Super-Resolution Imaging and Single Particle Tracking of Serotonin 5HT3A Receptor in Biomimetic Membranes

Abstract

The serotonin 5HT3 receptor is a member of the Cys-loop super family of ligand gated ion channels (LGIC). Like all members of this family, 5HT3 is composed of five independent subunits. The receptor has a large extracellular domain, with the ion channel being constructed from the second trans-membrane segment of each subunit, and there is a smaller intercellular region that is thought to interact strongly with cytoskeletal proteins. We have shown that the 5HT3 receptor can be efficiently incorporated into planar supported biomimetic membranes and the orientation can be controlled. The mobility of 5HT3 is greatly affected by both the orientation of the LGIC and the composition of the biomimetic assembly. We have used single molecule fluorescence imaging to track the mobility of individual proteins within the supported lipid bilayer and have used c-terminal labeling to determine the orientation of individual proteins. We have recently constructed a low temperature super-resolution fluorescence microscope and have carried out experiments in frozen assemblies in order to identify the individual subunits that compose 5HT3A (the homopentamer of 5HT3).