Substantial progress has been made in the last decade in defining the actions of general anaesthetic agents on ligand-gated ion channels, particularly in the areas of molecular biology, pharmacology and electrophysiology. The coming years will surely witness more major advances, perhaps most notably from the application of structural biology and gene-targeting approaches. The use of site-directed mutagenesis and chimeric receptors has proven very helpful in identifying regions of ligand-gated ion channels that play critical roles in modulation by general anaesthetics. However, more definite evidence of the existence of general anaesthetic binding pockets probably awaits the resolution of three-dimensional structures for the ligand-gated ion channels. Structural biology approaches have already been applied to the study of general anaesthetic interactions with model soluble proteins [20
], including the recent report of the 2.2-Å resolution three-dimensional structure of firefly luciferase complexed with the general anaesthetic bromoform [272
In common with other many integral membrane proteins, ligand-gated ion channels have proved recalcitrant to structural biology approaches. However, the crystallization and determination of a high-resolution structure for a bacterial potassium channel [273
] surely foreshadows the eventual determination of the three-dimensional structure of the ligand-gated ion channels. A more immediate possibility is the determination of the structure of limited domains of ligand-gated ion channels; indeed, researchers have very recently succeeded in resolving the structure of the extracellular domain of an ionotropic glutamate receptor complexed with kainate [274
]. Even in the absence of detailed structures, molecular modeling may be of use in making preliminary predictions that can be tested experimentally.
Targeted gene manipulations in mice will also provide hypothesis-driven tests of the in vivo roles of certain ligand-gated ion channels in mediating the diverse behavioral actions of general anaesthetics. As described above, researchers over the last 5 years have created ‘global knockout mice’ for various subunits of the ligand-gated ion channels. Given the abundance of ligand-gated ion channel knockout mice (and the commercial availability of some of these knockouts), it would be a logical step to test anaesthetic sensitivity in some or all of these mice. However, while knockout mice may provide initial clues as to the nature of anaesthetic targets, such mice can be very difficult to analyze for anaesthetic sensitivity if they exhibit grossly abnormal motor behavior, lethality or aberrations in neural development. These problems with knockout mice may be circumvented by ‘conditional’ gene knockouts, in which the gene of interest is disrupted only in limited brain regions and/or specified developmental time periods [172
Another elegant example of gene targeting is a ‘knock-in’ mouse. One possibility is the introduction of a mutated receptor subunit that is insensitive to anaesthetic modulation in place of the normal endogenous receptor subunit (e.g. see [175
]). This type of approach has recently been applied to the benzodiazepines. These studies utilized knock-in mice expressing a mutant GABAA
subunit that confers insensitivity to benzodiazepine modulation, in place of the benzodiazepine-sensitive wild-type α1
subunit. These preliminary studies have not only demonstrated the importance of the GABAA
subunit isoform for the behavioral actions of benzodiazepines but also have suggested that distinct GABAA
receptor α-subunit isoforms mediate different actions of the benzodiazepines, with the α1
subunit isoform necessary for sedative and anticonvulsant effects and other α-subunit isoforms critical for myorelaxant and anxiolytic actions (U. Rudolph, F. Crestani, H. Möhler, personal communication).
Knock-in mouse experiments potentially provide an elegant bridge between in vitro experiments and whole animal behavior. Ideally, the mutated receptor subunit would differ from the normal subunit only in terms of general anaesthetic modulation (i.e. agonist binding, channel gating, voltage dependence, kinetics etc. of the receptor would be relatively normal). Recently described mutations within TM2 and TM3 of GABAA
(see ) and glycine receptors, which confer insensitivity to volatile ether anaesthetics [206
], n-alkanols [219
], propofol [206
], trichloroethanol [227
], pentobarbitone [261
] and etomidate [228
] essentially fit this qualification, as do point mutations within GluR6 kainate receptors that abolish volatile anaesthetic potentiation [223
]. A complication to gene-targeting experiments is the presence of multiple subunit isoforms for the ligand-gated ion channels. For example, there are at least 17 gene products for GABAA
receptor subunits; if multiple GABAA
subunit isoforms play a role in general anaesthesia, then targeting of multiple genes may be required to obtain an unambiguous change in anaesthetic sensitivity. General anaesthetics produce a range of behavioral effects in animals and humans. It appears overly simplistic to ascribe all of these to a single receptor. Current and future research should eventually define the specific receptors that underlie each of the diverse behavioral actions of every class of general anaesthetics. The upcoming decade will undoubtedly be an exciting time for research into the molecular mechanisms of general anaesthetics.