Analysis of Secondary Structure Elements in the Intracellular Domain of GLIC Chimeras with ICD from Eukaryotic Cys-Loop Receptors

Abstract

Cys-loop receptors are pentameric ligand-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that forms the ion channel pore and an intracellular domain (ICD). All prokaryotic homologs of Cys-loop receptors, for example, GLIC and ELIC, lack an ICD that consists of a 50 to 270 amino acid long loop in eukaryotes. Very little is known about the structure of the ICD since none of the recently published X-ray structures of prokaryotic homologs contained an ICD, and the Torpedo nAChR cryo-EM structure resolved only 1/3rd of the ICD. We have recently constructed chimeras in which the ICD from eukaryotic serotonin (5-HT3A) receptors was inserted into GLIC (1).To extend our studies we generated GLIC chimeras with ICDs from other Cys-loop receptors (GlyR-α1, GABA-ρ1, nAChR-α7). All chimeras were expressed heterologously in Xenopus laevis oocytes to investigate the ion channel functionality by using two-electrode voltage clamp experiments. All functional chimeras were also expressed in E. coli membranes to enable overexpression and protein purification. Chimeric proteins were successfully purified by using Ni-NTA affinity and size exclusion chromatography (SEC). Limited proteolysis and spectroscopic methods (circular dichroism and fourier transform infrared spectroscopy) will be used to study conformational features and secondary structure elements of eukaryotic ICDs of the engineered GLIC-ICD chimeras. Our data will shed light on the structure and function of the intracellular domain of Cys-loop receptors, which will enable identification of potentially important drug targeting sites in intracellular domains for the treatment of neurodegenerative diseases.1. Goyal R. et al, (2011). J Biol Chem 286, 34635-42