Overexpression of the GABAA receptor ε subunit results in insensitivity to anaesthetics

The epsilon-subunit of the GABA(A) receptor was independently cloned and functionally characterised in recombinant expression systems by two groups (Davies, P.A. et al., Nature 385 (1997) 820; Whiting, P.J. et al., Journal of Neuroscience 17 (1997) 5027). Both groups showed that co-expression of ape-subunits produced functional receptors, however the sensitivity of these receptors to the potentiating effects of general anaesthetic agents differed. Co-expression of the two epsilon-constructs (hereafter referred to as epsilon(MRK) from Whiting, P.J. et al., Journal of Neuroscience 17 (1997) 5027) and epsilon(TIGR) from Davies et al., Nature 385 (1997) 820) with alpha1beta1 in Xenopus oocytes produced receptors that were sensitive (alpha1beta1epsilon(MRK)) and insensitive (alpha1beta(1)epsilon(TIGR)) to the potentiating effects of pentobarbitone, 5alpha-pregnan-3alpha-ol-20-one and etomidate. Both alpha1beta1epsilon(MRK) and alpha1beta1epsilon(TIGR) receptors were directly activated by these agents, however for pentobarbitone and 5alpha-pregnan-3alpha-ol-20-one this effect was greater on (alpha1beta1epsilon(TIGR) than alpha1beta1epsilon(MRK). alpha1beta1epsilon(TIGR) receptors were more sensitive to GABA and had a larger degree of constitutive activity than alp lemp.K. Insensitivity to the potentiating effects of anaesthetics was not due to the single amino acid difference between the two constructs nor to differences in the 5' and 3' untranslated regions. Transfer of epsilon(TIGR) from its original vector, pCDM8, into pcDNA1.1Amp and reduction in the amount Of EICR in pCDM8 relative to the amount of alpha1 and beta1 injected into the oocyte restored potentiation by pentobarbitone. Increased expression of epsilon(TIGR) protein compared to epsilon(MRK) was confirmed by Western blotting. We conclude that the differences in the potentiating effects of anaesthetic agents on al alpha1beta1epsilon(MRK/TIGR) receptors is due to overexpression of epsilon(TIGR) in the pCDMS vector, relative to the alpha1 and beta1-subunits, which may lead to an altered stoichiometry. (C) 2002 Elsevier Science Ltd. All rights reserved.