Detection and Binding Properties of GABAA Receptor Assembly Intermediates

Abstract

Density gradient centrifugation of native and recombinant γ-aminobutyric acid, type A (GABAA) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with α1, β3, and γ 2 subunits and cultured at 37 °C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from α1β3γ 2 transfected HEK cells cultured at 25 °C. In both systems, α1, β3, and γ2 subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABAA receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated α 1 and β3 or α1 and γ 2 subunits and coimmunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas α1γ2, α1Nγ2, α1γ 2N, and α1Nγ2N combinations exhibited specific [3H]Ro 15-1788 binding, specific [ 3H]muscimol binding could only be found in α 1β3 and α1β3N, but not in α1Nβ3 or α1Nβ 3N combinations. This seems to indicate that a full-length α1 subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.