title: Thermal unfolding of a mammalian pentameric ligand-gated ion channel proceeds at consecutive, distinct steps.
abstract: 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 α-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 (ligand binding, secondary structure, accessibility of Trp and Cys residues, and 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 the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation 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 create stabilized receptor proteins for structural and functional studies.
authors.affiliation: Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Menno B
authors.initials: MB
authors.lastname: Tol
authors.forename: Cédric
authors.initials: C
authors.lastname: Deluz
authors.forename: Gherici
authors.initials: G
authors.lastname: Hassaine
authors.forename: Alexandra
authors.initials: A
authors.lastname: Graff
authors.forename: Henning
authors.initials: H
authors.lastname: Stahlberg
authors.forename: Horst
authors.initials: H
authors.lastname: Vogel
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D006358:Hot Temperature; D058446:Ligand-Gated Ion Channels / Q000378:metabolism*; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D044348:Receptors, Serotonin, 5-HT2 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D003902:Detergents; D058446:Ligand-Gated Ion Channels; D008024:Ligands; D008051:Lipid Bilayers; D044348:Receptors, Serotonin, 5-HT2