The molecular mechanisms that underlie the rapid depression of central nervous system function by general anaesthetics remain to be identified. The strong correlation between simple indices of anaesthetic potency and the solubility of anaesthetics in fatty solvents established by Meyer and Overton has traditionally been held to indicate a hydrophobic site of action, commonly equated with the lipid bilayer of the nerve cell membrane. A relatively non-specific physicochemical interaction involving an alteration in the volume, thickness or phase behaviour of the lipid bilayer might, in principle, explain how agents that range from inert gases to complex steroidal structures can exert a similar end-effect (Little, 1996). However, not all anaesthetics act similarly. The pharmacological spectrum of an anaesthetic can include amnesia, immobility in response to a noxious stimulus, unconsciousness, analgesia and suppression of the stress response, but the balance of such effects are properties of the individual agent, an observation that is not readily explained by a unitary theory of anaesthesia (Eger et al. 1997). At a more fundamental level, general anaesthetics produce only modest changes in membrane structure that can be mimicked by small changes in temperature (Franks & Lieb, 1994). When coupled with the cut-off phenomenon and recently established violations of the Meyer-Overton rule, lipid theories of general anaesthesia appear less tenable (Franks & Lieb, 1994; Eger et al. 1997).
Neural proteins, particularly the major transmitter-gated channels mediating central inhibition (GABAA and glycine) and excitation (glutamate), have recently received considerable attention as alternative sites of anaesthetic action (Franks & Lieb, 1994; Harris et al. 1995). The spectrum of actions that contribute to a state of anaesthesia could potentially be explained by differential effects upon multiple transmitter systems, or subtypes of receptor responsive to a particular transmitter. A particularly persuasive case can be made for the involvement of the GABAA receptor in certain aspects of anaesthesia, such as the suppression of movement in response to noxious stimulation (Eger et al. 1997; Quinlan et al. 1998). Indeed although structurally diverse, the majority of clinically useful and experimental general anaesthetics at appropriate concentrations share the common feature of potentiating the actions of GABA at the GABAA receptor (Tanelian et al. 1993; Franks & Lieb, 1994; Lambert et al. 1998). Additionally, the enantioselectivity of a number of the general anaesthetics, such as isoflurane, etomidate, pentobarbitone and neuroactive steroids, is mirrored in their anaesthetic potency (Lambert et al. 1998). A direct link between anaesthetic mechanisms and GABAA receptor function has been established by the use of mice genetically engineered to lack the β3 GABAA receptor subunit. In comparison with wild-type mice, the null-allele animals demonstrated a change in anaesthetic requirement that was dependent upon the identity of the anaesthetic agent and the response quantified (i.e. loss of righting reflex or nocifensive reflex) (Quinlan et al. 1998).
In mammals, the GABAA receptor is composed of five polypeptide subunits drawn from the products of a multigene family (α1-6, β1-3, γ1-3, δ and ε). These are differentially expressed within the central nervous system (Smith & Olsen, 1995; Whiting et al. 1995) and recombinant expression studies have revealed that subunit composition influences both the physiological and pharmacological properties of the receptor (Sieghart, 1995). Determining the actions of anaesthetics at different GABAA receptor isoforms may help explain brain region-selective effects of general anaesthetics and may aid the identification of protein domains that form the anaesthetic binding pocket, or that are required for the alteration of GABA-gated chloride channel function by the anaesthetic (Smith & Olsen, 1995; Belelli et al. 1997; Mihic et al. 1997; Peters & Lambert, 1997).
In addition to enhancing the actions of GABA, a number of general anaesthetics possess GABA-mimetic activity (Tanelian et al. 1993; Lambert et al. 1998). This effect generally occurs at concentrations greater than those required for GABA enhancement. The differing concentration dependencies of the GABA-modulatory and GABA-mimetic activities of general anaesthetics are compatible with their respective mediation by high and low affinity anaesthetic binding sites on the GABAA receptor protein. The concept of distinct binding sites appears to be supported by our previous studies concerning the actions of the general anaesthetics propofol and pentobarbitone on an invertebrate recombinant GABA receptor (RDL) isolated from Drosophila melanogaster (ffrench-Constant et al. 1991; Chen et al. 1994) where these anaesthetics enhanced the actions of GABA but, in contrast to mammalian GABAA receptors, did not directly activate the receptor (Belelli et al. 1996). Therefore, either the RDL receptor truly lacks an anaesthetic activation site, or alternatively, the site is present but occupation by the anaesthetic is not transduced into channel opening. Independent of the underlying cause, the RDL subunit may be instructive in identifying those amino acids that are required for the GABA-mimetic actions of certain anaesthetics. Here we demonstrate that the nature of a single transmembrane amino acid governs, in a complementary manner, the GABA-mimetic actions of the general anaesthetics pentobarbitone and propofol at both mammalian and invertebrate GABA receptors. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction.
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A number of general anaesthetics exhibit both GABA-modulatory and GABA-mimetic actions at the GABAA receptor. The distinct concentration dependencies of these two effects may be explained by the presence of high (GABA-modulatory) and low (GABA-mimetic) affinity anaesthetic binding sites on the GABAA receptor protein (Harris et al. 1995). Consistent with this concept, the GABA-modulatory, but not the GABA-mimetic, effects of certain anaesthetics are reduced by the introduction of a δ- or ε-subunit into binary complexes of αβ-GABAA subunits (Zhu et al. 1996; Davies et al. 1997; cf. Whiting et al. 1997). Similarly, α4-subunit-containing receptors support GABA potentiation by propofol and pentobarbitone, but not direct receptor activation by these anaesthetics (Wafford et al. 1996). Further support for the existence of distinct modulatory and activation sites derives from observations made on the Drosophila GABA receptor, where the anaesthetics pentobarbitone and propofol clearly enhance the actions of GABA but are not GABA mimetic (Chen et al. 1994; Belelli et al. 1996).
The intriguing question remains as to how a variety of general anaesthetics, which exhibit little or no commonality in chemical structure to GABA, or between themselves, can nevertheless activate the GABAA receptor-channel complex. The results of interaction studies with binary combinations of anaesthetics, such as steroids and barbiturates (Belelli et al. 1996), indicate that despite sharing a common channel-gating effect, such agents may bind to distinct non-overlapping sites. Certainly, the direct gating effect occurs through a site different from that occupied by GABA, since numerous anaesthetics potentiate, rather than occlude, the binding of agonist ligands to the GABAA receptor (reviewed by Tanelian et al. 1993; Sieghart, 1995). In addition, mutations of the β-subunit that greatly diminish the potency of GABA have no effect upon the direct effects of pentobarbitone (Amin & Weiss, 1993). Moreover, the expression of β1- or β3-subunits in isolation results in the formation of homo-oligomeric receptor-channel complexes that are largely unresponsive to GABA, yet are activated by general anaesthetics that include etomidate, propofol and pentobarbitone (Sanna et al. 1995; Krishek et al. 1996; Wooltorton et al. 1997; reviewed in Lambert et al. 1997). However, the interpretation of those experiments is complicated by the fact that, in many cases, β-homomeric receptors appear spontaneously active in the absence of GABA (Krishek et al. 1996; Wooltorton et al. 1997).
The cardinal finding of the present study is that the direct gating of both a mammalian GABAA receptor (α6β3γ2L) and an invertebrate GABA receptor (RDL) by the structurally unrelated anaesthetics pentobarbitone and propofol is influenced in a complementary manner by a single transmembrane amino acid. Hence, these anaesthetics enhance the actions of GABA at both receptors, but only directly activate the chloride channel of the mammalian receptor (Belelli et al. 1996). However, the mutation of a single transmembrane amino acid residue (asparagine 289) of the β3-subunit to methionine (the homologous residue of the RDL receptor) abolished direct activation by propofol and substantially reduced the GABA-mimetic activity of pentobarbitone. In addition, the mutation reduced the GABA-modulatory effects of both pentobarbitone and propofol. The effects of this mutation could, of course, be non-specific, causing changes in the secondary, tertiary, or quaternary structure of the protein. Compelling evidence against such an interpretation is provided by the properties of the RDL receptor, where the transmembrane methionine residue was changed to the equivalent asparagine of the β2 and β3, or the serine of the β1-GABAA receptor subunit. Acting at RDLM314S receptors, either propofol or pentobarbitone, in the absence of GABA, directly activated the receptor channel complex in a picrotoxin-sensitive manner. This fundamental change was not restricted to general anaesthetics, as δ-HCH also directly activated the RDLM314S receptor, but was inert in this respect at the wild-type receptor. Indeed, the maximal current produced by these agents was similar to that evoked by a saturating concentration of GABA. Furthermore, analysis of their concentration-response relationships revealed Hill slopes substantially greater than 1, suggesting that co-operative drug binding was required to induce channel opening. Propofol and δ-HCH also activated the RDLM314N receptor-channel complex, although with a reduced maximal effect compared with that observed at the RDLM314S receptor. By contrast, pentobarbitone does not activate the RDLM314N receptor (McGurk et al. 1998). Interestingly, the appearance of the GABA-mimetic effects of propofol and pentobarbitone was also accompanied by an increase in the potency of these anaesthetics in enhancing the actions of GABA, suggesting these two effects are coupled, whereas the serine or asparagine mutation (RDLM314S, RDLM314N) revealed a GABA-mimetic action of δ-HCH, but with little effect on GABA-modulation. Should mutation of the TM2-located amino acid have resulted in an RDL receptor with an increased propensity for spontaneous channel openings, then the direct effects of these agents might merely reflect the promotion of background channel opening. Such a mechanism may contribute to the apparent direct effects of anaesthetics reported for homo-oligomeric receptors composed of β1- or β3-subunits (Krishek et al. 1996; Wooltorton et al. 1997). However, in the present study, the resting membrane conductance of both oocytes and S2 cells was not influenced by the expression of RDLM314S and RDLM314N receptors in comparison with the wild-type receptor. Furthermore, picrotoxin was without effect on such cells (cf. Krishek et al. 1996; Wooltorton et al. 1997) and therefore the direct gating of the channel by the anaesthetics does not result from this mechanism.
Therefore, the nature of this TM2-located amino acid exerts a considerable influence on the allosteric effects of at least three general anaesthetics. For etomidate, positive allosteric actions at both mammalian and invertebrate receptors are favoured by asparagine, reduced for serine and absent for methionine (Belelli et al. 1997; McGurk et al. 1998; see Table 1). By contrast, for propofol and pentobarbitone acting at RDL receptors, activity is favoured by serine, reduced for asparagine, but again is least active for the methionine residue. Similarly, the actions of these anaesthetics at mammalian receptors are limited by the methionine residue. However, propofol and pentobarbitone do not discriminate between β1 (serine)- and β2- or β3 (asparagine)-containing receptors (Hill-Venning et al. 1997) and hence the preference for the serine residue exhibited with the invertebrate receptor does not extend to the mammalian receptor.
For GABAA and glycine receptor α-subunits, residues occupying an equivalent position in TM2 exert a profound influence upon allosteric regulation by both volatile and intravenous anaesthetic agents. For the glycine receptor α1-subunit, modulation of receptor function by ethanol appears to be partly determined by the molecular volume of the amino acid which occupies this crucial position, potentiation being favoured by small amino acids and inhibition by relatively bulky residues (Ye et al. 1998). Whether the size of the amino acid residue is also an important determinant of anaesthetic action at the GABAA receptor has not been systematically investigated to date. The allosteric effects of general anaesthetics at both GABAA and glycine receptors are additionally influenced by the nature of an amino acid residue within TM3, which again is thought to be located near to the extracellular aspect of the membrane (Mihic et al. 1997; Krasowski et al. 1998a,b). Of particular interest is the observation that expression of the GABAAα2-subunit with a β1-subunit in which the methionine residue in TM3 of the wild-type protein has been exchanged for a tyrosine residue results in a receptor which is insensitive to the GABA potentiating effects of propofol, but the direct-gating of the receptor channel complex by the anaesthetic is unchanged (Krasowski et al. 1998a). Therefore, the GABA-modulatory and GABA-mimetic actions of propofol are dramatically influenced by amino acid residues in both TM2 and TM3.
In aggregate, the actions of a number of chemically disparate compounds acting at GABAA, RDL and glycine receptors are influenced by the nature of two transmembrane (TM2 and TM3) amino acids. This permissiveness of both receptors and drugs would appear incompatible with the identified amino acids contributing to the anaesthetic binding site and favours these residues playing a key role in transduction. In this respect it is of interest that mutation of a conserved leucine to a threonine residue in the TM2 domain of the nicotinic α7-subunit produced a dramatic change in the pharmacology of this transmitter-gated ion channel, since a number of structurally distinct antagonists acquire agonist character (Bertrand et al. 1992; Palma et al. 1998). However, it is important to note that the actions of steroidal anaesthetics at GABAA and RDL receptors are not affected and therefore not all classes of anaesthetics are similarly influenced (Belelli et al. 1997; McGurk et al. 1998; Rick et al. 1998).
In conclusion, both the GABA-modulatory and GABA-mimetic effects of two structurally distinct general anaesthetics at both mammalian and invertebrate GABA receptors are greatly influenced by the nature of an amino acid located within TM2. However, whether or not this amino acid participates directly in the binding of the anaesthetic to the receptor protein remains to be clarified. In this respect, two recent reports highlighting the importance of the N-terminal extracellular domain of the GABAA receptor α-subunit for the GABA-modulatory effects of propofol (Uchida et al. 1997) and the GABA-mimetic actions of pentobarbitone (Fisher et al. 1997) may prove to be pertinent. In addition to GABA and glycine receptors, key amino acids which dictate the interaction of certain anaesthetics with glutamate (Minami et al. 1998) and nicotinic receptors (Forman et al. 1995) have also been identified. Collectively, these observations demonstrate that far from being indiscriminate, the interaction of anaesthetics with transmitter-gated ion channels is highly specific. In the future, the generation of mice carrying such anaesthetic resistant mutations should permit a better evaluation of the molecular targets that mediate the behavioural effects of general anaesthetics.