Testing the Functionality of Mutant Chimerical Transmembrane Protein, Gloeobacter violaceus Ligand-Gated Ion Channel (GLIC), with Genetically Inserted Eukaryotic Intracellular Domain (ICD)

Cys-loop receptors are ligand-gated ion channels that are abundant in the central nervous system where they mediate fast synaptic chemical transmission. Cys-loop receptors are targets for various pharmaceutical drugs for the treatment of severe diseases such as epilepsy, anxiety, Alzheimer’s disease, schizophrenia, ADHD, depression, and psychosis. Eukaryotic Cys-loop receptors have three domains: extracellular domain (ECD), transmembrane domain (TMD) and intracellular domain (ICD), where prokaryotes lack the ICD. Both the ECD and TMD have been described and studied in great depth but little is known about the ICD. Thus, understanding of the function of the ICD will provide an insight into the design of ICD specific drugs. Homo-pentameric mutant chimeras were created through genetic engineering by inserting the ICD from human nicotinic acetylcholine receptor (nAChR) α7 subunits in the short cytosolic loop between transmembrane segments M3 and M4 of the prokaryotic Gloeobacter violaceus ligand-gated ion channel (GLIC). In this comprehensive study the functionality of chimeras is being compared to control (wild type GLIC) channels. The chimeras were expressed in Xenopus laevis oocytes and Two Electrode Voltage Clamp (TEVC) recordings were employed to measure the currents induced by application of agonist, protons. An initial electrophysiological test utilizing application of pH 5.5 showed 11 out of 12 mutants were functional. Further experiments will study the functionality of the GLIC-α7ICD chimeras in more detail.