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

Heba Al Khamici,Bruce Cornell,Stella Valenzuela,Khondker Hossain

doi:10.3390/membranes6040051

Heba Al Khamici, Bruce Cornell + Show 2 more

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https://doi.org/10.3390/membranes6040051

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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.

Highlights

The Chloride Intracellular Ion Channel (CLIC) family members contain no obvious transmembrane domain in their protein structure; they are capable of inserting into phospholipid membranes directly from their soluble state, where they can function as ion channels [1,2]
In a study using phospholipid vesicle chloride efflux assays by Tulk et al, 2002 [7], it was found that CLIC1 demonstrated no ion channel activity in membranes containing pure neutral lipid mixtures, while activity was greater in membranes containing 10% of a negatively charged phospholipid such as phosphotidylserine (PS) or phosphatidic acid (PA)
In order to study the effect of cholesterol on CLIC1’s ion channel function, recombinant CLIC1 (rCLIC1) were added to sealed tethered bilayer membranes (tBLMs) containing phospholipids and varied concentrations of cholesterol, where the initial conductance of all the freshly formed membranes was stabilised to a baseline value of less than 1 μS and with a capacitance ranging between 20 and 23 nF

Summary

Introduction

The Chloride Intracellular Ion Channel (CLIC) family members contain no obvious transmembrane domain in their protein structure; they are capable of inserting into phospholipid membranes directly from their soluble state, where they can function as ion channels [1,2]. 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]. A study by Singh, et al [10] using Langmuir-film monolayers and patch clamping techniques, shows that membranes containing POPE, POPS and cholesterol in a molar ratio of 4:1:1 induced CLIC1 membrane insertion and ion channel conductance

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