Breaking brains and barriers: A novel approach to studying neurotransmitter receptor protein-protein interactions.

Abstract

The Serotonin Receptor 3A (5-HT3A) is a member of the pentameric ligand-gated ion channel (pLGIC) superfamily and plays a role in numerous neuronal signaling pathways. However, relatively little remains known about its regulation. The intracellular domain (ICD) of the 5-HT3A receptor provides an attractive target for regulation studies, as the over 100 amino acid long sequence is exposed to the cell's internal milieu and is poorly conserved compared to that of other superfamily members. In this study, we set off to discover which proteins interact with the ICD of the 5-HT3A receptor, to identify potential regulatory partners. After developing a modified cellular fractionation technique using mouse brains, we were able to show, initially with western blotting, the presence of the 5-HT3A receptor in the endoplasmic reticulum (ER) where the receptor subunits assemble and the plasma membrane (PM) where the pentameric receptor is located. Once cellular localizations were preliminarily confirmed, proteins present in fractionated brain lysate fractions were identified using MALDI-TOF-MS/MS for further validation. Not only did this novel process work for serotonin receptors, it also worked for a variety of other key neurotransmitter receptors. These new techniques and findings provide a potential means of targeting regulatory proteins effecting numerous neuronal processes. This paves the way forward for progress in the study and treatment of neuropsychiatric disorders such as schizophrenia, Alzheimer's, and addiction.