Elucidating the Mechanism for Sterol Regulation of Chloride Intracellular Ion Channel Protein Interactions with Lipid Membranes

Abstract

Sterols have been reported to modulate conformation and function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family. These largely soluble proteins possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. To date, the structure of their membrane-bound form and factors influencing their auto-insertion remains largely unknown. We have performed Langmuir-film, x-ray, and neutron reflectivity experiments to study the interaction of wild-type or mutant versions of the protein CLIC1 with monolayers prepared from 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) alone, or mixtures of this lipid with either one of the following sterols: cholesterol, β-sitosterol, ergosterol, hydroxyecdysone or cholestane. In lipid monolayers lacking sterols, CLIC1 did not insert. However significant membrane insertion occurred when CLIC1 was added to membranes containing cholesterol. Substitution of membrane cholesterol with different sterols, not only increased CLIC1's membrane interaction but also increased its rate of insertion. However, CLIC1 showed no insertion into monolayers containing cholestane, which lacked the intact sterol 3β-OH group. Unlike CLIC1-wild-type and C24A-CLIC1 mutant protein, G18A and G22A mutants did not insert in monolayers containing cholesterol. These findings for the first time demonstrate that the spontaneous membrane insertion of CLIC1 is dependent on the presence of cholesterol in membranes, along with the presence of an intact 3β-OH group within the sterol structure, which is critical for CLIC1-membrane interactions. Our novel findings also extend to the identification of a cholesterol-binding domain-GXXXG motif within CLIC1 that facilitates the protein's membrane interaction and insertion.