Boileau Andrew J, Pearce Robert A, Czajkowski Cynthia
Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA.
J Neurosci. 2005 Dec 7;25(49):11219-30. doi: 10.1523/JNEUROSCI.3751-05.2005.
GABAergic synapses likely contain multiple GABAA 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 GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha1 subunit linked to a beta2 subunit. Cotransfection of the gamma2 subunit with alphabeta-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 alpha1 and beta2 subunits. Similarly, expression of alpha subunits together with alphabeta-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 alphabeta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha1beta2 and alpha1beta2gamma2 receptors. We used this method to test whether the accessory protein GABAA 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 alpha1beta2gamma2S 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 alphabetagamma receptors.
GABA能突触可能包含多种GABAA受体亚型，这使得对突触后电流的剖析变得困难。然而，即使在异源表达系统中，对由α、β和γ亚基组成的受体进行分析时，单独由α和β亚基表达的受体也可能会造成混淆。为了产生具有固定亚基化学计量比的重组GABAA受体，我们将单个亚基与连接到β2亚基的“串联”α1亚基共表达。在人胚肾293细胞中，γ2亚基与αβ串联亚基共转染产生的电流，其宏观动力学、单通道幅度和药理学特性与γ亚基与未连接的α1和β2亚基过表达产生的电流相似。同样，α亚基与αβ串联亚基一起表达产生的受体，其生理和药理学特性与α亚基与β亚基共转染密切匹配。在首次使用膜片钳和快速激动剂应用技术对串联GABAA亚基进行描述时，我们得出结论，αβ串联亚基的掺入可用于固定化学计量比，并确定α1β2和α1β2γ2受体的内在动力学特性。我们使用这种方法来测试辅助蛋白GABAA受体相关蛋白（GABARAP）是直接改变GABAA受体特性还是影响亚基组成。在具有固定化学计量比的重组受体中，GABARAP-增强型绿色荧光蛋白（EGFP）融合蛋白的共表达对GABA介导电流的脱敏、失活或地西泮增强作用没有影响。然而，在化学计量比未固定的α1β2γ2S转染中，GABARAP-EGFP改变了GABA介导电流的脱敏、失活和地西泮增强作用。数据表明，GABARAP不是直接改变受体动力学，而是通过促进αβγ受体的表面表达来实现的。
