4 AMINO-ACID EXCHANGES CONVERT A DIAZEPAM-INSENSITIVE, INVERSE AGONIST-PREFERRING GABA(A) RECEPTOR INTO A DIAZEPAM-PREFERRING GABA(A) RECEPTOR

Benzodiazepines (BZ) exert their effects through GABA(A) receptors, wh ich belong to the superfamily of ligand-gated ion channels. Coexpressi on of recombinant alpha, beta, and gamma subunits in a cell culture sy stem mimics the BZ binding sites. The alpha variants largely determine the nature of the BZ binding site in such alpha i beta j gamma k hete romultimers (i = 1-6; j = 1-3; k = 1-3). Notably, the alpha 1 and alph a 6 variants confer high and low affinity for BZ agonists to the resul ting receptor subtype, respectively. Glycine/glutamate and histidine/a rginine positions in the alpha subunits of alpha x beta 2 gamma 2 rece ptors are involved in BZ I versus BZ II type selectivity. We now ident ify four amino acids in alpha 6 which together increase the affinity o f the mutant alpha x beta 2 gamma 2 receptor for classical BZ receptor agonists above the level seen for any wild-type GABA(A)/BZ receptor. The most pronounced effect was due to an isoleucine to valine exchange . It simultaneously decreased the affinity for the BZ partial inverse agonist Ro 15-4513 20-fold and increased the affinity for diazepam 4-f old. The four amino acid residues stretch over most part of the N-term inal extracellular domain of the alpha subunit, suggesting that amino acids distant in the primary sequence form the BZ binding pocket.