MOLECULAR POLYPHARMACY: Modulation of GABAA Receptors

许多药物受体是变构蛋白，疗效反映了药物引起结构变化的程度。这项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，变构试剂通过这些特征影响受体的功能。就像药物相互作用研究导致了可饱和结合部位的确定一样，这项研究将通过研究通过不同结合部位作用于同一受体的药物之间的相互作用来寻找“变构饱和”的证据。如果两种制剂都影响一个共同的结构特征，那么一种足够有效的制剂的存在应该会减少第二种制剂的观察到的效果。将对GABAA受体进行研究，这是一种配体门控的氯离子通道，至少有五个独立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-孕烷-3α-o1-20-酮、H+和La3+)。瞬时转导GABAA受体基因的HEK 293细胞的由外向外的斑块将在存在或不存在单一调节剂的情况下暴露于不同浓度的GABA。将确定每个调节剂对受体激活、脱敏、失活和脱敏恢复的动力学的影响。然后，将结合使用调节器来确定一个调节器的存在如何影响另一个调节器的效力和功效。这项研究的结果最终可以用来指导对突变受体的研究，以确定与变构调节有关的结构特征。这项研究还将加深对GABAA受体的了解，GABAA受体是中枢神经系统中的主要抑制性神经递质受体，也是许多有毒和治疗药物的作用部位。

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.