Structural Elements Governing GABA-A Receptor Channel Activation and Drug Modulation

Abstract

Inhibition in the brain depends on rapid opening and closing of GABA-A receptor (GABAR) channels. Therapeutic drugs such as benzodiazepines (BZDs), barbiturates and general anesthetics modulate GABAR currents by binding to distinct allosteric sites that are far from the agonist binding sites. Structural mechanisms underlying how GABA binding promotes channel gating and how drug binding modulates channel function are still unclear. In a recent elements-based kinetic model of GABAR gating (JGP 144:27-39, 2014), intermediate transduction elements mediate the coupling between the two GABA binding sites to the channel activation gate and desensitization gates. Moreover, the BZD binding site also interacts with one of the transduction elements to promote BZD modulation of GABA-mediated currents. Whether specific regions of the GABAR are associated with these intermediate transduction elements is not known. We hypothesize that a region in the α subunit that physically links the BZD and GABA binding sites (α β4-β5 linker, K105-M111) is an important transduction element. Previously, when glycine residues were inserted into this linker, BZD modulation of GABA-mediated currents was significantly decreased (PNAS 287: 6714-6724,2012). Here, we expressed wild-type αβγ GABARs and mutant GABARs with 4-glycine insertions in the α β4-β5 linker (αGly4) in human embryonic kidney 293 cells and measured GABAR currents using outside-out patch clamp electrophysiology and ultra-rapid ligand exchange. Compared to wild-type GABARs, αGly4 currents were slow to activate and fast desensitization was eliminated indicating that this linker region is a key structural element that influences GABAR kinetics. Kinetic models will be used to explain αGly4 effects on GABAR macroscopic current responses and to identify the role the α β4-β5 linker plays in GABA activation and BZD potentiation.