Metamorphic Response of CLIC1 Chloride Intracellular Ion Channel upon Interaction with the Membrane

Abstract

The Chloride Intracellular Channel (CLIC) family can be characterized as ‘metamorphic’proteins; namely, capable of reversibly shifting between two or more different-folded conformations. The CLICs are expressed as soluble proteins but can also auto-insert into the membrane to form active ion channels. Such a conformational transition must involve large-scale structural rearrangement to confer favourable interactions with the membrane. To date, little is known about the process and cellular triggers for CLIC membrane insertion; although a unique feature of mammalian CLIC1 is its ability to undergo a dramatic structural rearrangement from a monomer to a dimer upon oxidation in solution. Whether this oxidation-induced metamorphosis in solution facilitates CLIC1 membrane insertion is unclear. We have sought to characterize the structural response of CLIC1 upon interaction with the membrane. A novel labeling scheme for CLIC1 was devised to enable site-directed-labeling of single native cysteines with either fluorophores or spin labels. Fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) revealed that the interaction of CLIC1 with the membrane results in a large-scale inter-domain movement. The two phases for membrane interaction, i.e. association followed by insertion, were also monitored by probing the local environment of an N-terminal transmembrane tryptophan residue (Trp35) using fluorescent quenching. A sucrose-loaded-vesicle sedimentation assay was also used to quantify membrane binding. Our results suggest oxidation of CLIC1 monomer in the presence of the membrane promotes CLIC1 binding and insertion. Our current model for the structural transitions and environmental triggers of CLIC1 membrane-induced metamorphosis will be discussed.