Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein

GABAergic synapses likely contain multiple GABA(A) receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of alpha, beta, and gamma subunits can be confounded by receptors expressed from alpha and beta subunits alone. To produce recombinant GABA(A) receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha 1 subunit linked to a beta 2 subunit. Cotransfection of the gamma 2 subunit with alpha beta-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the gamma subunit with nonlinked alpha 1 and beta 2 subunits. Similarly, expression of alpha subunits together with alpha beta-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of alpha with beta subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of alpha beta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha 1 beta 2 and alpha 1 beta 2 gamma 2receptors. We used this method to test whether the accessory protein GABA(A) receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in alpha 1 beta 2 gamma 2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of alpha beta gamma receptors.