Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors

GABA(A) receptors composed of alpha (1)beta (3)gamma (2) and alpha (1)beta (3) subunits were expressed in insect Sf9 cells and solubilized in 1% Trito n X100. In sucrose density gradients, [H-3] Ro15-1788 binding activity, in the case of alpha (1)beta (3)gamma (2), and [H-3]-muscimol binding activity , in the case of alpha (1)beta (3) containing receptors sedimented as a sin gle sharp peak suggesting the formation of receptors containing a defined n umber of subunits. When alpha (1)beta (3)gamma (2)-containing receptors wer e incubated with nn alpha -subunit specific antibody (bd24), a single class of antibody receptors wer incubated formed irrespective of the receptor-an tibody ratio. This is consistent with two alpha subunits cross-linked withi n the receptor by the antibody. Similar results were obtained using a beta -subunit specific antibody (bd17). Several classes of antibody-receptor com plex were formed when receptors were pre-incubated with a gamma specific an tibody (anti gamma (2) 1-15 Cys). This profile is consistent with the prese nce of a single gamma subunit in each complex. Experiments with alpha (1)be ta (3) subunit containing receptors and antibody bd24 produced a profile si milar to that seen with alpha (1)beta (3)gamma (2) receptors, consistent wi th two a subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with thre e beta subunits per receptor. This method permits the rapid determination o f subunit stoichiometries of homogeneous receptor populations.