Mutagenesis of the GABA(A) receptor alpha 1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine-site ligands

We have mutated several amino acids in the region of the GABA(A) receptor a lpha1 subunit predicted to form a small extracellular loop between transmem brane domains two and three to investigate its possible role in ligand sens itivity. The mutations were S275T, L276A, P277A, V279A, A280S and Y281F. Mu tant alpha1 subunits were co-expressed with beta2 and gamma2 subunits in ts A201 cells or Xenopus oocytes. Binding studies revealed that the only mutat ion that significantly affected [H-3]Ro15-4513 binding was the V279A substi tution which reduced the affinity for this ligand. Electrophysiological exa mination of mutant receptors revealed that L276A, P277A and V279A displayed rightward shifts of their GABA concentration-response curves, the largest occurring with the L276A mutant. The impact of these mutations on allosteri c modulation by benzodiazepine-site ligands was examined. V279A reduced the potency of both flunitrazeparn and Ro15-4513 but, in each case, their effi cacy was enhanced. A280S resulted in a decrease in flunitrazepam efficacy w ithout affecting its potency. Additionally, P277A and A280S resulted in Ro1 5-4513 losing its inverse agonist effect at these receptors. These results suggest that a domain within this small extracellular loop between TMII-TMI II plays a role in determining the sensitivity of GABA(A) receptors to both GABA and benzodiazepine-site ligands.