Topf Norbert, Jenkins Andrew, Baron Nicole, Harrison Neil L
Department of Anesthesiology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Anesthesiology. 2003 Feb;98(2):306-11. doi: 10.1097/00000542-200302000-00007.
Volatile anesthetics prolong inhibitory postsynaptic potentials in central neurons an allosteric action on the gamma-aminobutyric acid type A (GABA(A)) receptor, an effect that may underlie the hypnotic actions of these agents. Inhaled anesthetics such as isoflurane act to enhance responses to submaximal concentrations of GABA, but it is not clear whether their effect is mediated by an increase in the binding of the agonist or by changes in receptor gating behavior. To address this question, the authors studied the effects of isoflurane on a mutant GABA(A) receptor with a gating defect that decreases receptor sensitivity by lowering agonist efficacy. They then compared the effects of clinically relevant concentrations of isoflurane on the actions of GABA and piperidine-4-sulfonic acid (P4S), a partial agonist at the GABA(A) receptor.
The authors created a mutant of the GABA receptor alpha subunit (L277A) by site-directed mutagenesis. The mutant subunit was coexpressed with beta(2) and gamma(2S) subunits in HEK293 cells, and responses to GABA and P4S were recorded using the whole-cell patch clamp technique. EC values were determined for the full agonist GABA and the partial agonist P4S. The authors also determined the relative efficacy (epsilon) of P4S. These measurements were then repeated in the presence of isoflurane.
The concentration-response curve for GABA was shifted to the right (EC(50) = 278 microm) in the alpha(1)(L277A)beta(2)gamma(2S) mutant receptor, compared with the corresponding wild-type alpha(1)beta(2)gamma(2S) GABA(A) receptor (EC(50) = 16 microm). P4S is a partial agonist at both receptors, with a dramatically decreased relative efficacy at the mutant receptor (epsilon = 0.24). When the mutant receptor was studied in the presence of isoflurane, the concentration-response curves for both GABA and P4S were shifted to the left (EC(50) for GABA = 78 microm); the efficacy of P4S also increased significantly (epsilon = 0.40).
By studying a mutant GABA receptor with impaired gating, the authors were able to demonstrate clearly that isoflurane can increase the efficacy of a partial agonist, as well as increase agonist potency. These data suggest that the volatile anesthetic isoflurane exerts at least some of its effects on the GABA(A) receptor via alterations in gating rather than simply changing binding or unbinding of the agonist.
挥发性麻醉药通过对γ-氨基丁酸A型（GABA(A)）受体的变构作用延长中枢神经元的抑制性突触后电位，这一效应可能是这些药物催眠作用的基础。吸入麻醉药如异氟烷可增强对亚最大浓度GABA的反应，但尚不清楚其作用是由激动剂结合增加介导还是由受体门控行为改变介导。为解决这个问题，作者研究了异氟烷对一种具有门控缺陷的突变型GABA(A)受体的影响，该缺陷通过降低激动剂效力来降低受体敏感性。然后他们比较了临床相关浓度的异氟烷对GABA和哌啶-4-磺酸（P4S）（一种GABA(A)受体的部分激动剂）作用的影响。
作者通过定点诱变创建了GABA受体α亚基（L277A）的突变体。将突变亚基与β(2)和γ(2S)亚基在HEK293细胞中共表达，并使用全细胞膜片钳技术记录对GABA和P4S的反应。测定了完全激动剂GABA和部分激动剂P4S的EC值。作者还测定了P4S的相对效力（ε）。然后在异氟烷存在的情况下重复这些测量。
与相应的野生型α(1)β(2)γ(2S) GABA(A)受体（EC(50)=16μM）相比，α(1)(L277A)β(2)γ(2S)突变型受体中GABA的浓度-反应曲线向右移动（EC(50)=278μM）。P4S在两种受体上都是部分激动剂，在突变型受体上相对效力显著降低（ε=0.24）。当在异氟烷存在的情况下研究突变型受体时，GABA和P4S的浓度-反应曲线都向左移动（GABA的EC(50)=78μM）；P4S的效力也显著增加（ε=0.40）。
通过研究一种门控受损的突变型GABA受体，作者能够清楚地证明异氟烷可以增加部分激动剂的效力，以及增加激动剂的效能。这些数据表明，挥发性麻醉药异氟烷至少部分通过门控改变而非简单改变激动剂的结合或解离对GABA(A)受体发挥作用。
