Enhancement by GABA of the association rate of picrotoxin and tert‐butylbicyclophosphorothionate to the rat cloned α1β2γ2 GABAA receptor subtype
Enhancement by GABA of the association rate of picrotoxin and tert‐butylbicyclophosphorothionate to the rat cloned α1β2γ2 GABAA receptor subtype
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GABA 增强印防己毒素和叔丁基二环硫代磷酸酯与大鼠克隆 α1β2γ2 GABAA 受体亚型的关联率
DOI：
10.1111/j.1476-5381.1995.tb16368.x
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发表时间：
1995
影响因子：
7.3
通讯作者：
D. D. McKinley
中科院分区：
文献类型：
--
作者：
G. Dillon;W. Im;D. Carter;D. D. McKinley
1采用全细胞膜片钳技术，观察了γ-氨基丁酸对印防己毒素和硫代叔丁基二环磷酸盐与人胚胎肾细胞稳定表达的重组大鼠α-1-β-2-γ-2受体相互作用的影响。2GABA(5μM)作用于S 15时，印防己毒素和三苯氧胺呈剂量依赖性地加速GABA诱发的内向电流的衰减(在对称的C1−梯度下，保持电位为60 mV)。这些药物在与受体预先孵育时，也以预先孵育时间和浓度依赖的方式降低了初始电流幅度。这表明它们与GABA结合的受体和静止的受体都有相互作用。3在15 Sγ-氨基丁酸(5μM)脉冲开始时，印防己毒素和三苯氧胺的半数抑制浓度是脉冲结束时的数倍。因此，GABA似乎增强了印防己毒素和TBP的效力，但仅在导致高亲和力和低亲和力GABA位点占据的浓度下，即5μM。在200nM的GABA存在下，受体与药物预先孵育，导致仅占据α1β2γ2亚型的高亲和力GABA位点，对印防己毒素或TBP的效力没有明显变化。这表明它们优先与多配体受体相互作用，而不是与单配体受体相互作用，不像U-93631，它是一种新的印防己毒素位点的配体，与单配体和多配体受体的亲和力高于静止受体。4γ-氨基丁酸诱导的Cl-−电流的初始幅度随时间衰减和预孵育时间依赖于孵育时间呈单指数时间变化，且时间常数与药物浓度呈线性关系。用单一药物位点的动力学模型对数据进行分析，结果表明，5μM的GABA可使印防己毒素和TBP的结合率提高近100倍，但其解离速率仅为10倍。目前从印防己毒素或TBP块中回收得到的解离速率与上述分析结果几乎相同。5我们得出结论：印防己毒素和TBP与静息受体和GABA结合受体都有相互作用，但它们对后者的亲和力比前者高约10倍，这主要是由于它们与多配体受体(而不是单配体受体)的结合率显著增加。这项研究和我们早期对U-93631的研究提高了我们对γ-氨基丁酸和印防己毒素位点之间功能偶联的理解，这似乎有助于表征各种印防己毒素位点配体的相互作用模式。
1 We examined how y‐aminobutyric acid (GABA) influences interaction of picrotoxin and tert‐butylbicyclophosphorothionate (TBPS) with recombinant rat α1β2γ2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl− currents measured by the whole‐cell patch clamp technique. 2 During application of GABA (5 μM) for 15 s, picrotoxin and TBPS dose‐dependently accelerated the decay of inward GABA‐induced currents (a holding potential of −60 mV under a symmetrical C1− gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration‐dependent manner. This indicates their interaction with both GABA‐bound and resting receptors. 3 The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 μM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 μM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the α1β2γ2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U‐93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono‐ and multiliganded receptors than resting receptors. 4 The time‐dependent decay and preincubation time‐dependent reduction of initial amplitude of GABA‐induced Cl− currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 μM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis. 5 We conclude that picrotoxin and TBPS interact with both resting and GABA‐bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U‐93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.
非洲爪蟾卵母细胞中表达的荷包牡丹碱/巴氯芬不敏感的γ-氨基丁酸受体的表征。
影响因子：
3.6
作者：
Woodward,RM;Polenzani,L;Miledi,R
通讯作者：
Miledi,R
影响因子：
3.6
作者：
R. Squires;J. Casida;M. Richardson;E. Saederup
通讯作者：
R. Squires;J. Casida;M. Richardson;E. Saederup