Expression and Purification of the Intracellular Domain of a Cationic Pentameric Ligand-Gated Ion Channel

Abstract

Cationic pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily function as receptors for endogenous (e.g. acetylcholine, serotonin) and/or exogenous (e.g. nicotine) ligands. These receptors are the targets for a number of current clinical drugs used to treat nicotine addiction, Alzheimer's disease, Parkinson's disease, epilepsy, pain perception, anxiety, and depression. In order to develop new, more effective drugs and improve current treatments, research is imperative to identify and characterize the different structural elements that mediate each aspect of receptor function. From an early electron density map of a pLGIC of the Cys-loop superfamily to the most recent high-resolution crystal structures of mammalian Cys-loop receptors, their structural information has revealed that the overall architecture of these pLGICs is highly-conserved. The intracellular domain (ICD) presents the notable exception in that the functions, lengths, amino acids sequences, and likely also structural folds are vastly different between subunits. Based on its diversity, the ICD presents a target for developing subtype-selective drugs with the hope of fewer side effects than current drugs, all of which target the highly-conserved extracellular or transmembrane domains.We developed a protein expression and purification strategy to produce the ICD of a cationic pLGIC in sufficient quantity and quality. Chimeras were generated by adding the ICD of the α7 nicotinic acetylcholine receptor to a soluble protein, and conditions for optimal expression in E. coli were identified. A two-step purification process consisting of affinity chromatography followed by size-exclusion chromatography yielded 5 mg of purified protein per liter of bacterial culture. Thus, our results are consistent with the successful large-scale expression and purification of the α7-ICD chimera that is now amenable to structural studies.