Modulatory and direct effects of propofol on recombinant GABAA receptors expressed in Xenopus oocytes: Influence of α- and γ2-subunits

doi:10.1016/S0006-8993(97)01334-6

Brain Research

Volume 784, Issues 1–2, 16 February 1998, Pages 179-187

https://doi.org/10.1016/S0006-8993(97)01334-6 Get rights and content

Abstract

Propofol (2,6-diisopropylphenol) is an intravenous general anaesthetic which can directly activate and positively modulate the GABA_A receptor. The effects of propofol on human recombinant GABA_A receptors were studied in Xenopus oocytes expressing either α₁β₂, α₁β₂γ_2L, or α₂β₂γ_2L receptor isoforms. In all receptor isoforms tested, propofol was able to potentiate the GABA-activated currents in a concentration-dependent manner. Although propofol potentiated both α₁β₂ and α₁β₂γ_2L receptor isoforms with equal affinity, the efficacy of propofol potentiation was markedly greater in the α₁β₂ receptor isoform. In contrast, potentiation of the α₂β₂γ_2L receptor isoform by propofol occurred with higher affinity and lower efficacy than in the α₁β₂γ_2L receptor isoform. Propofol directly activated all three receptor isoforms in a concentration dependent manner. Addition of the γ_2L subunit subtype to the α₁β₂ receptor isoform decreased receptor sensitivity to direct activation by propofol. Replacement of the α₁-subunit subtype with the α₂-subunit subtype increased receptor sensitivity to propofol's direct effects. These results suggest that the α-and γ_2L-subunit subtypes each have the ability to influence both the direct and modulatory actions of propofol on GABA_A receptor function.

Introduction

The GABA_A receptor is an important target involved in inhibitory neurotransmission within the central nervous system (CNS). Many anaesthetic agents dramatically alter GABA_A receptor function either by enhancing GABA-mediated activation of the receptor or by directly activating the receptor in the absence of GABA 4, 11, 16, 17, 34, 44. These actions are thought to play a major role in the mechanism of general anaesthesia.

Within the GABA_A receptor class, there exists a great deal of receptor heterogeneity, as different protein subunits (α_1–6, β_1–3, γ_1–3, δ, ε) can assemble in various combinations to form different receptor isoforms. In addition, GABA_A receptor subunit subtypes are unevenly distributed in brain regions and cell types throughout the CNS 6, 9, 10, 13, 21, 22, 25, 26, 32, 48. Receptor heterogeneity has been shown to exist in vivo as well as in vitro, with the α₁β₂γ₂ and α₂β_2/3γ₂ receptor isoforms being the two most common subunit combinations found in vivo [for review see Ref. [28]]. The effects of anaesthetic agents such as barbiturates and neurosteroids are influenced by the subunit composition of the GABA_A receptor 5, 14, 35, 37, 43. Differential effects of anaesthetic agents on different GABA_A receptor isoforms may explain the unique pharmacological properties of different anaesthetics. Moreover, differences in subunit dependence for anaesthetic modulation of receptor function may also help to elucidate the sites and/or mechanisms by which these drugs act on the GABA_A receptor.

Propofol is a relatively new general anaesthetic which is structurally unrelated to other known anaesthetics. Due to its favourable clinical properties, propofol has become one of the most popular anaesthetics in use today 3, 12, 23, 24, 40. Although the ability of propofol to act at the GABA_A receptor is well-established, the influence of subunit composition on propofol action is not very well understood. The β-subunit appears to contribute to the direct actions of propofol 5, 35. Recently, work from this laboratory showed that the presence or absence of the γ₂-subunit significantly influenced both the potency and efficacy of propofol to potentiate GABA_A receptor activation in receptor isoforms composed of α₂β₂ and α₂β₂γ_2L subunit combinations [33]. The purpose of this study, therefore, was to further examine the effects of α- and γ₂-subunit subtypes on the modulatory and direct effects of propofol on the GABA_A receptor.

Section snippets

Preparation of oocytes

Female Xenopuslaevis frogs (Xenopus, Ann Arbor, MI) were anaesthetized in 0.2% 3-aminobenzoic acid ethyl ester (Sigma Chemical, St. Louis, MO) dissolved in ice cold water. A small incision was made in the lower abdomen and one to two lobes of ovary were removed. From these ovarian pieces, stage V and VI oocytes were isolated, rinsed and stored in a physiological buffer solution containing (in mM): 88 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, 2.4 NaHCO₃, 5 HEPES, 2 sodium pyruvate, 0.5 theophylline,

Xenopus oocytes express functional GABA_A receptors

Oocytes injected with combinations of RNA encoding GABA_A receptor subunits were voltage-clamped at −60 mV. Perfusion of these oocytes with GABA produced inward currents which increased in amplitude as the GABA concentration increased. Uninjected oocytes did not respond to GABA. In other control experiments, GABA was applied to oocytes expressing α₁β₂ and α₁β₂γ_2L receptor isoforms in the presence of Zn²⁺. Application of GABA in the presence of 10 μM Zn²⁺ reduced the GABA response by greater than

Discussion

Propofol belongs to a group of anaesthetic, anxiolytic, and sedative–hypnotic agents which have a profound effect on GABA_A receptor function. Similar to benzodiazepines, barbiturates, alcohols, and the anaesthetic class of steroids, propofol can enhance GABA-mediated activation of the GABA_A receptor 2, 15, 30, 34. In addition to allosteric modulation of GABA_A receptor activation, general anaesthetics such as barbiturates, steroids, and propofol share the ability to directly activate the GABA_A

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Research reportModulatory and direct effects of propofol on recombinant GABAA receptors expressed in Xenopus oocytes: Influence of α- and γ2-subunits

Abstract

Introduction

Section snippets

Preparation of oocytes

Xenopus oocytes express functional GABAA receptors

Discussion

Brain Res.

Neuron

Neuroscience

Biochem. Pharmacol.

Neuroscience

J. Mol. Biol.

TINS

J. Biol. Chem.

Eur. J. Pharmacol.

Brain Res.

Brain Res.

Neurosci. Lett.

Neuron

Assembly of GABAA receptor subunits: α1β1 and α1β1γ2S subunits produce unique ion channels with dissimilar single-channel properties

J. Neurosci.

Subhypnotic doses of propofol possess direct antiemetic properties

Anesth. Analg.

The general anesthetic propofol enhances the function of γ-aminobutyric acid-coupled chloride channel in the rat cerebral cortex

J. Neurochem.

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

Br. J. Pharmacol.

Chronic ethanol consumption differentially alters the expression of γ-aminobutyric acidA receptor subunit mRNAs in rat cerebral cortex: Competitive, quantitative reverse transcriptase-polymerase chain reaction analysis

Mol. Pharmacol.

γ-Aminobutyric acid gating of Cl− channels in recombinant GABAA receptors

J. Pharmacol. Exp. Ther.

Subpopulations of GABA neurons in the dentate gyrus express high levels of the α1 subunit of the GABAA receptor

Hippocampus

Porpofol and emesis

Anaesthesia

Role of the β subunit in determining the pharmacology of human γ-aminobutyric acid type A receptors

Mol. Pharmacol.

Enhancement by propofol of the γ-aminobutyric acidA response in dissociated hippocampal pyramidal neurons of the rat

Anaesthesiology

Different subunit requirements for volatile and nonvolatile anesthetics at γ-aminobutyric acid type A receptors

Mol. Pharmacol.

Research report
Modulatory and direct effects of propofol on recombinant GABA_A receptors expressed in Xenopus oocytes: Influence of α- and γ₂-subunits

Xenopus oocytes express functional GABA_A receptors

Assembly of GABA_A receptor subunits: α₁β₁ and α₁β₁γ_2S subunits produce unique ion channels with dissimilar single-channel properties

Modulation by general anaesthetics of rat GABA_A receptors comprised of α₁β₃ and β₃ subunits expressed in human embryonic kidney 293 cells

Chronic ethanol consumption differentially alters the expression of γ-aminobutyric acid_A receptor subunit mRNAs in rat cerebral cortex: Competitive, quantitative reverse transcriptase-polymerase chain reaction analysis

γ-Aminobutyric acid gating of Cl⁻ channels in recombinant GABA_A receptors

Subpopulations of GABA neurons in the dentate gyrus express high levels of the α₁ subunit of the GABA_A receptor

Enhancement by propofol of the γ-aminobutyric acid_A response in dissociated hippocampal pyramidal neurons of the rat