Insights into the expression and pharmacology of GABAA receptors: From an epilepsy mutant to determinants of benzodiazepine binding and efficacy.

摘要

The gamma-aminobutyric acid type A receptor (GABAR) mediates the majority of rapid inhibitory neurotransmission in the central nervous system. Mutations in the GABAR have been linked to idiopathic generalized epilepsies (IGE). Benzodiazepines (BZDs), pharmacological agents used to treat seizure and anxiety disorders, exert their therapeutic effects by modulating GABAR activity. The overarching goal of my thesis work has been to explore the role of GABARs in epilepsy, as well as to elucidate the structural elements in the GABAR protein that underlie BZD binding and efficacy.; To this end, I examined the effects of the GABAR mutation gamma 2R43Q linked to IGE on GABAR function. Using heterologous expression in HEK293 cells, radioligand binding, immunofluorescence, and immunoblotting, I determined that the gamma2R43Q mutation causes a reduction in the expression of alphabetagamma GABARs. Thus, gamma2R43Q likely results in diminished phasic GABAergic neurotransmission mediated by gamma 2 containing receptors, thereby contributing to seizure susceptibility and hence the pathogenesis of epilepsy.; Using heterolgously expressed GABARs, chimeragenesis, radioligand binding, and simulated molecular docking, I identified a structural element (loop F) at the BZD binding interface of the gamma2 subunit that is responsible for conferring selective high affinity binding of the popularly prescribed sleep-aid zolpidem (AmbienRTM) to alphabetagamma GABARs. These findings are the first to implicate gamma2 Loop F in the binding of a BZD-site ligand. Furthermore, these results suggest that the gamma 2 subunit may play a greater role in dictating the pharmacological specificity of BZDs than previously thought.; Finally, to help elucidate the structural pathway governing BZD efficacy, I used the substituted cysteine accessibility method (SCAM) to probe the dynamics of the GABA binding site interface during BZD binding. Little is known about how the GABA binding site interface, which is distinct from the BZD binding interface, may contribute to the conformational changes within the receptor that are necessary to properly transduce BZD binding to channel modulation. Through my research, I show that BZDs induce structural rearrangements at the GABA binding site interface. Understanding the molecular underpinnings of BZD binding and efficacy may have profound impact on the future development of pharmacologically and behaviorally selective BZD-site ligands.