Serafini R, Bracamontes J, Steinbach J H
Department of Anesthesiology Research Unit, Washington University School of Medicine, CB 8054, 660 S. Euclid Avenue, St Louis MO, 63110, USA.
J Physiol. 2000 May 1;524 Pt 3(Pt 3):649-76. doi: 10.1111/j.1469-7793.2000.00649.x.
This study was conducted to search for the residues of the beta3 subunit which affect pentobarbital action on the gamma-aminobutyric acid type A (GABAA) receptor. Three chimeras were constructed by joining the GABAA receptor beta3 subunit to the rho1 subunit. For each chimera, the N-terminal sequence was derived from the beta3 subunit and the C-terminal sequence from the rho1 subunit, with junctions located between the membrane-spanning regions M2 and M3, in the middle of M2, or in M1, respectively. In receptors obtained by the coexpression of alpha1 with the chimeric subunits, in contrast with those obtained by the coexpression of alpha1 and beta3, pentobarbital exhibited lower potentiation of GABA-evoked responses, and in the direct gating of Cl- currents, an increase in the EC50 together with a marked decrease in the relative maximal efficacy compared with that of GABA. Estimates of the channel opening probability through variance analysis and single-channel recordings of one chimeric subunit showed that the reduced relative efficacy for gating largely resulted from an increase in gating by GABA, with little change in efficacy of pentobarbital. A fit of the time course of the response by the predictions of a class of reaction schemes is consistent with the conclusion that the change in the concentration dependence of activation by pentobarbital is due to a change in pentobarbital affinity for the receptor. Therefore, the data suggest that residues of the beta3 subunit involved in pentobarbital binding to GABAA receptors are located downstream from the middle of the M2 region.
本研究旨在寻找影响戊巴比妥对γ-氨基丁酸A型（GABAA）受体作用的β3亚基残基。通过将GABAA受体β3亚基与rho1亚基连接构建了三种嵌合体。对于每种嵌合体，N端序列来自β3亚基，C端序列来自rho1亚基，连接点分别位于跨膜区M2和M3之间、M2中间或M1中。与α1和β3共表达所获得的受体相比，在α1与嵌合亚基共表达所获得的受体中，戊巴比妥对GABA诱发反应的增强作用较低，并且在Cl-电流的直接门控中，与GABA相比，EC50增加，相对最大效能显著降低。通过方差分析和对一个嵌合亚基的单通道记录对通道开放概率的估计表明，门控相对效能的降低主要是由于GABA门控增加，戊巴比妥的效能变化很小。用一类反应方案的预测对反应的时间进程进行拟合，与戊巴比妥激活浓度依赖性变化是由于其对受体亲和力改变这一结论一致。因此，数据表明参与戊巴比妥与GABAA受体结合的β3亚基残基位于M2区域中间的下游。
