Download The molecular mechanisms of general anaesthesia: dissecting the

The molecular mechanisms of
general anaesthesia: dissecting the
GABAA receptor
Cameron J Weir BSc(Hons) FRCA PhD
The mechanisms underlying the dramatic
clinical effects of general anaesthetics remain
elusive. This review summarizes the remarkable developments which have occurred in
general anaesthetic research over the past decade demonstrating that, rather than acting
nonspecifically to disrupt lipid membranes,
general anaesthetics target certain CNS
proteins in a highly selective manner.
Lipid theory of narcosis
Paul Ehrlich (1854–1915) first proposed the
concept of highly specific interactions between
drugs and receptors—corpora non agunt nisi
fixata (drugs do not act unless they are
bound). However, this concept cannot be easily applied to general anaesthetics because they
are chemically and structurally diverse and
they lack obvious structure–activity relationships. Such diversity led early researchers to
focus on nonspecific perturbation of central
nervous system neurones as the basis for the
clinical effects of general anaesthetic drugs.
For example, at the beginning of the 20th century HH Meyer and CE Overton independently reported a striking correlation between
the oil/water partition coefficients of a range of
anaesthetic compounds and their ability to
immobilize tadpoles, that is, the greater the
lipid solubility of the compound the greater
its anaesthetic potency. Meyer’s son (KH
Meyer) refined these observations in his
1937 publication ‘Contributions to the theory
of narcosis’.1
Anomalies with unitary
theories
The development of sophisticated scientific
techniques during the 20th century provided
the impetus for numerous theories of anaesthetic action including alterations to the volume or fluidity of lipid bilayers (critical volume
hypothesis or lateral phase separation theory)
or the disruption of neuronal activity by the
formation of anaesthetic microcrystals with
water molecules (clathrate theory). These
‘physicochemical’ theories accommodated
the diverse chemical nature of general anaesthetic drugs, but closer scrutiny reveals several
anomalies:
(i) Some compounds do not obey the Meyer
and Overton rule. For example, some
halogenated alkanes predicted to be
potent anaesthetics based on their lipid
solubility fail to suppress movement in
response to noxious stimulation at appropriate concentrations. These compounds
are therefore termed nonimmobilizers.
(ii) The ability of general anaesthetics to
perturb lipid membranes in vitro can be
reproduced by a temperature increase of
less than 1 C, a change well within the
physiological range and clearly not sufficient to induce loss of consciousness
per se.
(iii) Stereoisomers existing as mirror images
of each other are termed enantiomers
(e.g. the isomers of R-(þ)- and S-()etomidate). Enantiomers have identical
physicochemical effects in an achiral
environment (e.g. the lipid bilayer).
However, in vitro and in vivo studies
demonstrate that enantiomers of many
general anaesthetics do not produce
identical clinical effects. For example, the
Rþ isomer of etomidate is 10 times more
potent than its S isomer at potentiating
GABAA receptor activity. These differential effects suggest that the primary
site of action of such anaesthetics is not
the lipid bilayer and provide compelling
evidence for specific interactions with
stereoselective binding sites (i.e. within
proteins).
(iv) According to Meyer and Overton, the
addition of methylene groups to a homologous series of long chain alcohols,
or alkanes, should increase their lipid
solubility and thereby produce a corresponding increase in anaesthetic potency.
However, at a certain chain length
(n ¼ 10) addition of further methylene
groups does not produce the expected
doi 10.1093/bjaceaccp/mki068
Continuing Education in Anaesthesia, Critical Care & Pain | Volume 6 Number 2 2006
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Key points
Early theories of general
anaesthetic action focused on
nonspecific disruption of
neuronal cell membranes.
Compelling evidence now
demonstrates that certain
general anaesthetics act in a
highly specific manner upon
central nervous system
proteins.
Key amino acids within the a
subunit of GABAA receptors
may contribute to an
anaesthetic binding pocket for
volatile anaesthetic agents.
Genetically engineered mice
harbouring single amino acid
mutations at critical sites
within GABAA receptor
subunits lack certain
components of i.v. anaesthetic
activity in vivo.
GABAA receptors containing
specific subunits appear to
mediate the sedative
(b2 subtype) and anaesthetic
(b3 subtype) activity of i.v.
anaesthetics.
Cameron J Weir BSc(Hons) FRCA PhD
Consultant Anaesthetist/Senior Lecturer
Department of Anaesthesia
Ninewells Hospital and Medical School
Dundee
DD1 9SY
Scotland
UK
E-mail: [email protected]
49
The molecular mechanisms of general anaesthesia
increase in anaesthetic potency, i.e. there appears to be a
‘cut off’ effect above a certain molecular volume which is
indicative of anaesthetic agents interacting with binding
site(s) of finite dimensions.
In a landmark series of experiments in the early 1980s, Franks
and Lieb2 demonstrated that the relationship reported by Meyer
and Overton could be reproduced using a soluble protein. They
demonstrated that a range of general anaesthetics acted as competitive antagonists of the protein firefly luciferase. Remarkably,
inhibition of luciferase was directly correlated with anaesthetic
potency providing persuasive evidence that general anaesthetic
drugs could selectively interact with proteins. Of course, the next
major hurdle involved identifying which proteins within the mammalian CNS were responsible for mediating the dramatic behavioural effects of general anaesthetic drugs.
Transmitter-gated ion channels
Chemical synaptic transmission is the process whereby an electrical signal in the presynaptic neurone modulates the release of
transmitter into the synaptic cleft to produce a receptor-mediated,
electrical, or biochemical event in the postsynaptic neurone.
This remarkable feat is achieved by the integrated activity of a
range of different proteins including ion channels, enzymes,
second messengers, and transporters located in pre- and postsynaptic neurones and within the synaptic cleft. In vitro experiments demonstrate that general anaesthetics alter the activity of
many of these proteins, but when clinically relevant concentrations are considered the list of possible targets is greatly diminished. However, one group of proteins has emerged as a likely
target for some general anaesthetic agents. This group is known as
the transmitter-gated ion channel (TGIC) superfamily and
includes g-aminobutyric acid type A (GABAA), strychnine-sensitive glycine, neuronal nicotinic acetylcholine (nAch) and
5-hydroxytryptamine (5-HT3) receptors (Fig. 1).
TGICs are usually located within the postsynaptic membrane
of excitable neurones and they mediate the majority of fast excitatory and inhibitory neurotransmission within the CNS. Opening
of intrinsic ion channels by specific agonists (neurotransmitters)
allows selective transfer of ions across the nerve cell membrane.
For example, under normal conditions, activation of GABAA
receptors induces inward movement of mainly chloride ions
down their electrochemical gradient resulting in a hyperpolarization of the cell membrane, whereas activation of nicotinic
receptors produces a net inward movement of sodium ions and
depolarization of the cell membrane. Therefore, augmenting an
inhibitory signal, or inhibiting an excitatory signal, provides a
logical mechanism for general anaesthetic action. However, when
analysis is restricted to clinically relevant concentrations it
appears that inhibitory GABAA receptors are sensitive to modulation by the majority of inhalation and i.v. general anaesthetics
agents with ketamine and xenon as the most noticeable exception.
GABA-gated receptors
The small amino acid GABA is synthesized from glutamate by the
action of glutamic acid dehydrogenase (GAD). On release from
the presynaptic terminal GABA diffuses across the synaptic cleft
to activate GABAA and GABAB receptors. GABAA receptors are
classed as ionotropic receptors (linked to an intrinsic ion channel)
General Anaesthetics
Fig. 1 General anaesthetics modulate the activity of transmitter-gated ion channels to either enhance inhibitory, or inhibit excitatory neurotransmission.
At clinically relevant concentrations the majority of general anaesthetics augment the activity of the GABAA receptor.
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The molecular mechanisms of general anaesthesia
and are the principal mediators of fast inhibitory neurotransmission in higher centres of the CNS accounting for approximately 30% of all inhibitory synapses. GABAB receptors are
classed as metabotropic receptors (coupled to a G-protein),
but they are essentially insensitive to therapeutic anaesthetic
concentrations and will not be discussed further in this review.
Subunit composition and topology
GABAA receptors are composed of pentameric arrangements of
subunits around a central ion channel pore. Eighteen possible
subunits including a1–6, b1–3, g 1–3, d, e, p, r1–3 subtypes have
been identified in the mammalian genome. If there were no limitations to receptor assembly, then many thousands of different
GABAA receptors could exist within the central nervous system.
However, certain molecular rules do regulate the co-assembly of
subunits within a receptor and the actual number of GABAA
receptor isoforms is probably 30. Furthermore, the differential
expression pattern of each subunit can limit certain GABAA
receptor combinations to specific brain regions or even neurones.
For example, a6-containing GABAA receptors are exclusively
found within cerebellar granule cells whereas a5-containing
receptors are limited to the hippocampus and sub-thalamic areas.
Each subunit is composed of a polypeptide sequence of
approximately 450–630 amino acids (40–60 kDa) with large Nterminal and smaller C-terminal extracellular domains (Fig. 2).
Hydropathy plots (analysis of primary amino acid sequences
predicting regions with high hydrophobic, or hydrophilic nature)
suggest that individual subunits contain four distinct transmembrane (TM) domains, with the second transmembrane
domain (TM2) lining the channel lumen. A large intracellular
loop connects the TM3 and TM4 regions providing sites for
phosphorylation by a range of serine, threonine, and tyrosine
kinases.
Pharmacology of GABAA receptors
GABAA receptors are sensitive to a number of clinically useful
drugs, including general anaesthetics, benzodiazepines, and other
neuro-depressant agents including ethanol.3 Many bind at distinct sites within the receptor to ‘allosterically’ modulate the
action of GABA. For example, in the presence of general anaesthetics, the ability of GABA to open, or ‘gate’ the ion channel is
increased and, as a result, the overall inhibitory activity of the
receptor is enhanced. At supra-clinical concentrations some
anaesthetic agents open GABAA receptor ion channels directly
without the need for GABA indicating that two or more distinct
binding sites (modulatory and direct) might exist for these agents
on each receptor.
In vitro studies
Single amino acids determine i.v. anaesthetic activity
If GABAA receptors are important targets for general anaesthetics, are all receptors equally sensitive to their effects, or are certain
subunit combinations more sensitive than others? This question
can be addressed by examining the activity of the i.v. anaesthetic,
etomidate which demonstrates selective effects for receptors containing certain subtypes of the b subunit. For example, receptors
containing b2 or b3, but not b1 subunits are particularly sensitive
to the modulatory effects of this anaesthetic.4 Studies using a
combination of electrophysiological and molecular biological
techniques demonstrated that the selectivity of etomidate maps
NH2
β
γ α
α
GABA
β
COOH
TM2
Fig. 2 GABAA receptors are composed of pentameric arrangements of subunits arrange around an ion channel pore. The predominant subunit ratio within
the cerebral cortex is 2a:2b:g. Each subunit contains approximately 450 amino acid residues putatively forming four transmembrane domains. The
second transmembrane domain (TM2) of each subunit faces into the ion channel. The binding of GABA to residues at the interface of a and b subunits
induces a conformational change within the TM2 region to open the ion channel pore. Key amino acid residues (represented as closed circle) at the
extracellular end of the transmembrane domains of a and b subunits are crucial for volatile and i.v. anaesthetic activity, respectively.
Continuing Education in Anaesthesia, Critical Care & Pain | Volume 6 Number 2 2006
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The molecular mechanisms of general anaesthesia
to a single amino acid located within the second transmembrane
domain (TM2) of the b subunit (asparagine for b2 and b3, and
serine for b1 subunits). Exchanging the asparagine for serine
within etomidate sensitive b3-containing GABAA receptors
reduced the activity of etomidate, whereas the reciprocal change
in the b1 subunit, (serine ! asparagine) enhanced the activity of
the anaesthetic. Other residues within the neighbouring TM1 and
TM3 domains have additionally been shown to influence the
actions of propofol, alphaxalone, and pentobarbital. Such dramatic effects produced by altering single amino acids within a
large receptor complex provides a persuasive argument for i.v.
general anaesthetics interacting in a highly specific manner with
GABAA receptors.
A binding site for volatile anaesthetics?
The effects of volatile agents on GABAA receptor activity are also
influenced by a single amino acid. Remarkably, the amino acid
responsible (serine) is located within the TM2 region, not in the b
subunit, but at the equivalent position in the a subunit. Subsequent studies demonstrated that the volume of this amino
acid correlated with the activity of volatile anaesthetics on
GABAA receptors. Specifically, substitution with small volume
amino acids enhanced the potentiating activity of isoflurane,
halothane, and chloroform, whereas the presence of bulkier
amino acids reduced anaesthetic activity. Moreover, the volume
of neighbouring amino acids within the TM1, TM3, and TM4
regions also influenced inhalation anaesthetic actions.5 The combined approach of altering amino acid volume, together with the
use of volatile agents of differing size (isoflurane, 144 Å3;
halothane, 110 Å3, and chloroform, 90 Å3) led to the proposal
that the four amino acid residues from the extracellular end of
TM1, TM2, TM3, and TM4 domains of the a subunit contribute
towards an anaesthetic binding pocket for volatile agents of
approximately 250–370 Å3.
In vivo studies
Although in vitro models are invaluable for investigating detailed drug–receptor interactions, interpretation of such experiments
is limited because they do not represent a fully integrated nervous
system. In order to address this problem, several research groups
have created genetically engineered mice harbouring precise
mutations within their genotype. Using these mice allows comparison of the pharmacological responses of ‘mutant’ animals
with their ‘wild type’ (genetically normal) counterparts and provides a powerful tool for the identification of neuroactive drug
targets.6 For example, loss of drug activity after alteration of a
specific protein indicates that the altered protein might be
involved with mediating the pharmacological effects of the
drug. This remarkable technology allows comparison of genetically diverse populations of mice differentiated by only single
amino acids within their genome. Such ‘knock-in’ mice have
been used successfully to dissect out some receptor isoforms
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Table 1 Assigning various behavioural features of benzodiazepines to
individual GABAA receptor subtypes
GABAA receptor subunit
Pharmacological effect
a1
Sedation
Anticonvulsant activity
Anterograde amnesia
a2
Anxiolysis
a3
Myorelaxation
a5
Memory and learning
Tolerance to benzodiazepines
that mediate specific behavioural effects of benzodiazepines
and general anaesthetics.6
For example, mice harbouring a single amino acid substitution within the GABAA receptor a1 subunit are resistant to the
sedative and anterograde amnesic effects of diazepam, but they
retain the anxiolytic response to the benzodiazepine. In contrast,
mice harbouring the equivalent mutation within the a2 subunit
display reduced anxiolytic behaviour, but retain sensitivity to
the sedative and amnesic effects of diazepam. These landmark
observations provide compelling evidence for the a1 subunit
mediating the sedative and amnesic effects of benzodiazepines,
whereas the a2 subunit might mediate anxiolytic activity. Furthermore, it appears that the a5 subunit plays an important role in
learning and memory and may also be involved with development
of tolerance to benzodiazepines. The various behavioural features
of benzodiazepines to individual GABAA receptor subtypes are
summarized in Table 1.
The role of the b subunit in anaesthesia
Similar investigations using ‘knock-in’ mice have been instrumental in assigning the various behavioural facets of general
anaesthetic drugs to certain receptor subtypes. The unique
subunit selectivity of etomidate guided the development of a
strain of genetically engineered mice harbouring a single amino
acid mutation within the TM2 region of the b3 subunit.7 These
mice were found to be insensitive to some of the effects of etomidate and propofol, that is, the hind limb withdrawal reflex
(immobilization) was completely absent and the duration of
the loss of righting reflex (hypnosis) was greatly reduced.
In contrast, actions of the volatile anaesthetics enflurane and
isoflurane were relatively unaffected in the mutant mice. These
exciting experiments using whole animals concur with in vitro
work showing the actions of certain i.v. anaesthetics to be
mediated, in part, by the b3 subunit of the GABAA receptor.
Further analysis of etomidate-induced anaesthesia has been
carried out using genetically engineered mice harbouring a single
amino acid mutation of the GABAA b2 subunit.8 In these knockin mice, the native b2 subunit TM2 asparagine was replaced with a
serine residue found within the etomidate insensitive b1 subunit.
Intra-peritoneal injection of etomidate, propofol, or pentobarbital produced an anaesthetic effect (loss of righting reflex,
loss of pedal withdrawal) indistinguishable from that seen in
Continuing Education in Anaesthesia, Critical Care & Pain | Volume 6 Number 2 2006
The molecular mechanisms of general anaesthesia
genetically normal mice.8 However, the mutant mice were resistant to the sedative and ataxic effects produced by sub-anaesthetic
concentrations of etomidate and significantly improved locomotor activity was also observed. In other words, the mutant
mice were sensitive to the anaesthetic effects of etomidate presumably because the b3 subunit was still functional, but their
recovery profile was consistent with the b2 subunit mediating
the ‘hangover’ effects of this i.v. anaesthetic (i.e. drowsiness
and ataxia).
Anaesthetic targets
The chemical diversity of general anaesthetics has resulted in the
perception that they are ‘dirty’ drugs. In particular, the volatile
agents modulate the activity of many proteins including ion
channels and second messenger systems. However, before a
protein can be considered a target for general anaesthetic
drugs certain criteria must be met; the protein must be sensitive
at clinically relevant concentrations; it must be expressed at
appropriate anatomical sites within the CNS and, if the anaesthetic displays stereoselective activity, this should be seen both
in vitro and in vivo.9 Experimentally, general anaesthetics alter the
function of many of these potential targets, but most do not meet
the strict conditions set out above. However, certain GABAA
receptor subtypes satisfy most of the criteria. It is possible, therefore, that the range of GABAA receptor subunit combinations
displaying individual anaesthetic sensitivities together with their
distinct expression patterns within the CNS, provides a rationale
for the diverse clinical effects of these drugs.
Of course, it is unlikely that enhancement of GABAA receptor
activity is the only mechanism to account for the wide range of
behavioural effects of general anaesthetics. Increasing evidence
suggests that modulation of two-pore domain potassium channels, or voltage-gated sodium channels may account for some of
the actions of volatile anaesthetic agents. Alternatively, inhibition
of glutamate-gated NMDA (N-methyl-D-aspartate) receptors by
ketamine, xenon, and nitrous oxide provides a mechanism of
action in keeping with a predominant analgesic profile.
specific point mutations permits the assignment of certain general
anaesthetic and benzodiazepine-induced behaviours to particular
GABAA receptor isoforms. Using this information, it may be
possible to synthesize novel anaesthetic agents with improved
therapeutic profiles, that is, subunit selective compounds producing anaesthesia with a reduced propensity for side-effects (e.g.
cardio-respiratory depression, postoperative sedation, ataxia,
and nausea and vomiting). Furthermore, relating GABAA receptor isoforms to their expression patterns in the CNS may help
with identifying the anatomical locus and the neural networks
responsible for general anaesthesia.
Acknowledgements
The author was supported by a Research Fellowship from the
British Journal of Anaesthesia and a research grant from the
Royal College of Anaesthetists. Thanks also to Professor
J. A. Peters and Dr S. Humble for their help in preparing the
manuscript.
References
1. Meyer KH. Contributions to the theory of narcosis. Trans Faraday Soc 1937;
33: 1062–8
2. Franks NP, Lieb WR. Do general anaesthetics act by competitive binding
to specific receptors? Nature 1984; 310: 599–601
3. Whiting PJ. GABA-A receptor subtypes in the brain: a paradigm for CNS drug
discovery? Drug Discov Today 2003; 8: 445–50
4. Belelli D, Pistis M, Peters JA, Lambert JJ. General anaesthetic action at
transmitter-gated inhibitory amino acid receptors. Trends Pharmacol Sci
1999; 20: 496–502
5. Jenkins A, Greenblatt EP, Faulkner HJ, et al. Evidence for a common
binding cavity for three general anesthetics within the GABAA receptor. J
Neurosci 2001; 21: RC136
6. Rudolph U, Mohler H. Analysis of GABAA receptor function and dissection of
the pharmacology of benzodiazepines and general anesthetics through
mouse genetics. Annu Rev Pharmacol Toxicol 2004; 44: 475–98
7. Jurd R, Arras M, Lambert S, et al. General anesthetic actions in vivo
strongly attenuated by a point mutation in the GABAA receptor beta3
subunit. FASEB J 2003; 17: 250–52
8. Reynolds DS, Rosahl TW, Cirone J, et al. Sedation and anesthesia mediated
by distinct GABAA receptor isoforms. J Neurosci 2003; 23: 8608–17
Conclusions
Dramatic progress has been made since Meyer and Overton first
used olive oil and tadpoles to investigate the mechanisms of
general anaesthesia. Today, the creation of mice harbouring
9. Krasowski MD, Harrison NL. General anaesthetic actions on ligand-gated
ion channels. Cell Mol Life Sci 1999; 55: 1278–1303
Please see multiple choice questions 1–5.
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