Williams D B, Akabas M H
Department of Physiology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Neuropharmacology. 2001 Oct;41(5):539-45. doi: 10.1016/s0028-3908(01)00099-5.
Benzodiazepines allosterically modulate GABA(A) receptors to increase currents induced by submaximal GABA concentrations. Benzodiazepine-induced conformational changes in the transmembrane domain increase the reactivity of cysteines substituted for a subset of residues in the alpha(1) subunit M3 membrane-spanning segment. With the cysteine-substitution mutant alpha(1)F296Cbeta(1)gamma(2) we previously noted that p-chloromercuribenzenesulfonate (pCMBS(-)) modification in the presence of diazepam potentiated subsequent GABA-induced currents. In contrast, pCMBS(-) modification in the presence of GABA caused inhibition of subsequent responses. We now show that in the presence of diazepam, pCMBS(-) only reacts with the engineered cysteine in one of the two alpha subunits; whereas, in the presence of GABA, pCMBS(-) reacts with the cysteine in the other alpha subunit, or with both cysteines. This implies that the two alpha subunits have distinct conformations in the diazepam-bound state. Based on analysis of single channel kinetic data, others have hypothesized that diazepam only alters the GABA affinity of one of the two GABA binding sites. The results presented here provide structural evidence to support the hypothesis that diazepam binding only alters the conformation of one of the two alpha subunits in a GABA(A) receptor and provides new insights into the mechanism of allosteric potentiation by benzodiazepines.
苯二氮䓬类药物通过变构调节γ-氨基丁酸A型（GABA(A)）受体，以增加由亚最大浓度GABA诱导的电流。苯二氮䓬类药物诱导的跨膜结构域构象变化增加了替代α(1)亚基M3跨膜片段中一部分残基的半胱氨酸的反应性。利用半胱氨酸替代突变体α(1)F296Cβ(1)γ(2)，我们之前注意到在地西泮存在的情况下，对氯汞苯磺酸盐（pCMBS(-)）修饰增强了随后的GABA诱导电流。相反，在GABA存在的情况下，pCMBS(-)修饰会抑制随后的反应。我们现在表明，在地西泮存在的情况下，pCMBS(-)仅与两个α亚基之一中的工程化半胱氨酸反应；而在GABA存在的情况下，pCMBS(-)与另一个α亚基中的半胱氨酸反应，或与两个半胱氨酸都反应。这意味着在与地西泮结合的状态下，两个α亚基具有不同的构象。基于对单通道动力学数据的分析，其他人推测地西泮仅改变两个GABA结合位点之一的GABA亲和力。此处呈现的结果提供了结构证据，以支持地西泮结合仅改变GABA(A)受体中两个α亚基之一的构象这一假说，并为苯二氮䓬类药物变构增强作用的机制提供了新的见解。
