Individual properties of the two functional agonist sites in GABA(A) receptors

The members of the pentameric ligand-gated receptor channel family are involved in information transfer in synapses and the neuromuscular junction. They often contain several copies of the same subunit isoform. Here, we present a method to functionally dissect the role of individual subunits that occur in multiple copies in these receptors. Opening of the inherent chloride channel in the GABA(A) receptor is achieved through the binding of two agonist molecules; however, it has been difficult to obtain information on the contribution of the two individual binding sites. The sites are both located at beta+/alpha- subunit interfaces, suggesting similar properties. One pair of subunits is flanked by gamma and beta (site 1) and the other by alpha and gamma (site 2), the different environment possibly affecting the binding sites. Here, we used concatenated subunits and two point mutations of amino acid residues, each in alpha and beta subunits, both located in the agonist binding pocket, to investigate the properties of these two sites. The sites were individually mutated, and consequences of these mutations on GABA and muscimol-induced channel opening and its competitive inhibition by bicuculline were studied. A model predicts that opening also occurs for receptors occupied with a single agonist molecule but is promoted approximately 60-fold in those occupied by two agonists and that site 2 has an approximately threefold higher affinity for GABA than site 1, whereas muscimol and bicuculline show some preference for site 1.