CHIMERIC GABA(A) GLYCINE RECEPTORS - EXPRESSION AND BARBITURATE PHARMACOLOGY/

GABA(A) and glycine receptors are close relatives in the 'gene superfa mily' of ligand-gated ion channels, but have distinctly different phar macology. For example, barbiturates have two effects on GABA(A) recept ors (GABA(A)-R): at low micromolar concentrations (2-5 mu M), the anes thetic barbiturate methohexital potentiates submaximal chloride curren t responses to GABA; at higher concentrations (20-50 mu M), the barbit urate causes direct gating of the channel in the absence of agonist. N either of these barbiturate effects is seen an the glycine receptor (G ly-R). In order to study the structural parts of the GABA(A)-R involve d 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 fi rst site ('X') corresponded to the C-terminal end of the third transme mbrane domain (M3) in each subunit and enabled exchange of a C-termina l fragment of similar to 100 amino acids (which includes the large 'cy toplasmic 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-ter minal end of each subunit and enabled exchange of a small N-terminal f ragment between GABA(A)-R and Gly-R subunits. Several chimeric recepto r subunit cDNAs were constructed and the resulting receptors tested fo r 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 assoc iated with the enhancement or direct activation of the GABA(A)-R by MT X. These results demonstrate the viability of chimeric GABA(A)/Gly-R a nd 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.