Regulation of the Membrane Insertion and Conductance Activity of the Metamorphic Chloride Intracellular Channel Protein CLIC1 by Cholesterol

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

doi:10.1371/journal.pone.0056948

Cited by 36 publications

(76 citation statements)

References 36 publications

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“…Moreover, to confirm the cholesterol-dependent nature of pyolysin we performed pre-experiments where PLO was pre-incubated with different amounts of cholesterol prior to its addition to tethered membranes. This approach had previously been used by others who had shown that pre-incubation with cholesterol blocked the dielectric damage inflicted to tethered membranes [30]. Our results clearly showed that cholesterol is acting as a potential binding or docking site for PLO, confirming the requirement of cholesterol for the activity of this toxin.…”

Section: Discussionsupporting

confidence: 80%

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Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Preta

Jankunec

Heinrich

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs.

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Section: Discussionsupporting

confidence: 80%

“…cholesterol, already performed in literature [30]. As seen in Figure 1D, pre-incubation of rPLO protein with cholesterol prior to its addition to membranes, resulted in dose-dependent abrogation of rPLO-induced dielectric damage to tBLMs.…”

Section: Electrochemical Impedance Of Tblms Is Affected By Rplomentioning

confidence: 52%

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Preta

Jankunec

Heinrich

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…The CLIC proteins are redox sensitive and partition into lipid bilayers upon oxidation (Goodchild et al, 2009;Littler et al, 2005;Littler et al, 2004;Valenzuela et al, 2013), where they have ion-conducting properties that are blocked by IAA-94. Indeed, bovine CLIC5B (p64) was first isolated from kidney by affinity purification with IAA-94 (Landry et al, 1993;Redhead et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

Clustered phosphatidylinositol 4,5 bisphosphate accumulation and ezrin phosphorylation in response to CLIC5A

Al-Momany

Alexander

et al. 2014

Journal of Cell Science

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CLIC5A (encoded by CLIC5) is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] reporter RFP-PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA-PIP5Ka colocalized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Ka abrogated CLIC5A-dependent ERM phosphorylation. N-and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane, failed to colocalize with PIP5Ka, did not alter the abundance of PI(4,5)P 2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5-deficient mice, the phosphorylation of glomerular ezrin was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P 2 accumulation through an interaction of CLIC5A with PI(4,5)P 2 -generating kinases, in turn facilitating ezrin activation and actin-dependent cell surface remodeling.

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“…This has allowed for their ease of study using a range of artificial lipid membrane systems including tip-dip and tethered bilayer membranes (tBLMs) [3,4]. Reported conductance levels from single channel recordings of the ion channel activity of recombinant CLIC1 (rCLIC1) added to artificial membrane systems and from cells over-expressing rCLIC have been varied [5,6].…”

Section: Introductionmentioning

confidence: 99%

Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes

Khamici

Hossain

Cornell³

et al. 2016

Membranes

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The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we 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 and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1’s ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.

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Regulation of the Membrane Insertion and Conductance Activity of the Metamorphic Chloride Intracellular Channel Protein CLIC1 by Cholesterol

Cited by 36 publications

References 36 publications

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case

Clustered phosphatidylinositol 4,5 bisphosphate accumulation and ezrin phosphorylation in response to CLIC5A

Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes

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