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

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

doi:10.1371/journal.pone.0056948

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.

Highlights

Non-classical integral membrane proteins such as members of the Chloride Intracellular Ion Channel (CLIC) family of proteins challenge our traditional assumptions regarding the processes by which such proteins insert into membranes
In our current study using a tethered lipid bilayer membrane system, we demonstrate that addition of soluble CLIC1 protein causes increased membrane conductance and as such, membrane insertion is subsequently inferred
The current study demonstrates the regulatory effect of cholesterol on the ability of the spontaneously membrane inserting protein CLIC1 to interact and insert into a lipid membrane

Summary

Introduction

Non-classical integral membrane proteins such as members of the Chloride Intracellular Ion Channel (CLIC) family of proteins challenge our traditional assumptions regarding the processes by which such proteins insert into membranes. The CLIC family proteins do not contain a signal sequence or any obvious membrane spanning domains, but are instead capable of spontaneous insertion into lipid bilayer membranes from their soluble form, and by-pass the traditional route of integral membrane synthesis and processing. One of their functions when located in the membrane is to act as ion channels. Channel activity has been demonstrated for the invertebrate orthologues, EXC-4 from Caenorhabditis elegans and Dm-CLIC from Drosophila melanogaster [13]

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Conclusion