A Biochemical and Biophysical Investigation of 5-HT3 Receptor Stability

The 5-hydroxytryptamine 3 receptor (5-HT3R) is a member of the pentameric ligand-gated ion channel (pLGIC) family, that plays an important role in fast signal transduction and cell-cell communication in synapses: They convert a chemical signal from a neurotransmitter into the electrical signal that represents the action potential. Activation of 5-HT3R with serotonin triggers a range of molecular reorganizations that lead to channel opening and subsequent membrane depolarization. Because of their role, pLGICs are involved in a wide range of diseases and disorders and are therefore important drug targets. A typical pLGIC is composed of a β -sheet rich extracellular domain (ECD) where ligand binding takes place, a transmembrane domain that is composed of four α-helices (TM1-TM4). In vertebrates pLGICs often contain a third intracellular domain (ICD), located between TM3 and TM4. The 5-HT3R is active as a homopentameric receptor of five identical A subunits, or as a heteropentamer containing one or more of several subunits (B to E) together with an A subunit. The 5-HT3 receptor is naturally located in the cell membrane in low amounts. For in vitro experiments such as biophysical investigation, the receptor has to be overproduced and purified in a detergent-solubilized state. Therefore, a robust and scalable expression system for homo- and heteropentameric 5-HT3 receptors was developed, building upon a tetracycline inducible cell line and Strep-tag affinity purification technology. Production of solubilized and purified 5-HT3R is well suited for use in the structural and functional characterization of the receptor. The stability of the 5-HT3 receptor is an important parameter during structural and functional experimentation. Using thermal unfolding the stability of 5-HT3R was investigated in plasma membranes as well as during detergent-extraction, purification and reconstitution into artificial lipid bilayers. A large loss in thermostability was found that correlates with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of 5-HT3R occurred in consecutive steps at distinct protein locations: First a loss of ligand binding is detected, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally leads to the formation large receptor aggregates. It was also found that structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Thermal unfolding leads to the irreversible formation of aggregates which is known to prevent effective refolding. Therefore, an further investigation of 5-HT3R was made by unfolding the receptor in urea and sodium dodecyl sulfate (SDS). The results indicate iii iv ABSTRACT that, unlike thermal unfolding, unfolding in urea and SDS does not result in the formation of aggregates. Unfolding by urea shows a 50