A Soluble GABAAρ1 Intracellular Domain Chimera for Structural Studies

Abstract

The GABAAρ1 pentameric ligand-gated ion channel (pLGIC) is an anion-conducting receptor of the Cys-loop superfamily gated by the neurotransmitter γ-aminobutyric acid (GABA). GABAAρ1 and other anionic pLGICs are the targets of pharmacotherapy including general anesthetics, anti-epileptics (anti-convulsants), sedatives, anxiolytics, and muscle relaxants. In an effort to improve current treatments and develop new drugs, further studies characterizing the pLGICs and more specifically their structural elements that contribute to their function are essential. Each subunit of eukaryotic Cys-loop receptors is comprised of three domains: extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD). Several structural studies including X-ray crystallography of prokaryotic as well as eukaryotic pLGICs have elucidated the three-dimensional structures of both the ECD and TMD. However, the complete picture on the three-dimensional structure of the ICD is not available. This is partly due to the lack of the ICD in prokaryotic pLGICs, such as Gleoeobacter violaceus ligand-gated ion channel (GLIC). Furthermore, in order to obtain higher resolution X-ray crystallographs of mammalian Cys-loop receptors, the ICD is frequently modified. Of the three domains, the ICD of Cys-loop receptors in the animal kingdom has the most diversity in terms of amino acid sequence and length. Therefore, drugs selective for the ICD would be an ideal alternative to current pharmacotherapy ridden with many side effects that result from targeting the highly-conserved extracellular or transmembrane domains.Our project focused on the expression and large-scale purification of GABAAρ1 chimeras in amounts that would allow future structural studies. A soluble chimera containing the ICD was over-expressed in E. coli cells and purified to homogeneity using a two-step purification process. We were successful in producing approximately 3-4 mg of purified protein from a liter of bacterial culture.