Department of Neurobiology, Boston University, Boston, MA, USA.
Anesthesiology. 2012 Jun;116(6):1235-44. doi: 10.1097/ALN.0b013e3182567df3.
Etomidate is a potent hypnotic agent that acts via γ-aminobutyric acid receptor type A (GABA(A)) receptors. Evidence supports the presence of two etomidate sites per GABA(A) receptor, and current models assume that each site contributes equally and noncooperatively to drug effects. These assumptions remain untested.
We used concatenated dimer (β2-α1) and trimer (γ2-β2-α1) GABA(A) subunit assemblies that form functional α1β2γ2 channels, and inserted α1M236W etomidate site mutations into both dimers (β2-α1M236W) and trimers (γ2-β2-α1M236W). Wild-type or mutant dimers (D(wt) or D(αM236W)) and trimers (T(wt) or T(αM236W)) were coexpressed in Xenopus oocytes to produce four types of channels: D(wt)T(wt), D(αM236W)T(wt), D(wt)T(αM236W), and D(αM236W)T(αM236W). For each channel type, two-electrode voltage clamp was performed to quantitatively assess GABA EC(50), etomidate modulation (left shift), etomidate direct activation, and other functional parameters affected by αM236W mutations.
Concatenated wild-type D(wt)T(wt) channels displayed etomidate modulation and direct activation similar to α1β2γ2 receptors formed with free subunits. D(αM236W)T(αM236W) receptors also displayed altered GABA sensitivity and etomidate modulation similar to mutated channels formed with free subunits. Both single-site mutant receptors (D(αM236W)T(wt) and D(wt)T(αM236W)) displayed indistinguishable functional properties and equal gating energy changes for GABA activation (-4.9 ± 0.48 vs. -4.7 ± 0.48 kJ/mol, respectively) and etomidate modulation (-3.4 ± 0.49 vs. -3.7 ± 0.38 kJ/mol, respectively), which together accounted for the differences between D(wt)T(wt) and D(αM236W)T(αM236W) channels.
These results support the hypothesis that the two etomidate sites on α1β2γ2 GABA(A) receptors contribute equally and noncooperatively to drug interactions and gating effects.
依托咪酯是一种作用于γ-氨基丁酸受体 A 型（GABA(A)) 的强效催眠药物。有证据表明每个 GABA(A) 受体上存在两个依托咪酯结合位点，目前的模型假设每个位点对药物效应的贡献相等且无协同作用。这些假设尚未得到验证。
我们使用串联二聚体（β2-α1) 和三聚体（γ2-β2-α1) GABA(A) 亚基组装体，这些组装体能形成功能性的α1β2γ2 通道，并将α1M236W 依托咪酯结合位点突变插入到二聚体（β2-α1M236W) 和三聚体（γ2-β2-α1M236W) 中。野生型或突变型二聚体（D(wt) 或 D(αM236W)) 和三聚体（T(wt) 或 T(αM236W)) 在非洲爪蟾卵母细胞中共同表达，产生四种类型的通道：D(wt)T(wt)、D(αM236W)T(wt)、D(wt)T(αM236W) 和 D(αM236W)T(αM236W)。对于每种通道类型，我们都进行了双电极电压钳实验，以定量评估 GABA 的 EC(50)、依托咪酯的调制（左移）、依托咪酯的直接激活以及其他受 αM236W 突变影响的功能参数。
串联野生型 D(wt)T(wt) 通道显示出与由游离亚基形成的 α1β2γ2 受体相似的依托咪酯调制和直接激活。D(αM236W)T(αM236W) 受体也表现出与由游离亚基形成的突变型通道相似的 GABA 敏感性和依托咪酯调制改变。两种单一位点突变型受体（D(αM236W)T(wt) 和 D(wt)T(αM236W)) 的功能特性均无明显差异，且 GABA 激活（-4.9 ± 0.48 对 -4.7 ± 0.48 kJ/mol，分别）和依托咪酯调制（-3.4 ± 0.49 对 -3.7 ± 0.38 kJ/mol，分别）的门控能变化相等，这共同解释了 D(wt)T(wt) 和 D(αM236W)T(αM236W) 通道之间的差异。
这些结果支持以下假设：α1β2γ2 GABA(A) 受体上的两个依托咪酯结合位点对药物相互作用和门控效应的贡献相等且无协同作用。
