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

Abstract

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.

Keywords: CLIC; chloride intracellular ion channel proteins; cholesterol; ergosterol; tethered lipid membranes.

Figure 1

Conductance of CLIC1 in tBLMs containing varying amounts of cholesterol. 20 µg of CLIC1 oxidised dimer was pre-incubated with 2 mM H₂O₂; 20 µg of CLIC1 reduced monomer was pre-incubated with 0.5 mM TCEP in 100 µL of HEPES/KCl buffer (pH 6.5) prior to adding to tethered bilayer membranes containing 0, 6.25, 12.5, 25 and 50 mol % cholesterol concentrations. Control is membrane with 0 mol % cholesterol containing 100 µL HEPES/KCl buffer (pH 6.5) with no protein added. The error bars represent the standard error of three independent impedance spectroscopy conductance measurements (n = 3).

Figure 2

Conductance of different concentrations of CLIC1 in tBLMs containing 25 mol % cholesterol. Concentrations of 0, 10, 20, 40 and 60 µg of CLIC1 reduced monomer (pre-incubated with 0.5 mM TCEP) and CLIC1 oxidised dimer (pre-incubated with H₂O₂) in 100 µL of HEPES/KCl buffer (pH 6.5) were added into membranes containing 25 mol % cholesterol where the conductance was measured and linear fitting (as indicated in black for CLIC1 reduced monomer and red for oxidised dimeric CLIC1) and quadratic polynomial fits were generated using Microsoft Excel 2010 (y = −0.0005x² + 0.0924x + 0.3639, R² = 0.9985 for CLIC1 monomer and y = −0.0001x² + 0.0399x + 0.5761, R² = 0.9857). The error bars represent the standard error of three experimental repeats (n = 3).

Figure 3

Amino Acid Sequence Alignment of Human CLIC proteins showing the CRAC motif. Highlighted in red is the GXXXG motif and in Green highlighted the LWLK motif in human CLICs. CLIC1 (accession number: CAG46868.1), CLIC2 (accession number: CAA03948.1), CLIC3 (accession number: NP_004660.2), CLIC4 (accession number: CAG38532.1), CLIC5 (accession number: AAF66928.1), CLIC6 (accession number: NP_444507.1). The alignment was produced using Clustalw.

Figure 4

Conductance of CLIC1 mutants and EXC-4 in membranes containing 25 mol % cholesterol. 20 µg of CLIC1-C24A; CLIC1-C24S; CLIC1-C59A; EXC-4 and CLIC1 (WT) proteins in 100 µL HEPES/KCl buffer (pH 6.5) were reconstituted in tethered bilayer membranes containing 25 mol % cholesterol and the conductance was measured and analysis was performed using excel 2010 and Graph pad prism 6. Control sample is buffer only containing 0.5 mM TCEP with no protein added to membrane containing 25 mol % cholesterol. The error bars represent the standard error of three independent repeats of conductance measurements (n = 3).

Figure 5

Conductance and capacitance of membranes containing different concentrations of cholesterol or ergosterol. Bilayer lipid membranes were formed using 10% tethered lipids on a gold electrode representing the monolayer of membrane and zwitterionic lipids dissolved in ethanol containing different concentrations of cholesterol or ergosterol was used as the second layer for the membrane. Membranes were then rapidly flushed with HEPES/KCl buffer (pH 6.5) in order to remove the ethanol by solvent exchange method and enhance the formation of the bilayer lipid membranes. The conductance and the capacitance of membranes were measured using impedance spectroscopy. (A) Represents membrane conductance at different cholesterol or ergosterol concentrations and (B) Capacitance of membrane with different cholesterol or ergosterol concentrations. The error bars represent the standard error of three independent impedance spectroscopy conductance measurements.

Figure 6

Conductance of CLIC1 in tBLMs containing 25 mol % ergosterol. 20 µg of CLIC1 monomer (pre-incubated with 0.5 mM TCEP) and dimer (pre-incubated with 2 mM H₂O₂) in 100 µL of HEPES/KCl buffer (pH 6.5) were incorporated into membranes containing zwitterionic lipids and 25 mol % ergosterol. The error bars represent the standard error of three independent experimental repeats (n = 3).

Figure 7

Conduction of pre-incubated CLIC1 monomer with sterols in tBLMs containing 50 mol % cholesterol or ergosterol. CLIC1 (WT) monomeric protein (20 µg in 100 µL of HEPES/KCl buffer, pH 6.5) was incubated with 1% cholesterol or 1% ergosterol for ~1 h prior addition to tethered bilayer lipid membranes containing 25 mol % cholesterol or ergosterol. Conductance of pre-incubated CLIC1 monomer with sterols was then measured with impedance spectroscopy and compared to Controls: CLIC1 monomer not pre-incubated with sterols added into membranes containing 1% of cholesterol or ergosterol, listeriolysin-O (20 µg of LLO in 100 µL of HEPES/KCl buffer, pH 6.5) was also incubated with 1% cholesterol under the same conditions as CLIC1 monomer followed by addition to membranes with 50 mol % cholesterol. The error bars represent the standard error of three repeats of experimental measures (n = 3).