Ramakrishnan Latha, Hess George P
Department of Molecular Biology and Genetics, 216 Biotechnology Building, Cornell University, Ithaca, New York 14853-2703, USA.
Biochemistry. 2005 Jun 14;44(23):8523-32. doi: 10.1021/bi0477283.
The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.
γ-氨基丁酸A（GABA(A)）受体是一种主要的抑制性神经递质受体，属于膜结合蛋白家族，该家族调节神经系统中约10¹²个细胞之间的信号传递。它在包括癫痫在内的许多神经系统疾病中起主要作用。它是许多药物的作用靶点，包括惊厥剂印防己毒素。在此，我们展示了使用快速动力学技术结合全细胞电流记录研究印防己毒素对大鼠α1β2γ2L GABA(A)受体抑制作用的机制。通过使用瞬态动力学技术、细胞流动法和具有微秒至毫秒时间分辨率的激光脉冲光解（LaPP）技术获得了以下新结果。（i）印防己毒素对于受体开放通道形式的表观解离常数比关闭通道形式的约高5倍。（ii）印防己毒素使通道关闭速率常数（k(cl)）增加约4倍，而通道开放速率常数（k(op)）基本保持不变。（iii）该机制表明印防己毒素以比开放通道形式更高的亲和力结合到受体的变构位点，从而通过将其通道开放平衡常数降低4倍[Phi(I)( - )(1) = k(op(I))/k(cl(I))]来抑制受体。（iv）该机制进一步表明，与受体开放通道形式和关闭通道形式上的印防己毒素结合位点具有同等亲和力结合的化合物不会影响通道开放平衡，因此可以取代印防己毒素并防止印防己毒素对GABA(A)受体的抑制。此类化合物在对抗不利影响受体通道开放平衡的化合物和疾病的作用方面可能具有治疗用途。
