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1 The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant g-aminobutyric acid A (GABA A ) receptor subunits. 2 Currents mediated by recombinant receptors with the ternary subunit composition a x b y g 2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC 10 , were enhanced by etomidate in a manner that was dependent upon the identity of both the a and b subunit isoforms. 3 For the b 2 -subunit containing receptors tested, the EC 50 for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 mM) was little aected by the nature of the a subunit present within the hetero-oligomeric complex. However, replacement of the b 2 by the b 1 subunit produced a 9 ± 12 fold increase in the etomidate EC 50 (6 to 11 mM) for all a-isoforms tested. 4 For a 1 , a 2 and a 6 , but not a 3 -subunit containing receptors, the maximal potentiation of GABAevoked currents by etomidate was greater for b 2 -than for b 1 -subunit containing receptors. This was most clearly exempli®ed by receptors composed of a 6 b 1 g 2L compared to a 6 b 2 g 2L subunits, where a maximally eective concentration of etomidate potentiated currents evoked by GABA at EC 10 to 28+2% and 169+4% of the maximal GABA response, respectively. 5 For a 1 subunit-containing receptors, the potency and maximal potentiating eect of either pentobarbitone or propofol was essentially unaected by the b subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5a-pregnan-3a-ol-20-one was marginally higher for b 1 rather than the b 2 subunit-containing receptor, although its maximal eect was similar at the two receptor isoforms. 6 The GABA-mimetic action of etomidate was supported by b 2 -but not b 1 -subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either b isoform. For b 2 -subunit containing receptors, both the agonist EC 50 and the maximal current produced by etomidate were additionally in¯uenced by the a isoform. 7 It is concluded that the subtype of b-subunit in¯uences the potency with which etomidate potentiates GABA-evoked currents and that the b isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the a-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced in¯uences may aid the identi®cation of the site that recognises etomidate. More generally, these results provide a clear example of structural speci®city in anaesthetic action.

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1. The pioneering work of Hans Selye over 50 years ago demonstrated that certain steroid metabolites can produce a rapid depression of central nervous system activity. 2. Research during the last 10 years has established that such effects are mediated by a nongenomic and specific interaction of these steroids with the brain's major inhibitory receptor, the GABAA receptor. 3. Here we describe the molecular mechanism of action of such steroids and review attempts to define the steroid binding site on the receptor protein. The therapeutic potential of such neurosteroids is discussed.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

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SUMMARYIn Helix aspersa, activation of the cerebral giant serotonin neurones (GSNs) evokes a biphasic, excitatory synaptic response in the M neurones of the buccal ganglia. Local application of serotonin to the current-clamped M neurones also evokes fast and slow depolarizing responses.The slow response is thought to be dependent on calcium ions, whereas sodium ions have been implicated in the fast response. Here we provide further evidence that the slow response results from an increase in conductance to calcium ions, and show that okadaic acid, an antagonist of protein phosphatases 1 and 2A, potentiates the effect of serotonin, suggesting that the response is phosphorylation dependent. Further, agents known to activate protein kinase C, such as 1-oleoyl-2-acetyl-rac-glycerol and active phorbol esters (but not an inactive one) were found to increase the calcium current (actually carried by barium ions) of the M neurones. Such data suggest that the slow synaptic response mediated by serotonin can occur by activation of protein kinase C and phosphorylation of the affected voltage-sensitive calcium channels, or some closely associated protein(s).