Investigating the dual function of the chloride intracellular ion channel proteins
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The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans, CLIC1-CLIC6. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 in particular has challenged the widely held view that most proteins adopt one stable native structure essential for their biological function. In contrast, CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable soluble structural conformation. CLIC1 was also found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being soluble into a membrane bound protein have yet to be adequately described. Thus, the first objective of this thesis was to identify factors that are involved in CLIC1’s insertion and assembly into membranes using tethered bilayer lipid membranes and impedance spectroscopy as a novel system for the study of ion channel activity. Our findings demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1’s acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1’s membrane structure. Furthermore, our impedance spectroscopy results of CLIC1 mutants, suggest that residue Cys24 is not essential for CLIC1’s ion channel function however it is important for its optimal activity in membranes. Therefore oxidation and reduction may not be the only regulators of the ion channel activity of CLIC1. Structural studies have revealed that, soluble CLIC proteins adopt a glutathione S-transferase fold with a conserved glutaredoxin–like active site motif, similar to the GST-Ω class. Therefore the second aim of this project was to investigate the function of the soluble CLICs. Using the 2-hydroxyethyl disulfide enzyme assay, we have demonstrated for the first time that CLIC1, CLIC2 and CLIC4 possess “glutaredoxin-like” oxidoreductase activity. CLIC1 was found to catalyse the metabolism of the typical glutaredoxin substrates, sodium selenite and dehydroascrobic acid. As expected, the active site Cys24 was detected to be essential for the enzymatic activity of CLIC1 in vitro. Most importantly, indanyloxyacetic acid-94 and anthracene-9-carboxylic acid were found to also inhibit the enzymatic activity of CLIC1. Members of the CLIC protein family can now be classified as “moonlighting proteins” as they exhibit two independent functions; one as ion channels when in their membrane bound form and the other as oxidoreductase soluble enzymes.
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