Cholesterol Promotes Interaction of the Protein CLIC1 with Phospholipid Monolayers at the Air-Water Interface

Abstract

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.

Keywords: CLIC1; Langmuir monolayer film; POPC; POPE; POPS; cholesterol; membrane insertion; phospholipids.

Figure 1

Adsorption isotherm of CLIC1 to an air–water interface at 25 °C and final concentration of 2 µg·mL⁻¹ (number of experiments, n = 3).

Figure 2

(A) Percent area expansion profiles for CLIC1 insertion into cholesterol (orange line), POPC (green line), POPS (red line) and POPE (blue line) monolayers on buffer subphase. (B) Corresponding percent area expansion profiles of different lipids or cholesterol monolayers without (white bars) and with (black bars) recombinant CLIC1 wild-type protein after 3 h following protein injection into the subphase. The insertion of CLIC1 in the different lipid or sterol monolayers shows two-stages of interaction, an initial sharp increase in ΔA after a lag phase followed by a plateau and subsequent slow increase in ΔA over a period of 3 h. (n = 3)

Figure 3

Percent area expansion profiles of different lipid monolayers without (white bars) and with (black bars) cholesterol after 3 h following recombinant CLIC1 protein injection into the subphase. CLIC1 protein showed significant insertion and/or membrane interactions in monolayers containing cholesterol. (n = 3)

Figure 4

Percent area expansion profiles of monolayers made of different lipid combinations with/without cholesterol (black/white bars) after 3 h of CLIC1 protein injection into the subphase. CLIC1 protein showed significant insertion and/or membrane interactions in POPE:POPS monolayer containing cholesterol. (n = 3)

Figure 5

(A) Surface pressure-area isotherms of different lipid mixtures of 5:1 mole ratio (B) Surface pressure-area isotherms of the same lipid mixtures containing cholesterol in a 4:1:1 mole ratio measured with a Langmuir film balance (n = 3).

Figure 6

Percent area expansion profiles of POPC:Chol monolayer after 3 h without CLIC1 protein or after injection of recombinant CLIC1 wild-type and pre-incubated CLIC1 protein. CLIC1 was pre-incubated with cholesterol for an hour on ice prior to addition of the pre-incubated protein sample to the monolayer held at a constant surface pressure of 20 mN·m⁻¹. Pre-incubation of CLIC1 protein with cholesterol resulted in complete abrogation of CLIC1 insertion into the monolayer.