Overexpression of the GABAA receptor ε subunit results in insensitivity to anaesthetics
Introduction
The majority of anaesthetics have been shown to have dramatic effects on GABAergic neurotransmission in the central nervous system (Franks and Lieb, 1994, Tanelian et al., 1993), both potentiating responses to GABA and directly activating GABAA type receptors (Akaike et al., 1987, Parker et al., 1986, Adodra and Hales, 1995). It is proposed that modulation of GABAA receptor function is the major molecular mechanism for anaesthetic action in the mammalian CNS (Tanelian et al., 1993, Olsen, 1988). Recent studies have begun to reveal the molecular mechanisms for this interaction and have shown that these agents can interact directly with receptor protein to modify channel function (Mihic et al., 1997, Belelli et al., 1997, Amin, 1999, Mascia et al., 2000, Jenkins et al., 2001). γ-aminobutyric acidA receptors are ligand-gated ion channels made up of five subunits which assemble to form a chloride channel pore. The majority of GABAA receptors comprise of two α-subunits, two β-subunits and a γ-subunit (Chang et al., 1996, Tretter et al., 1997, Farrar et al., 1999, Knight et al., 2000) derived from subfamilies of α1–6, β1–3 and γ1–3. The γ- subunit can also be substituted by a δ-subunit, to generate a benzodiazepine insensitive form of the receptor while one copy of the β-subunit can be replaced by a θ-subunit. Recently the ε-subunit was identified as an additional novel substitute for the γ-subunit (Davies et al., 1997, Whiting et al., 1997). Located primarily in the hypothalamus, this subunit confers unique properties to the receptor, including benzodiazepine insensitivity, low sensitivity to zinc, rapid desensitisation and spontaneous channel activity. This subunit was initially reported to be unusual in conferring insensitivity to potentiation by anaesthetics while retaining direct activation (Davies et al., 1997), however conflicting data has recently been published showing that these receptors are potentiated by pentobarbitone, propofol, etomidate and the anaesthetic steroid, 5α-pregnan-3α-ol-20-one (Whiting et al., 1997, Neelands et al., 1999, Thompson et al., 1998). Here we have addressed this issue, and examined the anaesthetic sensitivity and properties of different forms of the ε-subunit clone expressed in Xenopus laevis oocytes. The conflicting data can be resolved in this study, where we show that over-expression of the ε-subunit can result in a form of the receptor showing insensitivity to modulation by anaesthetics.
Section snippets
Methods
Human GABAA receptor cDNAs. The cloning and sequencing of human α1, β1 and εMRK have been reported previously (Hadingham et al., 1993, Whiting et al., 1997). The α1 and β1-subunits were cloned into the expression vector pCDM8 while εMRK was cloned into pcDNA1.1Amp. A sample of εTIGR, which had been cloned into pCDM8, was kindly supplied by Ewan Kirkness (The Institute of Genomic Research, MD, USA). Throughout these studies the batch of α1 and β1 cDNA was kept constant so that any differences
Comparison of εMRK and εTIGR
Initial experiments compared the effects of three anaesthetic agents; pentobarbitone, 5α-pregnan-3α-ol-20-one and etomidate on the ε clone identified by Whiting et al. (1997) (εMRK) with that identified by Davies et al. (1997) (εTIGR) co-expressed with identical subunits α1β1 in an identical expression system, that of Xenopus oocytes. Single concentrations of each agent, which on α1β1γ2s receptors have previously been shown to elicit minimal direct activation but a significant degree of
Discussion
In this study we have attempted to account for the discrepancy in the literature regarding the pharmacological characteristics conferred on GABAA receptors by incorporation of the ε-subunit. The data suggests that in a side by side study using the same α1 and β1-subunits, the pharmacology with regard to potentiation by anaesthetics is reproducibly different between the two ε constructs and confirms the findings of Davies et al. (1997). The lack of potentiation on α1β1εTIGR by the steroid,
Acknowledgements
The authors thank Dr. E. Kirkness for kindly providing their ε-subunit clone and Dr. A.R. Rutter and S. Sandhu for technical assistance.
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