Harvey Victoria L, Duguid Ian C, Krasel Cornelius, Stephens Gary J
School of Pharmacy, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK.
J Physiol. 2006 Nov 15;577(Pt 1):127-39. doi: 10.1113/jphysiol.2006.112482. Epub 2006 Aug 31.
Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.
由异源分子亚基组成的离子型γ-氨基丁酸（GABA）受体是成年中枢神经系统中抑制性反应的主要介质。在此，我们使用据报道对含rho亚基的GABA(C)受体比对其他GABA受体具有更高亲和力的试剂，在小鼠小脑浦肯野细胞（PCs）中描述了一种新型离子型GABA受体。外源性应用偏好GABA(C)的激动剂顺式-4-氨基巴豆酸（CACA）可在PCs中诱发全细胞电流，而等摩尔浓度的GABA诱发的电流更大。CACA诱发的电流对选择性GABA(C)拮抗剂（1,2,5,6-四氢吡啶-4-基）甲基次膦酸（TPMPA）的敏感性高于GABA诱发的电流。局部应用激动剂产生了不同的反应模式；与GABA诱发的电流相比，CACA诱发的电流随着距PC胞体距离的增加衰减更为明显，达到峰值的时间更慢，脱敏作用更小。然而，CACA诱发的电流也被荷包牡丹碱完全阻断，荷包牡丹碱是一种GABA(A)受体的选择性试剂。因此，我们描述了一群具有混合GABA(A)/GABA(C)药理学特性的离子型GABA受体。TPMPA降低了中间神经元-浦肯野细胞（IN-PC）突触处的抑制性突触传递，导致微小抑制性突触后电流（mIPSC）的幅度和频率明显降低。联合应用NO-711（一种选择性GABA转运体亚型1（GAT-1）拮抗剂）和SNAP-5114（一种GAT-(2)/3/4拮抗剂）在PCs中诱导出强直GABA电导；然而，TPMPA对该电流没有影响。免疫组织化学研究表明，rho亚基主要在PC胞体和近端树突区表达，在更远端的树突中表达水平较低；这种选择性免疫反应性与PCs中GABA(A)α1亚基更均匀的分布形成对比。最后，免疫共沉淀研究表明，rho亚基可在小脑皮质中与GABA(A)受体α1亚基形成复合物。总体而言，这些数据表明rho亚基有助于形成功能性离子型受体，这些受体介导小脑IN-PC突触处阶段性抑制性GABA能传递的一个组成部分。
