Efficient expression of concatenated α1β2δ and α1β3δ GABAA receptors, their pharmacology and stoichiometry

Background and Purpose: GABA(A) receptors containing delta-subunits are notorious for being difficult to study in vitro due to heterogeneity of expressed receptor populations and low GABA-evoked current amplitudes. Thus, there are some published misconceptions and contradictory conclusions made regarding the pharmacology and stoichiometry of delta-containing receptors. The aim of this study was to obtain robust homogenous expression of alpha 1 beta delta receptors for in-depth investigation. Experimental Approach: Novel delta-containing pentameric concatenated constructs were designed. The resulting alpha 1 beta 2 delta and alpha 1 beta 3 delta GABA(A) receptor concatemers were investigated by two-electrode voltage-clamp electrophysiology using Xenopus laevis oocytes. Key Results: First, while homogenous alpha 1 beta delta GABA(A) receptor pools could not be obtained by manipulating the ratio of injected cRNAs of free alpha 1, beta 2/3, and delta subunits, concatenated pentameric alpha 1 beta 2 delta and alpha 1 beta 3 delta constructs resulted in robust expression levels of concatemers. Second, by using optimised constructs that give unidirectional assembly of concatemers, we found that the delta subunit cannot directly participate in GABA binding and receptor activation. Hence, functional delta-containing receptors are likely to all have a conventional 2 alpha:2 beta:1 delta stoichiometry arranged as beta alpha beta alpha delta when viewed counterclockwise from the extracellular side. Third, alpha 1 beta 2/3 delta receptors were found to express efficiently in X. laevis oocytes but have a low estimated open probability of similar to 0.5% upon GABA activation. Because of this, these receptors are uniquely susceptible to positive allosteric modulation by, for example, neurosteroids. Conclusion and Implications: Our data answer important outstanding questions regarding the pharmacology and stoichiometry of alpha 1 delta-containing GABA(A) receptors and pave the way for future analysis and drug discovery efforts.