Intracellular Ion Channels Research Articles

Viroporins: Structure, function, and their role in the life cycle of SARS-CoV-2.

Viroporins are indispensable for viral replication. As intracellular ion channels they disturb pH gradients of organelles and allow Ca2+ flux across ER membranes. Viroporins interact with numerous intracellular proteins and pathways and can trigger inflammatory responses. Thus, they are relevant targets in the search for antiviral drugs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underlies the world-wide pandemic of COVID-19, where an effective therapy is still lacking despite impressive progress in the development of vaccines and vaccination campaigns. Among the 29 proteins of SARS-CoV-2, the E- and ORF3a proteins have been identified as viroporins that contribute to the massive release of inflammatory cytokines observed in COVID-19. Here, we describe structure and function of viroporins and their role in inflammasome activation and cellular processes during the virus replication cycle. Techniques to study viroporin function are presented, with a focus on cellular and electrophysiological assays. Contributions of SARS-CoV-2 viroporins to the viral life cycle are discussed with respect to their structure, channel function, binding partners, and their role in viral infection and virus replication. Viroporin sequences of new variants of concern (α–ο) of SARS-CoV-2 are briefly reviewed as they harbour changes in E and 3a proteins that may affect their function.

The Plant Vacuole as Heterologous System to Characterize the Functional Properties of TPC Channels.

Human TPC channels are an emerging family of intracellular proteins fundamental for cell physiology and involved in various severe pathologies. Their localization in the membranes of endo-lysosomes, intracellular compartments of submicrometric dimensions, makes their study difficult with usual electrophysiological techniques. In this work, we show how the plant vacuole, a versatile organelle that can occupy up to 90% of the volume in mature plant cells, can be used as a heterologous system of expression for functional characterization. For this purpose, the use of vacuoles isolated from mesophyll cells of the Arabidopsis thaliana mutant lacking the endogenous TPC avoids unwanted interferences. The patch-clamp technique can be successfully applied to plant vacuoles in all different configuration modes; of note, the whole-vacuole configuration allows to study channel modulation by cytosolic factors. The combination of patch-clamp with fluorescence techniques, for example, by using fluorescent probes sensitive to specific ions of interest, represents a useful extension to investigate the selectivity properties of the channels. Therefore, the plant vacuole, similar to Xenopus oocytes for ion channels and transporters localized in the plasma membrane, has the capability to become a model system for functional studies on intracellular ion channels and transporters.

Identification of positive modulators of TRPM5 channel from a high-throughput screen using a fluorescent membrane potential assay.

Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.

Targeting the two-pore channel 2 in cancer progression and metastasis.

The importance of Ca2+ signaling, and particularly Ca2+ channels, in key events of cancer cell function such as proliferation, metastasis, autophagy and angiogenesis, has recently begun to be appreciated. Of particular note are two-pore channels (TPCs), a group of recently identified Ca2+-channels, located within the endolysosomal system. TPC2 has recently emerged as an intracellular ion channel of significant pathophysiological relevance, specifically in cancer, and interest in its role as an anti-cancer drug target has begun to be explored. Herein, an overview of the cancer-related functions of TPC2 and a discussion of its potential as a target for therapeutic intervention, including a summary of clinical trials examining the TPC2 inhibitors, naringenin, tetrandrine, and verapamil for the treatment of various cancers is provided.

Identification of a dihydropyridine scaffold that blocks ryanodine receptors

SummaryRyanodine receptors (RyRs) are large, intracellular ion channels that control Ca2+ release from the sarco/endoplasmic reticulum. Dysregulation of RyRs in skeletal muscle, heart, and brain has been implicated in various muscle pathologies, arrhythmia, heart failure, and Alzheimer's disease. Therefore, there is considerable interest in therapeutically targeting RyRs to normalize Ca2+ homeostasis in scenarios involving RyR dysfunction. Here, a simple invertebrate screening platform was used to discover new chemotypes targeting RyRs. The approach measured Ca2+ signals evoked by cyclic adenosine 5′-diphosphate ribose, a second messenger that sensitizes RyRs. From a 1,534-compound screen, FLI-06 (currently described as a Notch “inhibitor”) was identified as a potent blocker of RyR activity. Two closely related tyrosine kinase inhibitors that stimulate and inhibit Ca2+ release through RyRs were also resolved. Therefore, this simple screen yielded RyR scaffolds tractable for development and revealed an unexpected linkage between RyRs and trafficking events in the early secretory pathway.

Chloride intracellular channel protein 1 expression in normal and atherosclerotic human arteries. Challenges for a new therapeutic target in atheromatosis.

Background and Aims: Chloride intracellular ion channel protein 1 (CLIC1) is involved in inflammation from various disease and oxidative stress endothelial cells disfunction. Its role in atherosclerosis is scarcely suggested in the literature, being restricted to experimental induced atherosclerosis in mice. Its involvement in human atheroma plaque formation is completely unknown in this moment. AIM.To study CLIC1 expression in normal human fetal and adult aorta, on early steps of atheroma plaque development and also in complicated atherosclerotic lesions.

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.

P149 The intracellular chloride channel 4 (CLIC4) plays an important role in systemic sclerosis fibroblast activation

Abstract Background/Aims The intracellular chloride ion channel CLIC4 mediates the activation of cancer associated fibroblasts. Interestingly, systemic sclerosis (SSc) fibroblasts display a number of similar properties to cancer associated fibroblasts. Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Therefore in this study we investigated the role of CLIC4 in SSc fibroblast activation. Methods Dermal fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride ion channel inhibitors NPPB and IAA-94. Results CLIC4 was found to be expressed at significantly higher levels in SSc patients fibroblasts compared to healthy controls, at both the transcript (3.7 fold) and protein (1.7 fold) levels. Inhibition of the TGF-β signalling pathway led to reduced CLIC4 expression in SSc fibroblasts, confirming this pathway as the main driver of CLIC4 expression. Finally, treatment of SSc fibroblasts with small molecule inhibitors that target the channel led to reduced expression of the myofibroblast markers collagen type 1 and alpha-smooth muscle actin, suggesting a direct role for CLIC4 in SSc associated skin fibrosis. Conclusion We have identified a novel role for CLIC4 in SSc myofibroblast activation, which further strengthen the similarities between SSc fibroblasts and cancer associated fibroblasts. Furthermore this study highlights this channel as a novel target for therapeutic intervention. Disclosure C. Wasson: None. R. Ross: None. R. Morton: None. J. Mankouri: None. F. Del Galdo: None.

Perspective of Ion Channels in Prostate Cancer

Ion channels are membrane proteins, which play a great role in regulating cellular excitability. Alteration of ion channel may contribute to prostate cancer. This could be linked to inherited mutations of ion channel genes which alter channel’s biophysical properties, in a prostate cancer. It is an observed fact that genomic instability is the main cause as well as the major characteristics of prostate cancer. Prostate cancer cell genotypes are mainly characterized by uncontrolled metastasis, resistance to programmed cell death, sustained angiogenesis as well as tissue invasion and metastasis. It is known that genes encoding ion channels are affected in prostate cancer. The Membrane proteins which is involved in signaling in cell and among cells, for coupling of extracellular events with intracellular responses, and for maintaining intracellular ionic homeostasis ion channels which contribute to some extents to pathophysiological features of each prostate cancer.

Perspective of Ion Channels in Prostate Cancer

Ion channels are membrane proteins, which play a great role in regulating cellular excitability. Alteration of ion channel may contribute to prostate cancer. This could be linked to inherited mutations of ion channel genes which alter channel’s biophysical properties, in a prostate cancer. It is an observed fact that genomic instability is the main cause as well as the major characteristics of prostate cancer. Prostate cancer cell genotypes are mainly characterized by uncontrolled metastasis, resistance to programmed cell death, sustained angiogenesis as well as tissue invasion and metastasis. It is known that genes encoding ion channels are affected in prostate cancer. The Membrane proteins which is involved in signaling in cell and among cells, for coupling of extracellular events with intracellular responses, and for maintaining intracellular ionic homeostasis ion channels which contribute to some extents to pathophysiological features of each prostate cancer.

IP3R1 regulates Ca2+ transport and pyroptosis through the NLRP3/Caspase-1 pathway in myocardial ischemia/reperfusion injury

Intracellular ion channel inositol 1,4,5-triphosphate receptor (IP3R1) releases Ca2+ from endoplasmic reticulum. The disturbance of IP3R1 is related to several neurodegenerative diseases. This study investigated the mechanism of IP3R1 in myocardial ischemia/reperfusion (MI/R). After MI/R modeling, IP3R1 expression was silenced in myocardium of MI/R rats to explore its role in the concentration of myocardial enzymes, infarct area, Ca2+ level, NLRP3/Caspase-1, and pyroptosis markers and inflammatory factors. The adult rat cardiomyocytes were isolated and cultured to establish hypoxia/reperfusion (H/R) cell model. The expression of IP3R1 was downregulated or ERP44 was overexpressed in H/R-induced cells. Nifedipine D6 was added to H/R-induced cells to block Ca2+ channel or Nigericin was added to activate NLRP3. IP3R1 was highly expressed in myocardium of MI/R rats, and silencing IP3R1 alleviated MI/R injury, reduced Ca2+ overload, inflammation and pyroptosis in MI/R rats, and H/R-induced cells. The binding of ERP44 to IP3R1 inhibited Ca2+ overload, alleviated cardiomyocyte inflammation, and pyroptosis. The increase of intracellular Ca2+ level caused H/R-induced cardiomyocyte pyroptosis through the NLRP3/Caspase-1 pathway. Activation of NLRP3 pathway reversed the protection of IP3R1 inhibition/ERP44 overexpression/Nifedipine D6 on H/R-induced cells. Overall, ERP44 binding to IP3R1 inhibits Ca2+ overload, thus alleviating pyroptosis and MI/R injury.

A multi-level assessment of the bidirectional relationship between aging and the circadian clock.

The daily temporal order of physiological processes and behavior contribute to the wellbeing of many organisms including humans. The central circadian clock, which coordinates the timing within our body, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Like in other parts of the brain, aging impairs the SCN function, which in turn promotes the development and progression of aging‐related diseases. We here review the impact of aging on the different levels of the circadian clock machinery—from molecules to organs—with a focus on the role of the SCN. We find that the molecular clock is less effected by aging compared to other cellular components of the clock. Proper rhythmic regulation of intracellular signaling, ion channels and neuronal excitability of SCN neurons are greatly disturbed in aging. This suggests a disconnection between the molecular clock and the electrophysiology of these cells. The neuronal network of the SCN is able to compensate for some of these cellular deficits. However, it still results in a clear reduction in the amplitude of the SCN electrical rhythm, suggesting a weakening of the output timing signal. Consequently, other brain areas and organs not only show aging‐related deficits in their own local clocks, but also receive a weaker systemic timing signal. The negative spiral completes with the weakening of positive feedback from the periphery to the SCN. Consequently, chronotherapeutic interventions should aim at strengthening overall synchrony in the circadian system using life‐style and/or pharmacological approaches.

Disruption of transverse‐tubular network reduces energy efficiency in cardiac muscle contraction

Altered organization of the transverse-tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t-tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation-contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t-tubular network has a direct effect on cardiac mechano-energetics. Our aim was to understand the fundamental link between t-tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event. We exploited the "formamide-detubulation" technique to acutely disrupt the t-tubular network in rat left-ventricular trabeculae. We assessed the energy utilization by cellular Ca2+ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured. Following structural disorganization from detubulation, muscle heat output associated with Ca2+ cycling was reduced, indicating impaired intracellular Ca2+ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force-length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced. These results reveal the direct energetic consequences of disrupted t-tubular network, linking the energy disturbance and the t-tubular remodelling typically observed in heart failure.

Intracellular Chloride Channels: Novel Biomarkers in Diseases.

Ion channels are integral membrane proteins present on the plasma membrane as well as intracellular membranes. In the human genome, there are more than 400 known genes encoding ion channel proteins. Ion channels are known to regulate several cellular, organellar, and physiological processes. Any mutation or disruption in their function can result in pathological disorders, both common or rare. Ion channels present on the plasma membrane are widely acknowledged for their role in various biological processes, but in recent years, several studies have pointed out the importance of ion channels located in intracellular organelles. However, ion channels located in intracellular organelles are not well-understood in the context of physiological conditions, such as the generation of cellular excitability and ionic homeostasis. Due to the lack of information regarding their molecular identity and technical limitations of studying them, intracellular organelle ion channels have thus far been overlooked as potential therapeutic targets. In this review, we focus on a novel class of intracellular organelle ion channels, Chloride Intracellular Ion Channels (CLICs), mainly documented for their role in cardiovascular, neurophysiology, and tumor biology. CLICs have a single transmembrane domain, and in cells, they exist in cytosolic as well as membranous forms. They are predominantly present in intracellular organelles and have recently been shown to be localized to cardiomyocyte mitochondria as well as exosomes. In fact, a member of this family, CLIC5, is the first mitochondrial chloride channel to be identified on the molecular level in the inner mitochondrial membrane, while another member, CLIC4, is located predominantly in the outer mitochondrial membrane. In this review, we discuss this unique class of intracellular chloride channels, their role in pathologies, such as cardiovascular, cancer, and neurodegenerative diseases, and the recent developments concerning their usage as theraputic targets.

The MORN domain of Junctophilin2 regulates functional interactions with small-conductance Ca2+ -activated potassium channel subtype2 (SK2).

Small-conductance Ca2+ -activated K+ channel subtype2 (SK2) are stable macromolecular complexes that regulate myocardial excitability and Ca2+ homeostasis. Junctophilin-2 (JP2) is a membrane-binding protein, which provides functional crosstalk by physically linking with the cell-surface and intracellular ion channels. We previously demonstrated that the MORN domain of JP2 interacts with SK2 channels. However, the roles of the JP2 MORN domain in regulating the precise subcellular localization and molecular modulation of SK2 have not yet been incompletely understood. In the present study, in vitro and in vivo assays were used to confirm the physical interactions between the SK2 channel and JP2 in H9c2 and HEK293 cells, with a concentration on the association between the C-terminus of SK2 channels and the MORN domain of JP2. Furthermore, the membrane expression of SK2 were found to be significantly impaired by the mutation or knockdown of JP2. Using immunofluorescence staining along with Golgi/early endosome markers, we studied the mechanisms of JP2-regulated SK2 membrane trafficking, which indicates that the JP2 MORN domain is probably necessary for the retrograde trafficking of SK2 channels. The functional study demonstrates that whole cell SK2 current densities recorded from the HEK293 cells co-expressing the JP2-MORN domain with SK2 were significantly augmented, compared with cells expressing SK2 alone. Our findings suggest that the MORN domain of JP2 directly modulates SK2 channel current amplitude and trafficking, through its interaction with an overlapping region of the JP2 MORN domain on the SK2 C-terminus.

Intracellular Ion Channels in Pancreas Cancer.

The current basis of systemic treatment for pancreas cancer involves cytotoxic chemotherapy. Despite newer regimens, overall survival remains poor and this dilemma is further compounded by a lack of effective, novel therapeutic targets. Another challenge in treating pancreas cancer is the complex tumor microenvironment, which contains regulatory T cells, myeloid derived suppressor cells, and tumor associated macrophages that forms a barrier to standard therapies. Intracellular ion channels are ubiquitously expressed in all cells and their role in carcinogenesis is increasingly becoming elucidated. They play an integral role in each of the six "Hallmarks of Cancer" and are potential novel prognostic biomarkers and therapeutic targets for pancreas cancer. Although examined in various hematologic and gastrointestinal malignancies, there are limited data examining the prognostic role of specific ion channels in pancreas ductal adenocarcinoma. This review focuses on chloride (CLCA-1, CLIC1, CLIC3), calcium (TRPM7, TRPM8), and potassium (Kir3.1, KCa3.1, Kv11.1, Kv1.3) channels in pancreas cancer.

Functionalisation of human chloride intracellular ion channels in microfluidic droplet-interface-bilayers

Profiling ion flux through human intracellular chloride ion channels using live-cell based techniques, such as patch-clamp electrophysiology, is laborious and time-consuming. The integration of scalable microfluidic systems with automatable protocols based on droplet-interface-bilayers (DIBs) within which ion channels are incorporated circumvents several limitations associated with live-cell measurements and facilitates testing in controllable in vitro conditions. Here, we have designed and tested novel microfluidic layouts for the formation of arrays of DIBs in parallel and developed the first example of a miniaturised, DIB-based, fluorescence assays for Cl− fluxing, allowing the investigation of the functional properties of the human chloride intracellular ion channel 1 (CLIC1). The microfluidic protocols relied on passive geometries for droplet pairing and DIB formation. Using recombinantly expressed CLIC1, we identified the best conditions to maximise protein integration into a lipid bilayer and the oligomerisation of the protein into functional ion channels. Finally, CLIC1 ion channel functionality was assessed relative to α-Haemolysin into microfluidic DIBs using the same Cl− fluxing assay.

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

Differential Levels and Phosphorylation of Type 1 Inositol 1,4,5-Trisphosphate Receptor in Four Different Murine Models of Huntington Disease.

The intracellular ion channel type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) releases Ca2+ from the endoplasmic reticulum upon stimulation with IP3. Perturbation of IP3R1 has been implicated in the development of several neurodegenerative disorders, including Huntington disease (HD). To elucidate the putative role of IP3R1 phosphorylation in HD, we investigated IP3R1 levels and protein phosphorylation state in the striatum, hippocampus and cerebellum of four murine HD models. Quantitative immunoblotting with antibodies to IP3R1 protein and its phosphorylated serines 1589 and 1755 was applied to brain homogenates from R6/1 mice to study early-onset aggressive HD. To determine if IP3R1 changes precede overt pathology, we immunostained tissues from the regions of interest and several control regions for IP3R1 in tgHDCAG51n rats and BACHD and zQ175DNKI mice, all recognized models for late-onset HD. R6/1 mice had reduced total IP3R1 immunoreactivity, variably reduced serine1755-phosphorylation in all regions investigated, and reduced serine1589-phosphorylation in cerebellum. IP3R1 levels were decreased relative to cell-specific marker proteins. In tgHDCAG51n rats we found reduced IP3R1 levels in the cerebellum, but otherwise unchanged IP3R1 phosphorylation and protein levels. In BACHD and zQ175DNKI mice only age-dependent decline of IP3R1 was observed. The level and phosphorylation of IP3R1 is reduced to a variable degree in the different HD models relative to control, indicating that earlier findings in more aggressive exon 1-truncated HD models may not be replicated in models with higher construct validity. Further analysis of possible coupling of reduced IP3R1 levels with development of neuropathological responses and cell-specific degeneration is warranted.

A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction

BackgroundSterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol. MethodWe have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol. ResultsUnlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane.Conclusion: In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels. General significanceMembers of the CLIC family of proteins demonstrate unusual structural and dual functional properties – as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting activity and a novel regulatory role for cholesterol in such a process.