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. 2013;8(2):e56948.
doi: 10.1371/journal.pone.0056948. Epub 2013 Feb 14.

Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol

Stella M Valenzuela  1 , Heba Alkhamici, Louise J Brown, Oscar C Almond, Sophia C Goodchild, Sonia Carne, Paul M G Curmi, Stephen A Holt, Bruce A Cornell
Affiliations

Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol

Stella M Valenzuela et al. PLoS One. 2013.

Abstract

The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

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Conflict of interest statement

Competing Interests: Sonia Carne and Bruce A. Cornell are employed by Surgical Diagnostics Pty Ltd. who provided lipids for this study. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Membrane Conductance of α-hemolysin.
Representative impedance spectroscopy recordings of 50 nM α-hemolysin added to tethered membranes made from AM199 lipid containing varying amounts of cholesterol (mol% chol) (A) 0, (B) 10, (C) 20, (D) 30, (E) 40, (F) 50; [n = 3].
Figure 2
Figure 2. Membrane Conductance of Lysteriolsin.
Representative impedance spectroscopy recordings of (A-E) 2 µM Lysteriolysin and (F) buffer control, added to tethered membranes made from AM199 containing varying amounts of cholesterol (mol% chol) (A) 50, (B) 25, (C) 12.5, (D) 6.25, (E) 0, (F) 50; [n = 3].
Figure 3
Figure 3. Membrane Conductance of CLIC1.
A. Representative impedance spectroscopy recordings of (A-E) 7.4 µM CLIC1 and (F) buffer control, added to tethered membranes made from AM199 lipid containing varying amounts of cholesterol (mol% chol) (A) 50, (B) 25, (C) 12.5, (D) 6.25, (E) 0, (F) 50; [n = 3]. B. Representative impedance spectroscopy recordings of (A) buffer control, (B) 7.4 µM boiled CLIC1 and (C) 14.8 µM boiled CLIC1, added to tethered membranes made from AM199-PC lipid containing 25 mol% cholesterol; [n = 3].
Figure 4
Figure 4. Effects of Cholesterol on the Membrane Conductance of CLIC1.
Representative impedance spectroscopy recordings of (A) 7.4 µM CLIC1 (in 100 µL volume) pre-incubated with 2 µL ethanol (solvent control), (B) no protein added (buffer control), (C) 7.4 µM CLIC1 (in 100 µL volume) pre-incubated with 2 µL cholesterol in EtOH, and (D) 14.8 µM CLIC1 (in 100 µL volume) pre-incubated with 2 µL cholesterol in EtOH. Samples added to tethered membranes made from AM199 lipid containing 50 mol% cholesterol; [n = 3].
Figure 5
Figure 5. Insertion of CLIC1 into Lipid Monolayers.
Representative area traces of Langmuir films held at a surface pressure of 20 mN/m with 0.036 µM CLIC1 (A-C) and (D, E) no CLIC, added beneath POPC monolayers containing cholesterol (mol% chol) (A) 16.7, (B) 9.0, (C) 0.0, (D) 16.7, (E) 0.
See this image and copyright information in PMC

References

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