The Metamorphic CLIC1 Protein Requires Cholesterol for Optimal Conduction In Membranes — ASN Events
  1. Schedule
  2. Session Five - Poster Session A
  3. The Metamorphic CLIC1 Protein Requires Cholesterol for Optimal Conduction In Membranes

The Metamorphic CLIC1 Protein Requires Cholesterol for Optimal Conduction In Membranes (#124)

Heba Alkhamici 1 , Sonia Carne 2 , Louise Brown 3 , Bruce Cornell 2 , Stella Valenzuela 1
  1. Medical and Molecular Biosciences, University of Technology Sydney, Sydney, NSW, Australia
  2. Surgical Diagnostics Pty Ltd, St Leonards, NSW, Australia
  3. Department of Chemistry and Bimolecular Sciences, Macquarie University, Sydney, NSW, Australia

The Intracellular Ion Channels (CLIC) are a family of metamorphic human proteins consisting of six members that were found to exist as soluble in solution and as membrane bound proteins (1,2). CLIC1 protein has been reported to spontaneously insert into cell membranes to form chloride ion channels. However, factors that control the structural transition of CLIC1 from being soluble in solution into membrane bound protein in order to form functional ion channels have yet to be adequately described. Thus, the objectives of this work are to identify and characterize factors that are involved in protein insertion and assembly into bilayer lipid membranes.

Monomeric CLIC1 (WT) protein was expressed in E-Coli and purified in the presence of the reducing agent TCEP (0.5mM), first by His-tag Ni2+-NTA high affinity chromatography column followed by size exclusion chromatography. Control proteins (boiled CLIC1), Listeriolysin O (Sapphire Bioscience), α-Hemolysin (Sigma Aldrich) along with reduced CLIC1 (WT) (incubated with 0.5mM TCEP) and oxidised (incubated with 2mM H2O2) were added to tBLM (tethered bilayer lipid membrane) and the resultant conduction from each protein was measured by impedance spectroscopy (3). tBLMs were formed using Hepes buffer (pH 6.5) and neutral lipids mixed with 50mol%, 25mol%, 12.5mol%, 6.25mol% and 0mol% cholesterol.

Our findings suggest that in order for CLIC1 (WT) protein to insert into membranes and form ion conducting channels, cholesterol is required in the range10mole% to 50mole% relative to membrane phospholipids. A model is proposed in which CLIC1 (WT) protein inserts into bilayer membranes in a manner similar to the Cholesterol Dependent Cytolysin (CDC) protein family.

  1. Valenzuela, S. M. Martin, D. K. Por, S. B. Robbins, J. M. Warton, K. Bootcov, M. R. Schofield, P. R. Campbell, T. J. and Breit,S. N. (1997) Molecular cloning and expression of a chloride ion channel of cell nuclei, J Biol Chem 272, 12575-12582.
  2. Goodchild, S. C. Cordina, N. M. Littler, D. R. Breit, S. N. Curmi, P. M. Brown, L. J. (2009) Oxidation promotes insertion of the CLIC1 chloride intracellular channel into the membrane, Eur Biophys J 39, 129-138.
  3. Surgical Diagnostics Pty Ltd, St Leonards, Australia