Santhakumar Vijayalakshmi, Hanchar H Jacob, Wallner Martin, Olsen Richard W, Otis Thomas S
Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
J Neurosci. 2006 Mar 22;26(12):3357-64. doi: 10.1523/JNEUROSCI.4799-05.2006.
GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit alpha6 (Gabra6(100R) and Gabra6(100Q)). Depending on their subunit partners, these two variants of alpha6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6(100R/100R) and Gabra6(100Q/100Q) rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by delta subunit-containing GABARs in Gabra6(100Q/100Q) rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates alpha6/delta subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is approximately 160 nm in perfused slices. These results (1) demonstrate contributions of alpha6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that delta subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.
GABAA受体（GABARs）是由五个亚基组成的异源多聚体蛋白。特定的亚基组成决定了GABAR的关键特性，如药理学敏感性以及该受体是否参与突触或突触外形式的抑制作用。传统观点认为，突触GABARs而非突触外GABARs对苯二氮䓬有反应，而乙醇的情况则相反。为了在原位研究亚基组成对GABAR功能的影响，我们利用了大鼠GABAR亚基α6基因的两个天然等位基因（Gabra6(100R)和Gabra6(100Q)）。根据它们的亚基伙伴，α6的这两个变体对苯二氮䓬和乙醇可产生不同的敏感性。对来自Gabra6(100R/100R)和Gabra6(100Q/100Q)大鼠的小脑颗粒细胞中突触和突触外GABA介导的电流进行检测，发现了苯二氮䓬敏感性存在明显的等位基因依赖性差异。出乎意料的是，我们发现氟硝西泮和地西泮这两种苯二氮䓬增强了Gabra6(100Q/100Q)大鼠中含δ亚基的GABAR介导的突触外抑制作用。对重组GABARs进行的补充实验证实，在低于饱和[GABA]浓度时，氟硝西泮可增强含α6/δ亚基的GABARs的作用。基于这些数据和简单的理论分析，我们估计在灌注切片中突触外[GABA]的平均浓度约为160 nM。这些结果（1）证明了α6亚基对突触和突触外GABA反应均有贡献，（2）确定了在低于饱和[GABA]浓度时含δ亚基的GABARs对苯二氮䓬敏感，以及（3）提供了切片中突触外[GABA]的经验估计值。
