Detection and binding properties of GABA(A) receptor assembly intermediates

Density gradient centrifugation of native and recombinant gamma -aminobutyr ic acid, type A (GABA(A)) receptors was used to detect assembly intermediat es. No such intermediates could be identified in extracts from adult rat br ain or from human embryonic kidney (HEK) 293 cells transfected with alpha ( 1), beta (3), and gamma (2) subunits and cultured at 37 degreesC. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts fr om the brain of 8-10-day-old rats and from alpha (1)beta (3)gamma (2) trans fected HEK cells cultured at 25 degreesC. In both systems, alpha (1), beta (3), and gamma (2) , subunits could be identified in subunit dimers, indica ting that different subunit dimers are formed during GABA(A) receptor assem bly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha (1) and beta (3) or alpha (1) and gamma (2 ) subunits and coimmunoprecipitation with subunit-specific antibodies indic ated that even subunits containing no transmembrane domain can assemble wit h each other. Whereas alpha (1)gamma (2), alpha N-1 gamma (2), alpha (1)gam ma N-2, and alpha N-1 gamma N-2, combinations exhibited specific [H-3]Ro 15 -1788 binding, specific [H-3]muscimol binding could only be found in alpha (1)beta (3) and alpha (1)beta N-3, but not in alpha N-1 beta (3) or alpha N -1 beta N-3 combinations. This seems to indicate that a full-length alpha ( 1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.