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% loss of helical content while keeping the characteristic pentamer intact. Contrastingly, unfolding by SDS shows dissociation of the pentamer, but no noticeable change in secondary structure. Still, refolding from urea and SDS proved to be impossible. Two weak fluorophores, crystal violet (CrV) and ethidium bromide (EtBr), bind to the homologous nicotinic acetylcholine receptor (nAChR). Because the fluorescence of these compounds is also sensitive towards their environment they can be used as a molecular beacon for the functional state of a receptor. When applied to 5-HT3R, it was found that CrV and EtBr bind with micromolar affinity to the desensitized state of the receptor – similar to binding reported for the nAChR. It appeared that binding of CrV or EtBr to the 5-HT3R induces a certain functional state (resting, open or desensitized). Because no detectable change in fluorescence occurs, this prevents their use as molecular beacons. Still, non-competitive binding to the 5-HT3R was little reported on before and could be an important target in pharmacological research. The ICD of the 5-HT3R is predicted to contain a stretch of ∼60 disordered or flexible residues which might be involved in subunit assembly and membrane trafficking. These unstructured regions of a 5-HT3R were probed using limited proteolysis. Thereby two distinct fragments were formed that remain tightly associated with each other, while a small stretch of 35 residues between TM3 and TM4 was removed. Furthermore, it was shown that the thermal stability of the helical core is unchanged compared to untreated 5-HT3R, but that the extracellular ligand-binding domain is very sensitive to cleavage in the ICD. By probing the 5-HT3R with limited proteolysis a rigid core was identified, indicating the important role of the transmembrane domain for holding together the pLGIC scaffold. The results presented here contribute a significant body of knowledge to membrane proteins in general and to the 5-HT3 receptor in particular, upon which further research can be based leading to advances in the fields of biochemistry, biophysics and pharmacology.