Gamma-aminobutyric acid type A receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) that mediate neuronal inhibition in the brain. Defects in GABAAR signaling are associated with a variety of neurological diseases and psychiatric disorders. GABAARs are the target for many therapeutic drugs including benzodiazepines (BZDs), barbiturates, anesthetics and neurosteroids that allosterically modulate channel function (Braat and Kooy, 2015; Carver and Reddy, 2013; D'Hulst et al., 2009; Rudolph and Knoflach, 2011). Structural mechanisms underlying how agonist binding promotes channel gating and drug binding modulates channel function are still not well understood.

In this thesis, I used the substituted cysteine accessibility method and two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes to demonstrate that orthosteric and allosteric non-competitive agonists stabilize the same open channel pore structure via distinct allosteric mechanisms, and structural mechanisms underlying the effects of combining drug modulators are different than mechanisms underlying their actions alone. I discovered that TMD inter-subunit interfaces are hotspots that shape drug actions. I also used whole-cell recordings with rapid solution application in HEK 293 cells to present that the alpha-subunit beta4-beta 5 linker is important for GABAAR kinetics and BZD positive modulation and thus a potential structural correlate of the intermediate transduction element.

The results in this thesis provide new information about structural mechanisms underlying GABAAR activation and allosteric drug modulation. These data will provide useful information on targeting subunit interfaces in allosteric proteins for novel drug discovery and development.