Dynamics of the 5-ht3a serotonin receptor in living cells

Abstract

The 5-HT3 serotonin receptor belongs to the cys-loop ligand-gated ion channel (LGIC) family. It is located in the central and peripheral nervous system and is implicated in certain pathologic situations. The 5-HT3 receptor is investigated here, as a typical example of these LGIC's, to elucidate its structure and dynamics in living cells. Distribution, receptor-ligand interaction and low-resolution structural information of the 5-HT3 receptor were determined using a new method for protein labelling (chapter 2). This generic method does not require the fusion of bulky fluorophores or other moieties to the target molecule. Instead, small nickel-chelating nitrilotriacetate probes are used that bind specifically and reversibly to polyhistidine loops on the target proteins. The lateral mobility of the 5-HT3 in different functional states was investigated by single-molecule microscopy using the newly developed repetitive protein labelling method (chapter 3). A significant reduction of the lateral mobility of the desensitized receptor was observed indicating the role of serotonin in modulating the diffusion of the receptor, which might be important in the development of the synapse. Conformational transitions of the 5-HT3 receptor were investigated by tethering a fluorophore to a single cysteine side chain introduced at the 20' position in the TM2 region (chapter 4). During serotonin application, fluorescence decreased by 18% indicating a change of the fluorophore environment. Eighteen single cysteine receptors were characterized for functionality and cysteine accessibility. Finally, electrical current through single activated 5-HT3 mutant receptors was measured (chapter 5). Five different conductance levels were determined and noise analysis of current fluctuations in the open states of the activated receptor revealed a 1/fα dependence with α < 1. According to a proposed model, the oscillating domain responsible of the current fluctuations contains charged amino acids. These results contribute to a more comprehensive model of the dynamics both of the 5-HT3 receptor and 5-HT3 receptor within cells.