced by general anaesthetics to finally be revealed.
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