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 w ith GABA(A) (alpha 1, beta 2, and gamma 2S) or glycine (alpha 1, alpha 2, a nd 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 tw o 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 gl ycine alpha 2 subunits, GABA response properties were different from those of homomeric rho 1(T314A) receptors. Additionally, the sensitivity of heter omeric rho 1(T314A) and gamma 2S receptors to picrotoxinin (PTX) blockade o f GABA-evoked responses was altered compared to that of homomeric rho 1(T31 4A) receptors. Changes in GABA response properties and picrotoxinin sensiti vity were also observed when rho 1(T314A) subunits were coexpressed with wi ld-type rho 1 subunits. When rho 1(T314A/L317A) subunits were coexpressed w ith GABA gamma 2S, glycine alpha 1 or glycine alpha 2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of ho momeric rho 1(T314A/L317A) receptors. Recovery of the spontaneous current f rom inhibition by GABA for GABA rho 1(T314A/L317A)/gamma 2S heteromeric rec eptors displayed an additional component. Coinjection of wild-type rho 1 wi th gamma 2S cRNAs at a ratio of 1:1 resulted in a > 10-fold reduction in GA BA-evoked current. Furthermore, coexpression of wild-type rho 1 and gamma 2 S subunits was found to shift the GABA dose-response curve. Our results pro vide 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 i n vitro.