Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein
Tandem subunits effectively constrain GABAA receptor stoichiometry and recapitulate receptor kinetics but are insensitive to GABAA receptor-associated protein
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DOI：
10.1523/jneurosci.3751-05.2005
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发表时间：
2005-12-07
影响因子：
5.3
通讯作者：
Czajkowski, C
中科院分区：
文献类型：
--
作者：
Boileau, AJ;Pearce, RA;Czajkowski, C
GABA能突触可能含有多种GABA(A)受体亚型，使得突触后电流难以分析。然而，即使在异源表达系统中，对由α、β和伽马亚基组成的受体的分析也可能被仅由α和β亚基表达的受体所混淆。为了生产含有固定亚基化学计量比的重组GABA(A)受体，我们将单个亚基与一个与β2亚基相连的“串联”的α1亚基共表达。在人胚胎肾293细胞中，将γ2亚基与αβ串联亚基共转染产生的电流在宏观动力学、单通道幅度和药理学上与未连接的α1和β2亚基的伽马亚基过表达相似。同样，α亚基和αβ串联亚基的表达产生了具有生理和药理学特征的受体，该受体与α亚基和β亚基的共转染紧密匹配。在用膜片钳和快速激动剂应用技术测量的GABAA串联亚单位的第一个描述中，我们得出结论，αβ串联亚单位的掺入可用于固定化学计量学和建立α1β2和α1β2γ2受体的本征动力学性质。我们使用这种方法来测试辅助蛋白GABA(A)受体相关蛋白(GABARAP)是否直接改变GABAA受体的特性或影响亚基组成。在固定化学计量比的重组受体中，共表达GABARAP增强的绿色荧光蛋白(EGFP)融合蛋白对GABA介导的电流的脱敏、失活或安定增强没有影响。然而，在化学计量比不固定的α1β2伽马2S转基因中，GABARAP-EGFP改变了GABA介导的电流的脱敏、失活和安定增强。这些数据表明，GABARAP并不直接改变受体动力学，而是通过促进α-β-γ受体的表面表达。
GABAergic synapses likely contain multiple GABA(A) receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of alpha, beta, and gamma subunits can be confounded by receptors expressed from alpha and beta subunits alone. To produce recombinant GABA(A) receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha 1 subunit linked to a beta 2 subunit. Cotransfection of the gamma 2 subunit with alpha beta-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the gamma subunit with nonlinked alpha 1 and beta 2 subunits. Similarly, expression of alpha subunits together with alpha beta-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of alpha with beta subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of alpha beta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha 1 beta 2 and alpha 1 beta 2 gamma 2receptors. We used this method to test whether the accessory protein GABA(A) receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in alpha 1 beta 2 gamma 2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of alpha beta gamma receptors.