A novel site on γ3 subunits important for assembly of GABAA receptors

Abstract

γ-Aminobutyric acid, type A (GABAA) receptors are ligand-gated chloride channels and are the major inhibitory transmitter receptors in the central nervous system. The majority of these receptors is composed of two α, two β, and one γ subunits. To identify sequences important for subunit assembly, we generated C-terminally truncated and chimeric γ3 constructs. From their ability to associate with full-length α1 and β3 subunits, we concluded that amino acid sequence γ3(70-84) either directly interacts with α1 or β3 subunits or stabilizes a contact site elsewhere in the protein. The observation that this sequence contains amino acid residues homologous to γ2 residues contributing to the benzodiazepine-binding site at the α1/γ2 interface suggested that in α1β3γ3 receptors the sequence γ3(70-84) is located at the α1β3γ3 interface. In the absence of α1 subunits this sequence might allow assembly of β3 with γ3 subunits. Other experiments indicated that sequences γ3(86-95) and γ3(94-107), which are homologous to previously identified sequences important for assembly of γ2 subunits, are also important for assembly of γ3 subunits. This indicates that during assembly of the GABAA receptor, more than one N-terminal sequence is important for binding to the same neighboring subunit. Whether the three sequences investigated are involved in direct interaction or stabilize other regions involved in intersubunit contacts has to be further studied.