Characterization of the Interaction of the Intracellular Domain of 5-HT3A Receptors with the Chaperone Protein RIC-3

Abstract

Nicotinic acetylcholine (nACh), γ-aminobutyric acid type A (GABAA), glycine, and 5-HT3 (serotonin) receptors are members of the pentameric ligand-gated ion channel (pLGIC) superfamily. Each channel consists of five homologous subunits consisting of three domains, namely, extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). The ECD and TMD that display high sequence-similarity between different subunits are well-studied and their structures have been solved. The ICD on the contrary is extremely diverse between subunits and therefore may provide a specific target for future drug development. For both nAChR and 5-HT3A receptors the chaperone protein resistance to inhibitors of cholinesterase (RIC-3) modulates functional maturation. We have shown previously, that the replacement of the ICD in 5-HT3A by a heptapeptide abrogates this modulation by RIC-3. Additionally, we have shown, that adding either the 5-HT3A or nAChRα7 ICD to the prokaryotic Gloeobacter violaceus pLGIC (GLIC) confers RIC-3 sensitivity to otherwise RIC-3-insensitive GLIC. Together, this demonstrated that the ICD is required for the RIC-3 modulation. The present study is aimed at identifying the molecular identity within the 5-HT3A-ICD that interacts with RIC-3. To this end we have genetically engineered chimeras containing the 5-HT3A-ICD and expressed them in Escherichia coli (E. coli). These chimeras are then purified to homogeneity and their interaction with RIC-3, which is separately expressed and purified from E. coli, assessed. It is thought that RIC-3 associates with monomers and promotes pentamerization. Therefore, we investigate both 5-HT3A-ICD chimera constructs that form pentamers, but also those that form monomers. Additionally, we are using 5-HT3A-ICD chimera constructs to further identify interacting cytosolic proteins.