Date of Award
Doctor of Philosophy (PhD)
David A. Wagner
Edward Blumenthal, Michelle Mynlieff, Robert Peoples
The gamma-aminobutyric acid type A (GABAA) receptor is a member of the cys-loop family of ligand-gated ion channels, and plays a crucial role in normal brain function by providing inhibitory neurotransmission. The objective of my research is to establish the mechanisms that underlie the interaction between the GABAA receptor and GABA during binding, as well as to provide direct information about the architecture of the ligand binding pocket. To achieve this, a multitude of amino acid residues surrounding the GABA binding pocket were individually mutated and structure-function relationships were explored. Changes in EC50-GABA, macroscopic kinetics, GABA binding rates and GABA unbinding rates were assessed using patch-clamp recording, rapid-ligand application, and kinetic modeling techniques.
A state-dependent interaction bridging the β/α inter-subunit interface was identified between α1R120 and β2D163 by characterizing GABA binding and unbinding rates for alanine mutations at each residue. These results were subjected to double-mutant cycle analysis. Intriguingly, the residues appear to be completely independent when considering the binding of GABA, but they are coupled when looking at the unbinding of GABA. These results suggest that β2D163 and α1R120 do not interact in the unbound state but form an interaction upon binding of GABA.
A role for β2F200 at the GABA binding site was also revealed. Mutation of β2F200 to alanine caused a dramatic reduction in GABA affinity. This was the result of both an increase in the rate of GABA unbinding and a decrease in the GABA binding rate. β2F200 fits the profile of a residue that could directly interact with GABA.
Finally, three mutations of the β2 subunit (Y97A, Y157A, and D163A) have interesting effects on the functional expression of receptors. Mutation of these residues allowed the assembly of functional receptors when expressed with α1 and γ2, but not when expressed with α1 only. The aligned residues on the γ2 subunit were also mutated and found to have unique expression patterns. Each of the residues appears to be required for the assembly of the β(+)/β(-) interface, which is only present in αβ receptors; however, only the residue homologous to β2Y97 (γ2F112) is critical for assembly at the γ/β interface.