Approaches to Structural Characterization of a Heteromeric GABA(A)R

Abstract: Structural biology has become an important part of researching various diseases and drug development. In this thesis, I provide details on how I worked with approaches to structural characterization of a heteromeric GABA(A)R. These pentameric ligand gated ion channels take part in regulating inhibition of action potentials in nerve cells by allowing the passage of Cl- ions when bound by gamma-aminobutyric acid (GABA). They are formed by the assembly of five subunits which can be of various different types, denoted by greek letters and a number. Much is still unknown about how GABA and several other ligands bind to these ion channels and how that impacts function. Obtaining a structure of these proteins can aid in closing those knowledge gaps. It is reasonable to screen the proteins you have before you study their structures by Cryo-EM in order to get the best result, a methodology for which is described here. I have followed this methodology to screen two heteromeric GABA$_A$R that we wish to determine the structure of, alpha 5 beta 3 and rho 1 gamma 2. Neither of the combinations of genes we used to express these proteins proved to produce the desired fully assembled heteromeric protein. In the case of alpha 5 beta 3, we only witnessed building blocks, with no fully assembled channels. In rho 1 gamma 2, we instead only witnessed fully formed homomers of the rho 1 subunit. These findings then exclude the gene constructs used from further structural study, and the methodology described will inform the next steps to be taken.