Molecular volume determines the activity of the halogenated alkane bromoform at wild-type and mutant GABAA receptors

doi:10.1016/S0006-8993(02)03748-4

Brain Research

Volume 960, Issues 1–2, 17 January 2003, Pages 36-41

https://doi.org/10.1016/S0006-8993(02)03748-4 Get rights and content

Abstract

The GABA_A receptor is an important target for a variety of general anesthetics, including halogenated ethers such as isoflurane and halogenated ethers such as chloroform. Bromoform is a halogenated alkane that exhibits anesthetic properties and has been shown by X-ray crystallography to bind to model anesthetic targets. In this study we report the ability of bromoform to potentiate GABA-induced current in recombinant GABA_A receptors composed of α₁β₂γ_2s subunits. Recent studies have shown that specific point mutations in the transmembrane (TM) region of the GABA_A receptor α-subunit can selectively abolish the modulatory activity of specific general anesthetics, and that molecular volume is a key determinant of anesthetic activity. The action of bromoform was examined in a series of mutant receptors and compared with the activity profiles of three other volatile anesthetics. The pharmacological profile of bromoform at the mutant receptors used in this study was similar to that seen with halothane, and distinct from that observed for isoflurane and chloroform. The molecular volume of bromoform is closest to that of halothane, and therefore our data are consistent with the idea that molecular volume is an important determinant of inhaled anesthetic activity at the GABA_A receptor.

Introduction

The γ-aminobutyric acid_A receptor (GABA_A-R) is a heteropentameric ligand-gated ion channel that functions as the primary fast inhibitory receptor in the central nervous system. Clinically relevant concentrations of several different classes of volatile anesthetics, including halogenated ethers such as isoflurane and halogenated alkanes such as chloroform, potentiate GABA_A-R function [5], [6], [7], [8], [9], [10], [11], [12]. Multiple subunits of the GABA_A-R have been identified but only a few of the myriad possible configurations of the receptor are actually found in the mammalian brain. Of these, the most abundant receptor subtype is α₁β₂γ_2s[4].

It has been suggested that volatile anesthetics act at a common binding pocket within the transmembrane domain of the GABA_A-R α subunit [7]. The purpose of this study was to test this hypothesis further by examining the ability of bromoform to potentiate agonist-induced currents in a series of receptors harboring mutations within transmembrane regions 2, 3 and 4 (TM2,3,4) that have been shown to alter receptor modulation by a range of volatile anesthetics [6], [7], [9], [10], [11], [12]. Bromoform (CHBr₃) is a compound that exhibits anesthetic activity and has been shown to interact with model anesthetic target sites [3]. It provides a unique opportunity to examine the effect of molecular volume in determining anesthetic activity, as it is a chemical homologue of the halogenated alkane anesthetic chloroform but is similar in size to halothane (Table 1), two compounds that have been shown to have dissimilar pharmacological profiles [6], [7].

Section snippets

Site directed mutagenesis

Point mutations were created in the cDNA encoding the human GABA_A receptor α1 subunit using the quikchange method, as previously described, and sequences of the mutated cDNA inserts were confirmed by automated fluorescent DNA sequencing of the complete receptor subunit insert [6] (Cornell University, Ithaca, NY and Rockefeller University, NY, USA). The mutagenic primer sequences are available upon request. GABA_A receptors were expressed in HEK 293 cells; these were maintained and transfected as

Modulation of agonist-induced chloride currents by bromoform in recombinant wild-type GABA_A receptors

Full GABA concentration–response curves were determined in cells expressing the α₁β₂γ_2s wild-type GABA_A-R. A typical response from a single cell is shown in Fig. 1A. The currents activated by at least seven concentrations of GABA were expressed as a fraction of the maximal GABA response, and these normalized values were fitted by a Hill equation. The EC₅₀ value for GABA on the wild-type α₁β₂γ_2s receptor was 23±3 μM. The ability of bromoform to modulate GABA-induced currents was quantified by

Discussion

In the present study, we assessed the ability of the halogenated alkane, bromoform, to potentiate agonist-induced current in wild-type α₁β₂γ_2s GABA_A receptors and in a series of receptors harboring mutations in regions that have been shown to be critical for anesthetic sensitivity [9], [10], [11], [6].

Conclusion

A comparison of the activity of the four anesthetic compounds studied across the panel of four mutants suggests that molecular size is the key molecular determinant for activity of these agents in the GABA_A-R (Fig. 1C), since bromoform closely resembles halothane rather than chloroform in this respect. The ability of bromoform to potentiate GABA_A receptors is of interest. not only with respect to its anesthetic actions, but should also prove illuminating once a high resolution structure of the

Acknowledgements

We acknowledge James R. Trudell for calculation of the polarizability of the compounds used in this study and Sunil Singh for converting the analysis macro.

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Research reportMolecular volume determines the activity of the halogenated alkane bromoform at wild-type and mutant GABAA receptors

Abstract

Introduction

Section snippets

Site directed mutagenesis

Modulation of agonist-induced chloride currents by bromoform in recombinant wild-type GABAA receptors

Discussion

Conclusion

Acknowledgements

Biophys. J.

Neurosci. Lett.

Biophys. J.

Neuropharmacology

Neuropharmacology

GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits

J. Comp. Neurol.

Research report
Molecular volume determines the activity of the halogenated alkane bromoform at wild-type and mutant GABA_A receptors

Modulation of agonist-induced chloride currents by bromoform in recombinant wild-type GABA_A receptors

GABA_A-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits