John A. Peters

Subunit-dependent interaction of the general anaesthetic etomidate with the γ-aminobutyric acid type A receptor

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 γ‐aminobutyric acidA (GABAA) receptor subunits. Currents mediated by recombinant receptors with the ternary subunit composition αxβyγ2L (where x=1,2,3 or 6 and y=1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the α and β subunit isoforms. For the β2‐subunit containing receptors tested, the EC50 for the potentiation of GABA‐evoked currents by etomidate (range 0.6 to 1.2 μm) was little affected by the nature of the α subunit present within the hetero‐oligomeric complex. However, replacement of the β2 by the β1 subunit produced a 9–12 fold increase in the etomidate EC50 (6 to 11 μm) for all α‐isoforms tested. For α1, α2 and α6, but not α3‐subunit containing receptors, the maximal potentiation of GABA‐evoked currents by etomidate was greater for β2‐ than for β1‐subunit containing receptors. This was most clearly exemplified by receptors composed of α6β1γ2L compared to α6β2γ2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28±2% and 169±4% of the maximal GABA response, respectively. For α1 subunit‐containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the β subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5α‐pregnan‐3α‐ol‐20‐one was marginally higher for β1 rather than the β2 subunit‐containing receptor, although its maximal effect was similar at the two receptor isoforms. The GABA‐mimetic action of etomidate was supported by β2‐ but not β1‐subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either β isoform. For β2‐subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the α isoform. It is concluded that the subtype of β‐subunit influences the potency with which etomidate potentiates GABA‐evoked currents and that the β isoform is a crucial determinant of the GABA‐mimetic activity of this compound. The nature of the α‐subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

Modulation of GABAa receptor activity by alphaxalone

1 The modulation of the γ‐aminobutyric acidA (GABAA) receptor by alphaxalone has been investigated by use of voltage‐clamp recordings from enzymatically isolated bovine chromaffin cells maintained in cell culture. 2 Alphaxalone (> 30 nm) reversibly and dose‐dependently potentiated the amplitude of membrane currents elicited by locally applied GABA (100 μm). The potentiation was not associated with a change in the reversal potential of GABA‐evoked currents and was not influenced by the benzodiazepine receptor antagonist, Ro15‐1788 (300 nm). 3 At relatively high concentrations (> 1 μm), alphaxalone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor activation since they were reversibly suppressed by bicuculline (3 μm), dose‐dependently enhanced by phenobarbitone (100–500 μm), and had a similar reversal potential (∼ 0 mV) to currents elicited by GABA. Additionally, on outside‐out membrane patches, alphaxalone activated single channel currents with amplitudes and a reversal potential similar to those evoked by GABA. 4 Alphaxalone (30 nm‐1 μm) had no effect upon the amplitude of membrane currents elicited by locally applied acetylcholine (ACh) (100 μm). However, higher concentrations of alphaxalone (10–100 μm) reversibly suppressed ACh‐evoked currents, the IC50 for blockade being 20 μm. 5 The β‐hydroxy isomer of alphaxalone, betaxalone (100 nm‐1 μm), did not potentiate GABA‐induced currents, nor did higher concentrations of the steroid (10–100 μm) directly evoke a membrane current. However, over the latter concentration range, betaxalone suppressed the amplitude of currents elicited either by GABA or ACh. 6 The relevance of the present results to the anaesthetic action of alphaxalone is discussed together with the broader implications of steroidal modulation of the GABAA receptor.

Cloning and fnctional expression of an apparent splice variant of the murine 5-HT3 receptor A subunit

The polymerase chain reaction has been employed to isolate a cDNA encoding a functional 5-HT3 receptor subunit from the murine neuroblastoma cell line N1E-115. Overall, the amino acid sequence predicted from this clone demonstrates a 98% homology with the 5-HT3 receptor A subunit cloned from NCB-20 hybridoma cells. A deletion of 6 amino acid residues located within the putative large intracellular loop, which may result from alternative splicing, represents the principal difference between the two clones. Upon expression in Xenopus oocytes, the homo-oligomeric receptor displayed pharmacological properties which define it as a functional 5-HT3 receptor.

Recent advances in the electrophysiological characterization of 5-HT3 receptors

5-HT3 receptors are ligand-gated, cation-selective ion channels, mediating membrane depolarization and neuronal excitation. Established and potential therapeutic applications of selective 5-HT3 receptor antagonists, coupled with the localization of this receptor subtype within discrete areas of the CNS, have resulted in an intensification of research in this area. In this review, Jeremy Lambert and colleagues summarize recent developments in the electrophysiological characterization of 5-HT3 receptors, and comment upon the unresolved issue of 5-HT3 receptor heterogeneity.

The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques

1 The characteristics of transmembrane currents evoked by 5‐hydroxytryptamine (5‐HT) in the neuroblastoma x Chinese hamster brain cell line NCB‐20 and neuroblastoma clonal cell line N1E‐115 have been studied under voltage‐clamp conditions by the whole‐cell recording and outside‐out membrane patch modes of the patch‐clamp technique. 2 In 73% of NCB‐20 cells examined (n = 221), and all N1E‐115 cells studied (n = 80), 5‐HT (10 μm) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5‐HT reversed in sign at a potential of approximately −2mV and demonstrated inward rectification. 3 The reversal potential of 5‐HT‐induced currents (E5‐HT) recorded from either NCB‐20 or N1E‐115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E‐115 cells, reducing internal K+ concentration from 140 to 20 mm produced a positive shift in E5‐HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mm was associated with a negative shift in E5‐HT of about 37 mV. A large reduction in internal Cl− concentration (from 144 to 6 mm) had little effect on E5‐HT. 4 5‐HT‐induced currents of NCB‐20 cells were unaffected by methysergide (1 μm) or ketanserin (1 μm), but were reversibly antagonized by GR38032F (0.1–1.0 nm) with an IC50 of 0.25 nm. GR 38032F (0.3 nm) reduced 5‐HT‐induced currents in N1E‐115 cells to approximately 26% of their control value. 5 On outside‐out membrane patches excised from both NCB‐20 and N1E‐115 cells, 5‐HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nm) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5‐HT (1 μm) in N1E‐115 cells suggests that 5‐HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside‐out membrane patches to 5‐HT cannot at present be resolved into clear single channel events.

The interaction of general anaesthetics and neurosteroids with GABAA and glycine receptors

The positive allosteric effects of four structurally distinct general anaesthetics (propofol, pentobarbitone, etomidate and 5alpha-pregnan-3alpha-ol-20-one [5alpha3alpha]) upon recombinant GABA(A) (alpha6beta3gamma2L), invertebrate GABA (RDL) and glycine (alpha1) receptors expressed in Xenopus laevis oocytes have been determined. Propofol and pentobarbitone enhanced agonist (GABA or glycine as appropriate) evoked currents at GABA(A), glycine, and RDL receptors, whereas etomidate and 5alpha3alpha were highly selective for the GABA(A) receptor. Utilizing site-directed mutagenesis, we demonstrate that the nature of the interaction of propofol, pentobarbitone and etomidate (but not 5alpha3alpha) with mammalian and invertebrate ionotropic GABA receptors depends critically upon the nature of a single amino acid located in the second transmembrane region (TM2) of these receptors. These data are discussed in relation to the specificity of action of general anaesthetics.

Complementary regulation of anaesthetic activation of human (α6β3γ2L) and Drosophila (RDL) GABA receptors by a single amino acid residue

1 The influence of a transmembrane (TM2) amino acid located at a homologous position in human β1 (S290) and β3 (N289) GABAA receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2 GABA‐evoked currents mediated by human wild‐type (WT) α6β3γ2L or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated α6β3γ2L, but not RDL, receptors. 3 GABA‐evoked currents mediated by human mutant GABAA receptors expressing the RDL methionine residue (i.e. α6β3N289Mγ2L) were potentiated by propofol or pentobarbitone with ≈2‐fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4 Incorporation of the homologous GABAAβ1‐subunit residue in the RDL receptor (i.e. RDLM314S) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous β3‐subunit residue (i.e. RDLM314N) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5 The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (α6β3γ2L) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed.

An electrophysiological investigation of the properties of 5‐HT3 receptors of rabbit nodose ganglion neurones in culture

1 The biophysical and pharmacological properties of 5‐hydroxytryptamine (5‐HT)‐evoked currents in rabbit nodose ganglion neurones in culture have been determined by use of the whole‐cell and outside‐out membrane patch recording modes of the patch‐clamp technique. 2 In 49% of cells investigated the bath application of 10−5 m 5‐HT at negative holding potentials elicited an inward current. The whole‐cell response to 5‐HT reversed in sign (E5‐HT) at approximately − 2 mV and exhibited inward rectification. 3 The influence of various ion substitutions upon E5‐HT established that the 5‐HT‐evoked current is mainly mediated by a mixed Na+, K+ cation conductance with little or no contribution from Cl− ions. The omission of Ca2+ and Mg2+ from the extracellular solution enhanced the amplitude of the 5‐HT‐induced current. 4 On isolated outside‐out membrane patches, the bath application of 10−6 m 5‐HT induced single channel currents with a chord conductance of approximately 17 pS at − 70 mV and an average slope conductance of 19 pS over the range − 100 to − 40 mV. The 5‐HT‐induced single channels exhibited modest inward rectification and were reduced in frequency, but not amplitude, by the 5‐HT3 receptor antagonist metoclopromide (10−6 m). 5 The bath application of 5‐HT (3 × 10−7 − 3 × 10−5 m) to whole cells voltage clamped at − 60 mV produced dose‐dependent inward currents which were mimicked by 2‐methyl‐5‐HT and 1‐phenylbiguanide with equipotent molar ratios, relative to 5‐HT, of 2.5 and 32 respectively. 6 Whole‐cell inward currents produced by the local pressure application of 5‐HT (10−5 m) were unaffected by 10−6 m methysergide, 10−6 m ketanserin or 10−6 m citalopram, but were concentration‐dependently antagonized by the selective 5‐HT3 receptor antagonists tropisetron (IC50 = 4.6 × 10−11 m) ondansetron (IC50 = 5.7 × 10−11 m), and bemesetron (IC50 = 3.3 × 10−10 m). The response to 5‐HT was also blocked by the non‐selective antagonists metoclopramide (IC50 = 1.2 × 10−8 m), cocaine (IC50 = 8.3 × 10−8 m) and (+)‐tubocurarine (IC50 = 1.6 × 10−7 m).

Pharmacological characterization of the apparent splice variants of the murine 5-HT3 R-A subunit expressed in Xenopus laevis oocytes

The actions of 5-hydroxytryptamine3 (5-HT3) receptor agonists and antagonists have been determined on the recombinant murine 5-HT3 R-A and an apparent splice variant of this subunit, termed 5-HT3 R-AS. When expressed in Xenopus laevis oocytes, both forms of the subunit functioned as a homo-oligomeric complex and exhibited inward current responses to bath applied 5-HT. Analysis of the 5-HT concentration-response curve obtained with either homo-oligomer gave Hill coefficients greater than two, suggesting positive co-operativity within the receptor complex. The rank order of potency of a range of 5-HT3 receptor agonists [m-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT (2-Me-5-HT) > or = phenylbiguanide] was identical for both subunits. Indeed, with the exception of 2-Me-5-HT, for the agonists tested there was little difference across the subunits in either their potency, or the maximal current response that they elicited relative to 5-HT. Although 2-Me-5-HT exhibited a similar potency for both subunits, the maximal response evoked by this agonist at the 5-HT3 R-AS subunit was much reduced when compared to the 5-HT3 R-A subunit. The 5-HT-induced current mediated by either form of the subunit was inhibited by the 5-HT3 receptor selective antagonists BRL 46470, granisetron and ondansetron and the non-selective antagonists (+)-tubocurarine, metoclopramide and cocaine in a reversible and concentration-dependent manner. These antagonists did not discriminate between the subunits and their potencies were similar to those reported previously for 5-HT3 receptors native to murine neuronal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

MODULATION OF HUMAN RECOMBINANT GABAA RECEPTORS BY PREGNANEDIOLS

Utilising two point voltage-clamp techniques on Xenopus laevis oocytes expressing human (alpha 1 beta 1 gamma 2L) recombinant GABAA receptors, the GABA modulatory actions of six naturally occurring neurosteroids have been determined and compared with those of known positive allosteric modulators. The anaesthetic steroids 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one produced a concentration-dependent enhancement of the GABA-evoked current. The maximal enhancement of the agonist-induced response produced by these steroids was intermediate between that of pentobarbitone and diazepam, but much greater than that caused by bretazenil. For both the 5 alpha and 5 beta steroid a reduction of the 20 ketone group to form either the corresponding 20 alpha or 20 beta hydroxy steroid produced, in all cases, a reduction in potency and a decrease in the maximal effect. The relationship of steroid structure to these two parameters is considered. The influence of the alpha subtype (alpha x beta 1 gamma 2L, where x = 1, 2 or 3) for the behaviourally active 5 alpha-pregnan-3 alpha,20 alpha-diol is also determined. Although the maximal effect of the steroid is not influenced by the alpha subtype, the alpha 2-containing receptor exhibits a modest decrease (approximately 6-fold) in potency compared to alpha 1- and alpha 3-containing receptors. The results described here are discussed in relation to the distinct behavioural actions of the neurosteroids.