Hossain Khondker R, Al Khamici Heba, Holt Stephen A, Valenzuela Stella M
School of Life Sciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia.
Bragg Institute, Australian Nuclear Science and Technology Organisation (ANSTO), New South Wales 2234, Australia.
Membranes (Basel). 2016 Feb 11;6(1):15. doi: 10.3390/membranes6010015.
CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion-channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre-incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1-cholesterol pre-complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels.
CLIC1是一种氯离子细胞内离子通道蛋白，它以两种形式存在，一种是在细胞质中呈可溶状态，另一种是作为膜结合蛋白。CLIC家族成员大多是可溶蛋白，具有一种有趣的特性，即能自发插入磷脂双层以形成整合膜离子通道。先前已使用阻抗谱评估了胆固醇在束缚脂质双层中对CLIC1离子通道活性的调节作用。在此，我们通过评估胆固醇对CLIC1自发插入到由1-棕榈酰-2-油酰磷脂酰胆碱、1-棕榈酰-2-油酰-sn-甘油-3-磷酸乙醇胺和1-棕榈酰-2-油酰-sn-甘油-3-磷酸-L-丝氨酸单独或与胆固醇组合制备的朗缪尔膜单层中的影响，扩展了这项研究。结果表明，CLIC1的自发膜插入依赖于膜中胆固醇的存在。此外，在将CLIC1添加到朗缪尔膜之前先用胆固醇预孵育，结果显示即使在含有胆固醇的单层膜中也没有膜插入，这表明形成了CLIC1 - 胆固醇预复合物。因此，我们的结果表明，CLIC1与膜的相互作用涉及CLIC1与膜中胆固醇结合以进行初始对接，随后插入。蛋白质随后可能还需要进行结构重排以及寡聚化以形成功能性离子通道。
