The effects of a point mutation of the β2 subunit of GABAA receptor on direct and modulatory actions of general anesthetics

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Abstract

The γ-aminobutyric acid type A receptor (GABAA receptor) sites involved in the direct and modulatory actions of general anesthetics remain to be elucidated. The mutation of tyrosine at position 157 in the β2 GABAA receptor subunit was reported to reduce sensitivity to activation by GABA, but not pentobarbital. We examined whether this mutation of the β2 subunit (Tyr157→Phe) affects the direct and modulatory actions of other general anesthetics such as propofol and etomidate. Using the two-electrode voltage clamp method, we recorded Cl current in Xenopus oocytes expressing α1β2γ2s and α1-mutated β2γ2s subunits. The mutation of the β2 subunit reduced the apparent affinity for propofol. However, the mutation had no effect on both the direct actions of pentobarbital and etomidate or on the modulatory actions of pentobarbital, propofol and etomidate. These results suggest that unique loci may exist for the direct action of propofol and that the GABA binding site may not mediate the modulatory actions of general anesthetics at GABAA receptors.

Introduction

The γ-aminobutyric acid type A receptor (GABAA receptor) is a major inhibitory receptor in the central nervous system and has attracted lots of interest as an important site of action of sedatives and anesthetics (Keane and Biziere, 1987; Sieghart, 1995; Whiting et al., 1995). GABA, the inhibitory neurotransmitter, induces an inward Cl current which results in membrane hyperpolarization and reduced neuronal excitability on binding to GABAA receptors. The major sedatives, such as benzodiazepines and general anesthetics, enhance the action of GABA at GABAA receptors (Franks and Lieb, 1993; Tanelian et al., 1994; Sieghart, 1995). The intravenous anesthetics, such as barbiturates, etomidate, propofol and alphaxalone, and volatile anesthetics, like isoflurane and enflurane, directly activate GABAA receptors at high concentrations and at lower concentrations, these drugs positively modulate the action of GABA as seen with benzodiazepines (Orser et al., 1994; Sieghart, 1995). Accumulated evidence suggests that the sites for the modulatory actions of these anesthetics may be distinct from the GABA binding sites and that these anesthetics exert their actions by binding to allosteric sites at GABAA receptors (DeLorey and Oslen, 1992; Tanelian et al., 1994; Sieghart, 1995; Lavoie and Twyman, 1996; Davies et al., 1997a).

The GABAA receptor is presumably a hetero-pentameric receptor and 19 genes of the subunits have been identified (Barnard et al., 1987; Wang et al., 1994; Sieghart, 1995; Whiting et al., 1995; Chang et al., 1996; Davies et al., 1997b). Although molecular biology studies have revealed the heterogeneity of GABAA receptors, the sites of action of GABA and anesthetic drugs remain to be elucidated. By using recombinant techniques and molecular engineering to change the primary structure of the cloned GABAA receptor subunit, it is possible to deduce the subunit and even the specific sites of subunits involved in the action of general anesthetics.

It has been reported that point mutations in the amino-terminal region of the β2 subunit result in impaired activation of GABAA receptors by GABA but not by pentobarbital, suggesting that GABA and pentobarbital occupy distinct sites to cause the direct activation of GABAA receptors (Amin and Weiss, 1993). In order to clarify whether the site involved in the action of GABA is related to the site at which anesthetics other than pentobarbital exert a direct action and also to the site of modulatory action of anesthetics, we electrophysiologically examined the effects of a point mutation of the β2 subunit (tyrosine to phenylalanine at position 157 of the amino acid sequence) on the agonistic as well as modulatory actions of anesthetic drugs. The α1β2γ2s or α1-mutated β2(mβ22s subunit of the GABAA receptor was expressed in Xenopus oocytes. First, the effects of the mutation on the agonistic action of GABA, as well as on the direct action of anesthetics, such as pentobarbital, propofol and etomidate, were studied. Secondly, the effects of the mutation on the modulatory action of the anesthetics on GABA-induced Cl currents were analyzed.

Section snippets

Expression vectors and in vitro transcription

Mouse cDNAs encoding for α1, β2, and γ2s GABAA receptor subunits were kindly provided by Dr. J. Yang (University of Rochester, Rochester, USA). All subunits were subcloned into transcription vector, pBluescriptMXT, in which multiple cloning sites were flanked by β-globins of Xenopus laevis for better expression in Xenopus oocytes. The cloned DNAs were confirmed as coding for each subunit by identification of the size of the fragments digested by restriction enzymes. The pBluescriptMXT

Results

Tyrosine at position 157 in the β2 subunit was mutated to phenylalanine. Either α1β2γ2s (wild-type) or α12γ2s GABAA receptor subunit combinations were expressed in oocytes. The expression of functional recombinant receptors was confirmed by the presence of GABA-induced Cl currents which were blocked by bicuculline and picrotoxin (Sieghart, 1995; Whiting et al., 1995). The expression of the γ2s subunit in the recombinant receptors was confirmed by the positive modulation of the GABA-activated

Discussion

General anesthetics are known to directly activate and modulate GABAA receptors at different concentration ranges; however, the exact sites of these dual actions are still not known (MacDonald, 1994; Zimmerman et al., 1994). Amin and Weiss (1993)showed that the mutation of the tyrosine (Tyr) residue to phenylalanine (Phe) in the β2 subunit at position 157 of the amino-terminal region dramatically reduced GABA sensitivity, but had no effect on pentobarbital sensitivity. The results suggest that

Acknowledgements

This work was supported by the Grant-in-Aid from the Ministry of Education, Science, Sport and Culture in Japan. The authors are grateful to Drs. Jay Yang and Kyeong Tae Min for providing the mouse GABAA receptor subunit clones and for helping with this project.

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