Highly Pure Soluble Chimeras of the Intracellular Domain of Anionic Pentameric Ligand-Gated Ion Channels

Abstract

The past few years have seen remarkable advances in structural information gleaned from atomic resolution X-ray crystallographs of important anionic and cationic pentameric ligand-gated ion channels (pLGIC) from the Cys-loop superfamily. The highly conserved extracellular domain (ECD) and transmembrane domain (TMD), and the diverse intracellular domain (ICD) constitute each subunit of eukaryotic pLGICs. The ECD and TMD of different Cys-loop receptors are prime targets for several clinical drugs. These drugs are thus notorious for binding indiscriminately to undesired receptors within the superfamily resulting in a wide range of unwanted side-effects. Intriguingly, the marked variability of ICDs between different subtypes can be exploited to aid in the development of newer drugs with refined specific binding. However, the ICD of any Cys-loop receptor has yet to be fully characterized. The two prominent anion-conducting pLGICs, glycine-α1 (Gly-α1) and γ-amino butyric acid type A-ρ1 (GABAA-ρ1) receptors, of the Cys-loop superfamily are critical in mediating postsynaptic inhibition in the central nervous system. Dysfunction of these pLGICs has been implicated in numerous serious neuropsychiatric and neurological diseases. The goal of our current project was to develop a strategy for protein expression and purification of the ICD of Gly-α1 as well as GABAA-ρ1 receptors. Here, we generated chimeras containing maltose-binding protein (MBP) fused to the N-terminus of the ICD of either Gly-α1 receptor or that of GABAA-ρ1 receptor and separated by a short alanine linker. We expressed these chimeras in E. coli and were able to purify both proteins of interest to homogeneity by employing 3-step purification. Further characterization to determine size using size exclusion chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) revealed the oligomeric state. Experiments are underway to gain detailed structural insights.