Effects of 5-HT3A Intracellular Domain Modifications on Oligomerization

Abstract

The 5-Hydroxytryptamine type 3 (5-HT3) receptor is a cation-selective pentameric ligand-gated ion channel (pLGIC) that contributes to fast synaptic neurotransmission in the central and peripheral nervous system. Targeting these receptors could be a potential treatment for anxiety, depression, and an alternative to antipsychotics drugs. Two of the three eukaryotic pentameric domains, the extracellular and transmembrane domain of the 5-HT3A, have been studied and fully crystallized. However, the intracellular domain (ICD), which is the most divergent domain of the pLGIC superfamily, has only been partially resolved, and its function remains mainly unknown. The ICD of homomeric 5-HT3A has been shown to form a pentameric quaternary structure in the absence of the two other domains. When mutations are introduced into this domain, alterations in receptor conductance have been observed. Based on this knowledge, we investigated if such alterations would also alter the oligomeric state of the receptor, and ultimately, resolve its structure. Chimeras of the ICD were created and a modified maltose-binding protein (MBP) was attached to its N-terminus by a short alanine linker, expressed in Escherichia coli, and the resulting protein construct was purified using amylose column purification and size-exclusion chromatography (SEC). To further analyze the oligomeric state of the ICD, we employed SEC in line with Multi-Angle Light Scattering (SEC-MALS) to measure the absolute molecular weight. Additionally, the constructs are used for spectroscopic studies of their stability and secondary structure content (DSC, CD), as well as for high-throughput crystallization trials.