Modulation of a recombinant invertebrate γ‐aminobutyric acid receptor‐chloride channel complex by isoflurane: effects of a point mutation in the M2 domain

1 Inhalational anaesthetics modulate ligand‐gated ion channels at clinical concentrations. In this paper we address submolecular mechanisms for γ‐aminobutyric acid (GABA) receptor modulation by isoflurane. 2 Wild‐type Drosophila melanogaster homo‐oligomeric GABA receptors were characterized and compared with an ion‐channel mutant (alanine substituted to a serine in M2) by means of two‐electrode voltage‐clamp in membrane‐invariant Xenopus oocytes. 3 Both channel receptor isoforms generated outwardly rectifying, bicuculline‐insensitive currents with reversal potentials characteristic of a chloride current. 4 As previously shown, the point mutation in the M2 domain conferred a profound resistance to the blocking action of 10 μ M picrotoxinin (PTX): circa 7 fold reduction at the GABA EC 20 . 5 Isoflurane, 195–389 μ M , enhanced GABA conductance in both receptor variants by significantly increasing the affinity of the agonist for its receptor without changing Hill slope or maximal response. Relative potencies were statistically indistinguishable. 6 Isoflurane concentration‐response curves (on circa GABA EC 25 ) demonstrated that enhancement was effected at around 100–195 μ M for both receptor subtypes, but a dramatic divergence was evident at concentrations above 400 μ M : wild‐type receptors exhibited concentration‐dependent block, whilst mutant conductances continued to increase over the same concentration range, showing no tendency to saturate (up to 3330 μ M ). 7 The above divergence was not attributable to differential desensitization: neither wild‐type nor mutant conductance desensitized significantly ( P >0.05) in the absence or presence of anaesthetic. 8 This work demonstrates that modulatory sites for anaesthetic are present on a relatively primitive insect ion channel. 9 The depression of GABA response at high isoflurane concentrations, in WT receptors, (typical of a variety of anaesthetic agents) may reflect low affinity channel block via the PTX site. 10 The non‐saturable enhancement of chloride conductances, when the PTX site is mutated, is not consistent with topical proposals that inhalational anaesthetics (stereoselectively) occupy a finite number of sites on these membrane spanning proteins.British Journal of Pharmacology (1997) 122 , 726–732; doi: 10.1038/sj.bjp.0701417