Pan Z H, Zhang D, Zhang X, Lipton S A
CNS Research Institute, Brigham & Women's Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.
Eur J Neurosci. 2000 Sep;12(9):3137-45. doi: 10.1046/j.1460-9568.2000.00198.x.
Functional coassembly of gamma-aminobutyric acid (GABA)C rho1 subunits with GABAA (alpha1, beta2, and gamma2S) or glycine (alpha1, alpha2, 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 rho1 subunits, rho1(T314A) and rho1(T314A/L317A). When the rho1(T314A) subunit was coexpressed with GABA gamma2S, glycine alpha1 or glycine alpha2 subunits, GABA response properties were different from those of homomeric rho1(T314A) receptors. Additionally, the sensitivity of heteromeric rho1(T314A) and gamma2S receptors to picrotoxinin (PTX) blockade of GABA-evoked responses was altered compared to that of homomeric rho1(T314A) receptors. Changes in GABA response properties and picrotoxinin sensitivity were also observed when rho1(T314A) subunits were coexpressed with wild-type rho1 subunits. When rho1(T314A/L317A) subunits were coexpressed with GABA gamma2S, glycine alpha1 or glycine alpha2 subunits, suppression by GABA of spontaneously active current was reduced compared to that of homomeric rho1(T314A/L317A) receptors. Recovery of the spontaneous current from inhibition by GABA for GABA rho1(T314A/L317A)/gamma2S heteromeric receptors displayed an additional component. Coinjection of wild-type rho1 with gamma2S cRNAs at a ratio of 1 : 1 resulted in a > 10-fold reduction in GABA-evoked current. Furthermore, coexpression of wild-type rho1 and gamma2S subunits was found to shift the GABA dose-response curve. Our results provide functional evidence that the GABAC rho1 subunit can coassemble with the GABAA gamma2S subunit, and, at least in its mutated form, rho1 can also form heteromeric receptors with glycine alpha1 or alpha2 subunits in vitro.
在非洲爪蟾卵母细胞表达系统中，利用双电极电压钳记录技术研究了γ-氨基丁酸（GABA）C ρ1亚基与GABAA（α1、β2和γ2S）或甘氨酸（α1、α2和β）亚基的功能性共组装。为便于此项研究，我们利用了两种突变型ρ1亚基rho1(T314A)和rho1(T314A/L317A)独特的门控和药理学特性。当rho1(T314A)亚基与GABA γ2S、甘氨酸α1或甘氨酸α2亚基共表达时，GABA反应特性不同于同源rho1(T314A)受体。此外，与同源rho1(T314A)受体相比，异源rho1(T314A)和γ2S受体对荷包牡丹碱（PTX）阻断GABA诱发反应的敏感性发生了改变。当rho1(T314A)亚基与野生型rho1亚基共表达时，也观察到了GABA反应特性和荷包牡丹碱敏感性的变化。当rho1(T314A/L317A)亚基与GABA γ2S、甘氨酸α1或甘氨酸α2亚基共表达时，与同源rho1(T314A/L317A)受体相比，GABA对自发激活电流的抑制作用减弱。GABA rho1(T314A/L317A)/γ2S异源受体的自发电流从GABA抑制中恢复表现出一个额外的成分。以1:1的比例共注射野生型rho1和γ2S cRNAs导致GABA诱发电流降低10倍以上。此外，发现野生型rho1和γ2S亚基的共表达会使GABA剂量反应曲线发生偏移。我们的结果提供了功能性证据，表明GABAC ρ1亚基可以与GABAA γ2S亚基共组装，并且至少在其突变形式下，rho1在体外也可以与甘氨酸α1或α2亚基形成异源受体。
