Evidence for coassembly of mutant GABA(c) rho 1 with GABA(A) gamma 2S, glycine alpha 1 and glycine alpha 2 receptor subunits in vitro

Functional coassembly of gamma-aminobutyric acid (GABA)(C) rho 1 subunits with GABA(A) (alpha 1, beta 2, and gamma 2S) or glycine (alpha 1, alpha 2, and beta) 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 rho 1 subunits, rho 1(T314A) and rho 1(T314A/ L317A). When the rho 1(T314A) subunit was coexpressed with GABA gamma 2S, glycine alpha 1 or glycine alpha 2 subunits, GABA response properties were different from those of homomeric rho 1(T314A) receptors. Additionally, the sensitivity of heteromeric rho 1(T314A) and gamma 2S receptors to picrotoxinin (PTX) blockade of GABA-evoked responses was altered compared to that of homomeric rho 1(T314A) receptors. Changes in GABA response properties and picrotoxinin sensitivity were also observed when rho 1(T314A) subunits were coexpressed with wild-type rho 1 subunits. When rho 1(T314A/L317A) subunits were coexpressed with GABA gamma 2S, glycine alpha 1 or glycine alpha 2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of homomeric rho 1(T314A/L317A) receptors. Recovery of the spontaneous current from inhibition by GABA for GABA rho 1(T314A/L317A)/gamma 2S heteromeric receptors displayed an additional component. Coinjection of wild-type rho 1 with gamma 2S cRNAs at a ratio of 1:1 resulted in a > 10-fold reduction in GABA-evoked current. Furthermore, coexpression of wild-type rho 1 and gamma 2S subunits was found to shift the GABA dose-response curve. Our results provide functional evidence that the GABA(C) rho 1 subunit can coassemble with the GABA(A) gamma 2S subunit, and, at least in its mutated form, rho 1 can also form heteromeric receptors with glycine alpha 1 or alpha 2 subunits in vitro.