The local application of GABA (10 µM, 1·4 × 10⁵ Pa, 3-20 ms, 0·033 Hz) evoked an inward current response (holding potential, V_h = -60 mV) on 44 % (n = 50), 24 % (n = 85) and 48 % (n = 70) of S2 cells previously transfected with RDL, RDL_M314S or RDL_M314N receptor subunit cDNAs, respectively (Fig. 1). For all receptor constructs examined, the reversal potential (V_rev) of the GABA-evoked current was close to zero millivolts (RDL, 0·8 ± 0·6 mV, n = 6; RDL_M314S, 1 ± 0·5 mV, n = 7; RDL_M314N, 0·9 ± 0·8 mV, n = 6) as expected for an anion selective channel under the present recording conditions ([Cl^-]_o = 148·8 mM; [Cl^-]_i = 146 mM). Similarly, the V_rev values determined for GABA acting at RDL (-34 ± 2 mV, n = 6), RDL_M314S (-33 ± 3 mV, n = 6) and RDL_M314N (-32 ± 2 mV, n = 5) receptors expressed in oocytes (not shown) are in accord with the equilibrium potential for chloride ions in these cells (Chen et al. 1994). The similarity in V_rev values across wild-type and mutant receptors suggests that the permeabilty of Cl^- relative to other ions present is not significantly affected by the mutations.

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Figure 1. GABA-evoked responses recorded under whole-cell voltage-clamp from Drosophila S2 cells transiently transfected with the cDNAs encoding the wild-type RDL and mutant RDL GABA receptors Mean current-voltage (I-V) relationship for GABA (top), together with exemplar current records (bottom), obtained from wild-type (A), RDLM314N (B) and RDLM314S (C) GABA receptors. The graphs depict normalized peak current amplitude (relative to the current obtained at -60 mV, ordinate) as a function of holding potential (abscissa). The recordings were performed with an approximately symmetrical distribution of Cl- ions across the cell membrane ([Cl-]o = 148·8 mM; [Cl-]i = 146 mM). Note that in each instance, currents invert in sign at a holding potential of approximately 0 mV and that the I-V relationship displays modest outward rectification. Data points represent the means of observations made from 6-7 S2 cells. Vertical lines, where exceeding the size of the symbol, indicate the S.E.M. In the representative current traces, leakage currents in the absence of GABA have been subtracted.

Changing the nature of TM2 located amino acids may result in a receptor which exhibits an increased probability of spontaneous channel openings in the absence of GABA. Such spontaneously open channels may be subject to positive allosteric regulation by certain modulators, as documented for homomeric receptors formed by murine ₃ GABA_A receptor subunits (Wooltorton et al. 1997). Under such circumstances, the distinction between the GABA-modulatory and GABA-mimetic activities of anaesthetics is blurred. However, in the present study, the average resting conductance of stage V or VI oocytes expressing RDL (0·81 ± 0·08 µS, n = 4), RDL_M314S (0·83 ± 0·09 µS, n = 6), or RDL_M314N (1·15 ± 0·19 µS, n = 6) was not significantly different (two-way ANOVA, P = 0·21) from that of sham injected oocytes (1·08 ± 0·2 µS, n = 5). Similarly, the average resting membrane conductances of S2 cells expressing RDL (1·88 ± 0·56 nS, n = 6), RDL_M314S (2·16 ± 0·77 nS, n = 5), or RDL_M314N (2·0 ± 0·18 nS, n = 6) receptors, did not differ appreciably. Moreover, the application of picrotoxin (1 µM) to oocytes or to S2 cells expressing wild-type or mutant RDL receptors did not induce an outward current (V_h = -60 mV), or reduce the resting membrane conductance of the cell. Collectively, these observations suggest that upon expression, both wild-type and mutant invertebrate receptors have a low probability of channel opening in the absence of GABA and therefore that GABA-mimetic activity can be unambiguously discerned (see below).

Propofol

Propofol (0·3-60 µM) produced a concentration-dependent enhancement of GABA-evoked currents recorded from oocytes expressing _632L receptors with a calculated EC₅₀ of 8·7 ± 0·5 µM and an E_max of 180 ± 26 % (n = 7; Fig. 2 and Table 1). Higher concentrations (100-300 µM) of propofol were associated with a reduced enhancement of the GABA-evoked response resulting in a bell-shaped concentration- response relationship (Fig. 2). That the peak current in the presence of a maximally effective concentration of propofol exceeded that evoked by a saturating concentration of GABA alone may imply that the latter is a partial agonist. However, it should be recognized that the kinetics of agonist application in the oocyte system are relatively slow (see Methods). Receptor desensitization during the rising phase of the inward current response to GABA would be likely to result in the truncation of the response to GABA at high concentrations, decreasing the apparent efficacy of the agonist. Whether the bell-shaped concentration-response curve to propofol (and indeed other anaesthetics, see below) can be attributed to enhanced desensitization or channel blockade cannot be resolved from the present data.

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Figure 2. The influence of a transmembrane amino acid (residue 289) on the allosteric effects of propofol at mammalian GABAA receptors A, traces illustrating the marked potentiation by propofol (60 µM) of inward currents evoked by GABA at EC10 (0·8 µM) recorded from an oocyte expressing human 632L GABAA receptors. Note that the pre-application of propofol alone is associated with an inward current (GABA-mimetic) response. B, records depicting the greatly reduced effect of propofol (60 µM) upon current responses evoked by GABA (EC10 = 1·8 µM) acting at human mutant 63N289M2L GABAA receptors. In addition, the GABA-mimetic activity of propofol is absent. In this and all subsequent figures, the period of drug application is indicated by horizontal bars above the current records. C, graphical depiction of the relationship between the concentration of propofol in the medium (abscissa, logarithmic scale) and the peak amplitude of the GABA evoked current (ordinate, linear scale) expressed relative to maximal current evoked by a saturating concentration (1 mM) of GABA for 632L () and 63N289M2L () GABAA receptors. Also plotted are the peak current responses evoked by propofol alone at 632L () and 63N289M2L () GABAA receptors. Each point represents the mean of data obtained from 4-7 oocytes and vertical lines indicate the S.E.M. Note that the EC50 values for propofol were obtained curve fits constrained to the ascending limb of the concentration-effect relationship; illustrated curves are 'free-hand' and have no theoretical significance.

In the absence of GABA, propofol (10-300 µM) induced a concentration-dependent inward current response which we have previously demonstrated to result from the anaesthetic directly gating the GABA_A receptor-channel complex (Belelli et al. 1996). At the highest concentration tested (300 µM), propofol induced a current amounting to 41 ± 4 % (n = 7) of the current induced by a saturating concentration of GABA (Fig. 2).

Propofol (EC₅₀ = 8 µM) is also a positive allosteric modulator of a splice variant form of the RDL GABA receptor (DRC 17-1-2) expressed in oocytes (Chen et al. 1994; Belelli et al. 1996). Acting at the RDL receptor, this anaesthetic is approximately threefold less potent (EC₅₀ = 25 ± 2 µM; n = 4) than at DRC 17-1-2 and human _632L receptors and exhibits a reduced maximum effect (E_max = 85 ± 6 %) in comparison with the human receptor (Fig. 3; Table 1). In contrast to its effects on mammalian GABA_A receptors, propofol ( 300 µM) does not directly gate the invertebrate RDL GABA receptor (Fig. 3). This result is in accord with that previously reported for the DRC 17-1-2 receptor (Belelli et al. 1996). The interaction of the general anaesthetic etomidate with both mammalian GABA_A receptors and the Drosophila GABA receptor is greatly influenced by the nature of a single amino acid located within the TM2 domain, the putative channel forming region of mammalian -subunits (₁, S290; ₂, N289; ₃, N289) and the RDL receptor (RDL _M314) (Belelli et al. 1997; McGurk et al. 1998). In the present study, the co-expression in oocytes of ₆- and _2L-subunits with a ₃-subunit in which the transmembrane asparagine residue had been mutated to methionine (the homologous residue in the RDL receptor) resulted in a receptor at which propofol as a GABA modulator was modestly less potent (EC₅₀ = 17 ± 2 µM, n = 4) and substantially less efficacious (E_max = 39 ± 2 %, n = 4) than with the wild-type (_632L) receptor (Fig. 2 and Table 1). Furthermore, high concentrations of propofol ( 300 µM) did not directly gate the _63N289M2L receptor (Fig. 2). Hence, in the latter respect, the mutant mammalian receptor now resembled the wild-type RDL receptor.

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Figure 3. The influence of a transmembrane amino acid (residue 314) on the allosteric effects of propofol acting at an invertebrate (RDL) GABA receptor A, traces illustrating the enhancement by propofol (100 µM) of inward currents evoked by GABA at EC10 (5·5 µM) recorded from an oocyte expressing the wild-type RDL GABA receptor. Note that the pre-application of propofol does not produce an inward current (GABA-mimetic) response. B, records depicting the effects of propofol (100 µM) at mutant RDLM314N receptors. Potentiation of responses evoked by GABA (EC10 = 1·4 µM) is apparent and additionally, upon pre-application, propofol exerts a direct GABA-mimetic effect. C, records depicting the effects of propofol at mutant RDLM314S receptors. Note the pronounced activation of the receptor complex by the pre-application of a relatively low concentration (30 µM) of propofol. As a consequence of the large GABA-mimetic activity, the potentiation of currents evoked by GABA (EC10 = 1·2 µM) is reduced in comparison with wild-type and RDLM314N GABA receptors. D, graphical depiction of the relationship between the concentration of propofol in the medium (abscissa, logarithmic scale) and the peak amplitude of GABA- or propofol-evoked current responses (ordinate, linear scale) expressed relative to maximal current evoked by a saturating concentration (1 mM) of GABA at RDL (circles), RDLM314N (triangles) and RDLM314S (squares) GABA receptors. Filled symbols relate to the propofol-induced enhancement of peak inward current responses to GABA, whereas open symbols indicate the GABA-mimetic activity of the anaesthetic. Each point represents the mean of data obtained from 3-4 oocytes and vertical lines indicate the S.E.M. Note that the EC50 values for the modulatory and agonist activities of propofol were obtained curve fits constrained to the ascending limb of the appropriate concentration-effect relationship; illustrated curves are 'free-hand' and have no theoretical significance.

The reciprocal mutation of the methionine residue of the invertebrate receptor to an asparagine (RDL_M314N) or a serine residue (RDL_M314S; the homologous residue of the mammalian ₁-subunit) increased the potency of propofol as a GABA receptor modulator (assessed in oocytes) by approximately 4- and 17-fold, respectively (RDL_M314N, EC₅₀ = 5·9 ± 0·5 µM; RDL_M314S, EC₅₀ = 1·5 ± 0·1 µM; versus RDL EC₅₀ = 25 ± 2 µM; all n = 4), but had little or no effect on the magnitude of the maximum potentiation produced (RDL_M314N, E_max = 71 ± 3 %; RDL_M314S, E_max = 83 ± 5 %; versus RDL E_max = 85 ± 6 %; all n = 4; see Fig. 3 and Table 1). However, these mutations produced a more fundamental change in the effects of propofol at the RDL GABA receptor. Hence, when applied to oocytes expressing mutant RDL receptors, propofol (3-300 µM) evoked concentration-dependent inward currents in the absence of GABA (Fig. 3). In the case of the RDL_M314S receptor, the maximal response to propofol was comparable to that evoked by a saturating concentration (1 mM) of GABA (EC₅₀ = 14·6 ± 0·3 µM, E_max = 104 ± 11 %, n_H = 2·1 ± 0·1, n = 4). The effects of propofol at the RDL_M314N receptor were less pronounced, both in terms of potency and maximal effect (EC₅₀ = 63 ± 4 µM, E_max = 25 ± 2 %, n_H = 1·8 ± 0·3, n = 4; see Table 1 and Fig. 3). The Hill slopes associated with anaesthetic activation of these mutant receptors suggest the co-operative involvement of at least two molecules of propofol in this effect. The ability of propofol to activate the mutant RDL receptors was confirmed in additional experiments conducted upon S2 cells. In this system, the brief local application of propofol (10 µM for RDL_M314S, 100 µM for RDL_M314N, 1·4 × 10⁵ Pa, 0·017 Hz, V_h = -60 mV) evoked transient inward currents from cells directed to express either the RDL_M314S or the RDL_M314N receptor, whereas the wild-type receptor was refractory to activation by the anaesthetic (Fig. 4). In both oocytes and S2 cells, the anaesthetic-induced currents mediated by the mutant RDL receptors were blocked by the co-application of the GABA antagonist picrotoxin (1 µM; Fig. 5). We have previously demonstrated that the GABA-evoked current associated with the activation of the RDL receptor in oocytes is mediated by the transmembrane flux of chloride ions (Chen et al. 1994 ). For oocytes expressing the RDL_M314S (Fig. 5) or the RDL_M314N receptor (not shown), the reversal potential of the currents induced by propofol (10-100 µM) was -34 ± 0·05 mV (n = 4) and -27 ± 3 mV (n = 4), respectively. These values are very similar to those determined for the appropriate control obtained with 2 µM GABA (i.e. RDL_M314S = -33 ± 3 mV, n = 6; RDL_M314N = -32 ± 2 mV, n = 5; Fig. 5). Additionally, the reversal potential of propofol-evoked currents recorded from S2 cells expressing either RDL_M314S or RDL_M314N receptors (-0·25 ± 1 mV, n = 4 and 0·6 ± 0·6 mV, n = 4, respectively) corresponded to those of GABA-evoked currents (1 ± 0·5 mV, n = 7 and 0·9 ± 0·8 mV, n = 6, respectively) and are consistent with chloride ions mediating these anaesthetic-evoked currents (see above). Collectively, these observations suggest that propofol can directly gate the mutant receptors.

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Figure 4. Propofol-evoked responses recorded under whole-cell voltage-clamp from Drosophila S2 cells transiently transfected with the cDNAs encoding mutant RDLM314N (A) and RDLM314S (B) GABA receptors The panels present the mean current-voltage (I-V) relationship for propofol (top), together with current records obtained from individual S2 cells (bottom). The graphs depict normalized peak current amplitude (relative to the current obtained at -60 mV, ordinate) as a function of holding potential (abscissa). The recordings were performed with an approximately symmetrical distribution of Cl- across the cell membrane ([Cl-]o = 148·8 mM; [Cl-]i = 146 mM). Note that in each instance, the reversal potential of the propofol-induced current is approximately 0 mV and that the I-V relationship displays modest outward rectification. Data points represent the mean of observations made from 4 S2 cells. Vertical lines, where exceeding the size of the symbol, indicate the S.E.M. In the representative current traces, leakage currents in the absence of propofol have been subtracted.

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Figure 5. Propofol directly activates mutant invertebrate RDLM314S GABA receptors A, traces illustrating inward current responses elicited by propofol (30 µM) acting at RDLM314S receptors expressed in Xenopus laevis oocytes. The control response to propofol (left) was strongly suppressed, in a reversible manner, by the bath application of picrotoxin (1 µM; right). B, current-voltage relationships for current responses to GABA or propofol mediated by the RDLM314S GABA receptor expressed in oocytes. The concentrations of agonist used produced well-maintained inward current responses at a holding potential of -60 mV. A voltage ramp (-90 to +10 mV) applied during the agonist-evoked response yielded the I-V relationship. Leakage currents obtained in the absence of GABA, or for propofol in the presence of a saturating concentration of picrotoxin, have been subtracted from the appropriate total currents. Note the currents elicited by GABA and propofol share a common reversal potential (~-34 mV) that corresponds to ECl for Xenopus laevis oocytes.

Pentobarbitone

Acting at wild-type _632L GABA_A receptors expressed in oocytes, pentobarbitone (3 µM to 100 µM) produced a concentration-dependent enhancement of GABA (EC₁₀)-evoked currents with a calculated EC₅₀ of 38 ± 2 µM and an E_max of 143 ± 20 % (n = 7; see Fig. 6 and Table 1). Higher concentrations of pentobarbitone (300-600 µM) were associated with a reduced enhancement of the GABA-evoked response, resulting in a bell-shaped concentration- effect relationship (Fig. 6). In the absence of GABA, pentobarbitone (30 µM to 1 mM) induced a concentration-dependent inward current (Fig. 6) which we have previously demonstrated to result from the barbiturate directly gating the receptor-channel complex (Belelli et al. 1996). The calculated EC₅₀ for the agonist effect of the barbiturate was 175 ± 9 µM (n = 7) with an E_max of 135 ± 52 % and an n_H of 2 ± 0·2 indicative of co-operative binding (see Table 1).

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Figure 6. The influence of a transmembrane amino acid (residue 289) on the allosteric effects of pentobarbitone at mammalian GABAA receptors A, traces illustrating the enhancement by pentobarbitone (300 µM) of inward currents evoked by GABA at EC10 (1 µM) recorded from an oocyte expressing human 632L GABAA receptors. The pre-application of pentobarbitone alone is associated with a clear inward current (GABA-mimetic) response. B, pentobarbitone also potentiates GABA (EC10 = 5·0 µM)-evoked currents mediated by mutant 63N289M2L GABAA receptors. However, the GABA-mimetic activity of pentobarbitone is absent. C, graphical depiction of the relationship between the concentration of bath applied pentobarbitone (abscissa, logarithmic scale) and the peak amplitude of the GABA-evoked current (ordinate, linear scale) expressed relative to maximal current evoked by a saturating concentration (1 mM) of GABA for 632L () and 63N289M2L () GABAA receptors. Also plotted are the peak current responses evoked by pentobarbitone alone at 632L () and 63N289M2L () GABAA receptors. Each point represents the mean of data obtained from 4-7 oocytes and vertical lines indicate the S.E.M. The EC50 values for pentobarbitone were obtained from curve fits constrained to the ascending limb of the concentration-effect relationship; illustrated curves are 'free-hand' and have no theoretical significance.

It is already documented that pentobarbitone is also a positive allosteric modulator of the invertebrate RDL GABA receptor expressed in Xenopus laevis oocytes (Chen et al. 1994; Belelli et al. 1996). However, pentobarbitone does not directly gate the RDL receptor, a feature which differs from the well established GABA-mimetic effect of this anaesthetic at the mammalian GABA_A receptor (Chen et al. 1994; Belelli et al. 1996). In the present study, the potency with which pentobarbitone enhanced GABA-evoked currents mediated by _63N289M2L GABA_A receptors expressed in oocytes was slightly reduced (EC₅₀ = 95 ± 7 µM, n = 4) compared with the wild-type receptor (Fig. 6 and Table 1). Maximal enhancement of GABA by the anaesthetic at wild-type and mutant (E_max = 143 ± 4 %) receptors was identical. However, the -subunit mutation exerted a profound effect upon the GABA-mimetic actions of pentobarbitone. At _63N289M2L receptors, pentobarbitone (1 mM) produced a current amounting to only 10 ± 3 % (n = 4) of that evoked by a saturating concentration of GABA. By comparison, current activation to 135 ± 52 % (n = 7) of the GABA maximum was produced by 600 µM pentobarbitone acting at _632L receptors. Hence, in this respect this mammalian mutant GABA_A receptor now more closely resembles the RDL GABA receptor.

For the RDL receptor, the exchange of the transmembrane methionine to asparagine (RDL_M314N) influences the actions of propofol and etomidate, but has no effect on the actions of pentobarbitone (McGurk et al. 1998). However, the interaction of pentobarbitone with RDL_M314S receptors was quite distinct from that observed for either RDL or RDL_M314N receptors. This mutation enhanced the GABA-modulatory potency of pentobarbitone approximately fivefold, but had little effect on the maximum potentiation produced (RDL_M314S, EC₅₀ = 179 ± 12 µM, E_max = 81 ± 6 %, n = 4; versus RDL, EC₅₀ = 837 ± 64 µM, E_max = 72 ± 7 %, n = 4; see Fig. 7 and Table 1).

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Figure 7. The influence of a transmembrane amino acid (residue 314) on the allosteric effects of pentobarbitone acting at an invertebrate (RDL) GABA receptor A, traces illustrating the enhancement by pentobarbitone (1 mM) of inward currents evoked by GABA at EC10 (8 µM) recorded from an oocyte expressing the wild-type RDL GABA receptor. Note that there is no inward current (GABA-mimetic) response associated with the pre-application of pentobarbitone. B, records depicting the effects of pentobarbitone (1 mM) at mutant RDLM314S receptors. Potentiation of responses evoked by GABA (EC10 = 0·5 µM) is retained and additionally, the pre-application of pentobarbitone elicits a direct GABA-mimetic effect. C, graphical depiction of the relationship between the concentration of pentobarbitone in the medium (abscissa, logarithmic scale) and the peak amplitude of GABA- (filled symbols) or pentobarbitone-evoked (open symbols) current responses (ordinate, linear scale) expressed relative to maximal current evoked by a saturating concentration (1 mM) of GABA at RDL (circles), RDLM314N (triangles) and RDLM314S (squares) GABA receptors. A GABA-mimetic effect of pentobarbitone was only observed for the RDLM314S receptor. Each point represents the mean of data obtained from 4 oocytes and vertical lines indicate the S.E.M. Note that the EC50 values for the modulatory and agonist activities of pentobarbitone were obtained curve fits constrained to the ascending limb of the appropriate concentration-effect relationship; illustrated curves are 'free-hand' and have no theoretical significance.

Additionally, pentobarbitone acquired agonist activity, evoking concentration-dependent (30 µM to 6 mM) inward current responses which, at the highest concentrations of anaesthetic tested, were comparable in amplitude to those elicited by a saturating concentration (1 mM) of GABA (pentobarbitone EC₅₀ = 1·5 ± 0·05 mM, E_max = 79 ± 4 %, n = 4; see Fig. 7 and Table 1). As for propofol this effect was associated with a Hill coefficient greater than 1 (n_H = 2·8 ± 0·01). In oocytes, such pentobarbitone-evoked currents were blocked by the co-application of picrotoxin (1 µM) and reversed in sign at a potential (-28·5 ± 2·2 mV, n = 4) similar to that of GABA-evoked responses (-33 ± 3 mV; not shown). Hence pentobarbitone can now gate this mutated receptor.

5-Pregnan-3-ol-20-one (53) and -hexachlorocyclohexane (-HCH)

Propofol and pentobarbitone are more potent allosteric modulators of mammalian GABA_A than invertebrate RDL receptors, and etomidate is inactive at the latter (McGurk et al. 1998). To determine whether the crucial residue may additionally influence the actions of other positive allosteric modulators, we quantified the actions of 53, an anaesthetic neurosteroid which exhibits a clear preference for mammalian versus invertebrate receptors, and an isomer of the insecticide -hexachlorocyclohexane (lindane), namely -HCH, which we have previously demonstrated to have a greater apparent efficacy at invertebrate than at mammalian GABA receptors (Belelli et al. 1996).

Acting at wild-type _632L receptors, 53 (10 nM to 3 µM) produced a concentration-dependent enhancement of currents evoked by GABA at EC₁₀ with a calculated EC₅₀ of 370 ± 10 nM and an E_max of 90 ± 10 % (n = 7, see Table 1). By contrast, we have previously reported that this steroid is much less effective at the RDL receptor, with the relatively high concentration of 10 µM increasing the amplitude of the current induced by an EC₁₀ concentration of GABA to only 25 % of the GABA maximum (McGurk et al. 1998; Table 1). However, mutation of the ₃ asparagine to the homologous methionine residue of the RDL receptor had little effect on the GABA-modulatory effects of the neurosteroid at _{63N289 M2L} receptors (EC₅₀ = 290 ± 40 nM, E_max 125 ± 21 %, n = 5). Similarly, the mutation of the transmembrane methionine of the RDL receptor to asparagine or serine did not influence the modest GABA-enhancing actions of 10 µM 53 (McGurk et al. 1998 and Table 1).

-HCH enhances GABA at both mammalian and invertebrate GABA receptors, but is not GABA mimetic (Belelli et al. 1996). In the present study, -HCH enhanced GABA (EC₁₀)-evoked currents recorded from oocytes expressing the RDL receptor with an EC₅₀ of 3·1 ± 0·1 µM and an E_max of 98 ± 9 % (n = 3, Fig. 8). These values are similar to those reported for the DRC 17-1-2 receptor and hence this compound does not discriminate between the splice variants of the invertebrate receptor (Belelli et al. 1996). Relatively high concentrations of -HCH did not induce an inward current.

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Figure 8. The influence of a transmembrane amino acid (residue 314) on the allosteric effects of -hexachlorocyclohexane (-HCH) acting at an invertebrate (RDL) GABA receptor A, traces illustrating the enhancement by -HCH (10 µM) of inward currents evoked by GABA at EC10 (7 µM) recorded from an oocyte expressing the wild-type RDL GABA receptor. Note that there is no inward current (GABA-mimetic) response associated with the pre-application of -HCH. B, records depicting the effects of -HCH (10 µM) at mutant RDLM314S receptors. Potentiation of responses evoked by GABA (EC10 = 0·7 µM) is retained and additionally, the pre-application of -HCH elicits a large direct GABA-mimetic effect. C, graphical depiction of the relationship between the concentration of -HCH in the medium (abscissa, logarithmic scale) and the peak amplitude of GABA- (filled symbols) or -HCH-evoked ( for RDLM314S or for RDLM314N) current responses (ordinate, linear scale) expressed relative to maximal current evoked by a saturating concentration (1 mM) of GABA at RDL (), RDLM314S () and RDLM314N () GABA receptors. A GABA-mimetic effect of -HCH was only observed for the RDLM314S and RDLM314N receptors. Each point represents the mean of data obtained from 4 oocytes and vertical lines indicate the S.E.M. Note that the EC50 values for the modulatory and agonist activities of -HCH were obtained curve fits constrained to the ascending limb of the appropriate concentration-effect relationship; illustrated curves are 'free-hand' and have no theoretical significance.

Mutation of the transmembrane methionine to a serine residue produced a dramatic change in the effects of -HCH acting at the Drosophila receptor. Concentrations of -HCH 1 µM acting upon oocytes expressing the RDL_M314S receptor evoked a concentration-dependent (1-300 µM) inward current response (EC₅₀ = 10·5 ± 0·3 µM, n_H = 3·3 ± 1·1; E_max = 79 ± 9 %, n = 3) that was completely blocked by picrotoxin (10 µM) suggesting that -HCH has a GABA-mimetic action (Fig. 8). Similar to the effects of propofol and pentobarbitone (Figs 3 and 7), the direct activation of this receptor by -HCH was associated with a steep Hill coefficient, indicative of channel activation requiring more than one drug molecule. The acquisition of the GABA-mimetic activity by pentobarbitone and propofol acting at the mutant RDL receptors was accompanied by an increased apparent affinity of these anaesthetics for the 'GABA-modulatory site'. However, by contrast this mutation had little effect on the GABA-modulatory actions of -HCH (RDL wild-type, EC₅₀ = 3·1 ± 0·1 µM, E_max = 98 ± 9 %, n = 3; RDL_M314S, EC₅₀ = 1·1 ± 0·05 µM, E_max = 57 ± 9, n = 3), the modest change in EC₅₀ and the reduction of the apparent E_max being a consequence of the juxtaposition of the GABA-modulatory and GABA-mimetic actions (Fig. 8).

The GABA_A modulatory EC₅₀ of -HCH acting at the RDL_M314N receptor was little changed (EC₅₀ = 3·9 ± 0·1 µM, n = 3) versus the wild-type RDL receptor, although the maximum potentiation produced was reduced (E_max = 56 ± 2 %, n = 3; Fig. 8 and Table 1). Acting at oocytes expressing RDL_M314N receptors, -HCH, in the absence of GABA, induced a small inward current (for 30 µM -HCH, E_max = 6 ± 2 %, n = 3) which was completely blocked by the co-application of 1 µM picrotoxin (Fig. 8 and Table 1). Hence, -HCH can directly activate this form of the receptor but with a much reduced apparent efficacy versus the RDL_M314S receptor.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

A number of general anaesthetics exhibit both GABA-modulatory and GABA-mimetic actions at the GABA_A 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 GABA_A 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 - or -subunit into binary complexes of -GABA_A subunits (Zhu et al. 1996; Davies et al. 1997; cf. Whiting et al. 1997). Similarly, ₄-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 GABA_A 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 GABA_A receptor (reviewed by Tanelian et al. 1993; Sieghart, 1995). In addition, mutations of the -subunit that greatly diminish the potency of GABA have no effect upon the direct effects of pentobarbitone (Amin & Weiss, 1993). Moreover, the expression of ₁- or ₃-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, -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 GABA_A receptor (_632L) 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 ₃-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 ₂ and ₃, or the serine of the ₁-GABA_A receptor subunit. Acting at RDL_M314S 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 -HCH also directly activated the RDL_M314S 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 -HCH also activated the RDL_M314N receptor-channel complex, although with a reduced maximal effect compared with that observed at the RDL_M314S receptor. By contrast, pentobarbitone does not activate the RDL_M314N 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 (RDL_M314S, RDL_M314N) revealed a GABA-mimetic action of -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 ₁- or ₃-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 RDL_M314S and RDL_M314N 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 ₁ (serine)- and ₂- or ₃ (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 GABA_A and glycine receptor -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 ₁-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 GABA_A receptor has not been systematically investigated to date. The allosteric effects of general anaesthetics at both GABA_A 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 GABA_{A 2}-subunit with a ₁-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 GABA_A, 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 ₇-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 GABA_A 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 GABA_A receptor -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.

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