Evidence for coassembly of mutant GABACρ1 with GABAAγ2S, glycine α1 and glycine α2 receptor subunits in vitro

Abstract

Functional coassembly of γ-aminobutyric acid (GABA)_Cρ1 subunits with GABA_A (α1, β2, and γ2S) or glycine (α1, α2, and β) subunits was examined using two-electrode voltage-clamp recordings in the Xenopus laevis oocyte expression system. To facilitate this study, we took advantage of the unique gating and pharmacological properties of two mutant ρ1 subunits, ρ1(T314A) and ρ1(T314A/L317A). When the ρ1(T314A) subunit was coexpressed with GABA γ2S, glycine α1 or glycine α2 subunits, GABA response properties were different from those of homomeric ρ1(T314A) receptors. Additionally, the sensitivity of heteromeric ρ1(T314A) and γ2S receptors to picrotoxinin (PTX) blockade of GABA-evoked responses was altered compared to that of homomeric ρ1(T314A) receptors. Changes in GABA response properties and picrotoxinin sensitivity were also observed when ρ1(T314A) subunits were coexpressed with wild-type ρ1 subunits. When ρ1(T314A/L317A) subunits were coexpressed with GABA γ2S, glycine α1 or glycine α2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of homomeric ρ1(T314A/L317A) receptors. Recovery of the spontaneous current from inhibition by GABA for GABA ρ1(T314A/L317A)/γ2S heteromeric receptors displayed an additional component. Coinjection of wild-type ρ1 with γ2S cRNAs at a ratio of 1 : 1 resulted in a > 10-fold reduction in GABA-evoked current. Furthermore, coexpression of wild-type ρ1 and γ2S subunits was found to shift the GABA dose–response curve. Our results provide functional evidence that the GABA_Cρ1 subunit can coassemble with the GABA_Aγ2S subunit, and, at least in its mutated form, ρ1 can also form heteromeric receptors with glycine α1 or α2 subunits in vitro.