TY - JOUR T1 - Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage Clamp Fluorometry JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.119.116657 SP - mol.119.116657 AU - Lachlan Munro AU - Lucy Kate Ladefoged AU - Vithushan Padmanathan AU - Signe Andersen AU - Birgit Schiott AU - Anders Skov Kristensen Y1 - 2019/01/01 UR - http://molpharm.aspetjournals.org/content/early/2019/10/03/mol.119.116657.abstract N2 - The 5-HT3 receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is a clinical target in a range of diseases. In the nervous system, the 5-HT3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand-binding that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT3 receptor, many important aspects of the mechanistic basis of 5-HT3 receptor function and modulation by drugs remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and desensitization. In the present study, we used voltage clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT3A (h5-HT3A) receptor during binding of 5-HT and different classes of 5-HT3A receptor ligands. VCF utilizes parallel measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can in real-time report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C-, D-, and E-regions that surround the orthosteric binding site in the h5-HT3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand class. Interpretation of these results within the framework of current models of 5-HT3A and Cys-loop receptor mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating.SIGNIFICANCE STATEMENT The 5HT3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5HT3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5HT3 receptor to identify intra-receptor motions during binding of a range of 5HT3 receptor agonists and antagonists. ER -