Chimeric GABA(A)/glycine receptors: Expression and barbiturate pharmacology

GABA(A) and glycine receptors are close relatives in the 'gene superfamily' of ligand-gated ion channels, but have distinctly different pharmacology. For example, barbiturates have two effects on GABA(A) receptors (GABA(A)-R): at low micromolar concentrations (2-5 mu M), the anesthetic barbiturate methohexital potentiates submaximal chloride current responses to GABA; at higher concentrations (20-50 mu M), the barbiturate causes direct gating of the channel in the absence of agonist. Neither of these barbiturate effects is seen an the glycine receptor (Gly-R). In order to study the structural parts of the GABA(A)-R involved in this barbiturate pharmacology, two unique restriction sites were introduced into the cDNAs encoding the alpha 2 and beta 1 subunits of the human GABA(A)-R and the alpha 1 subunit of the human gly-R. The first site ('X') corresponded to the C-terminal end of the third transmembrane domain (M3) in each subunit and enabled exchange of a C-terminal fragment of similar to 100 amino acids (which includes the large 'cytoplasmic loop' and M4 segment) between GABA(A)-R and Gly-R subunits. The second site ('S') was similar to 30 amino acids 3'- from the N-terminal end of each subunit and enabled exchange of a small N-terminal fragment between GABA(A)-R and Gly-R subunits. Several chimeric receptor subunit cDNAs were constructed and the resulting receptors tested for their ability to respond to GABA and glycine and for sensitivity to the barbiturate methohexital (MTX). The results show that neither the large C-terminal fragment nor the smaller N-terminal fragment is associated with the enhancement or direct activation of the GABA(A)-R by MTX. These results demonstrate the viability of chimeric GABA(A)/Gly-R and suggest that the method will be suitable for further investigation of the molecular basis of the barbiturate pharmacology of the GABA(A)-R. Copyright (C) 1996 Elsevier Science Ltd.