MOLECULAR POLYPHARMACY--CONTROL OF GABA ALPHA RECEPTOR

许多药物受体是变构蛋白，并且功效反映了药剂引起结构变化的程度。 本研究的长期目标是了解药物疗效的分子本质。 中心假设是受体具有有限数量的结构特征，变构剂通过这些结构特征影响它们的功能。 正如药物相互作用研究导致识别饱和结合位点，本研究将通过研究通过不同结合位点作用于相同受体的药物之间的相互作用来寻找“变构饱和”的证据。 如果第二种药物影响共同的结构特征，则存在足够有效的药物应降低观察到的第二种药物的作用。将对GABAA受体进行研究，GABAA受体是一种配体门控氯离子通道，具有至少5个单独的变构调节剂结合位点。 将研究四种结构不同的调节剂（苯巴比妥、5 β-葡聚糖-3 α-o 1 -20-酮、H+和La 3+）。 在存在或不存在单一调节剂的情况下，将来自用GABAA受体基因瞬时转染的HEK 293细胞的外向贴片暴露于各种浓度的GABA的快速应用。 将确定每种调节剂对受体活化、脱敏、失活和从脱敏恢复的动力学的影响。 然后将调节剂组合应用以确定一种调节剂的存在如何影响另一种调节剂的效力和功效。 这项研究的结果最终可用于指导突变受体的研究，以确定负责变构调节的结构特征。 这项研究还将增加对GABAA受体的理解，GABAA受体是中枢神经系统中的主要抑制性神经递质受体，也是许多毒性和治疗剂的作用部位。

Many drug receptors are allosteric proteins and efficacy reflects the extent to which agents cause structural changes. The long term goal of this study is to understand the molecular nature of drug efficacy. The central hypothesis is that receptors posses a limited number of structural features by which allosteric agents influence their function. Just as drug interaction studies led to the identification of saturable binding sites, this study will look for evidence of "allosteric saturation" by studying the interactions between agents which act on the same receptor through distinct binding sites. The presence of a sufficiently efficacious agent should reduce the observed effect of a second agent if both affect a common structural feature. Studies will be conducted on the GABAA receptor which is a ligand-gated chloride channel with at least five separate allosteric modulator binding sites. Four structurally distinct modulators (phenobarbital, 5beta-pregnan-3alpha-o1-20-one,H+ and La3+) will be studied. Outside-out patches from HEK 293 cells transiently transfected with GABAA receptor genes will be exposed to rapid application of various concentrations of GABA in the presence or absence of a single modulator. The effect of each modulator on the kinetics of receptor activation, desensitization, deactivation and recovery from desensitization will be determined. Modulators will then be applied in combination to determine how the presence of one modulator affects the potency and efficacy of another. Results from this study can eventually be used to guide studies on mutated receptors in order to identify the structural features responsible for allosteric modulation. This study will also add to the understanding of the GABAA receptor which is the major inhibitory neurotransmitter receptor in the central nervous system and the site of action of many toxic and therapeutic agents.