Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet structured extracellular domain opens an ion channel in the transmembrane -helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (5-HT3R) (ligand binding, secondary structure, accessibility of Trp and Cys residues, aggregation), in plasma membranes as well as during detergent-extraction, purification and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating 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. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification, and can be used to stabilize these membrane proteins for structural and functional studies.