Recombinant Bovine Chloride intracellular channel protein 1 (CLIC1)

What is the molecular structure of CLIC1 and how does it differ from other chloride channels?

CLIC1 belongs to the Chloride Intracellular Channel family and contains one GST C-terminal domain, reflecting its structural relationship to the glutathione S-transferase (GST) superfamily. Unlike conventional transmembrane channel proteins, CLIC1 exists in dual states - as a soluble cytoplasmic protein and as a membrane-inserted channel. The protein is defined by an approximately 240 conserved amino acid sequence at the C-terminus that enables this remarkable structural flexibility .

The protein forms a voltage-dependent, chloride-selective channel with a rectifying current-voltage relationship. Electrophysiological studies demonstrate that CLIC1 exhibits single channel conductances of 161 ± 7.9 and 67.5 ± 6.9 picosiemens in symmetric 300 mM and 150 mM KCl solutions, respectively . This distinguishes CLIC1 from other chloride channels that typically maintain fixed transmembrane structures.

What is the molecular weight and sequence information for bovine CLIC1?

Recombinant CLIC1 has a calculated molecular mass of approximately 29.0 kDa, though the apparent molecular mass observed in SDS-PAGE is typically around 33 kDa, suggesting possible post-translational modifications or structural properties affecting migration . The full protein sequence comprises 241 amino acids (Met1-Lys241), and recombinant versions often include fusion tags (such as N-terminal 6His-tags) to facilitate purification .

What is the ion selectivity profile of CLIC1 channels?

Electrophysiological studies of reconstituted CLIC1 channels reveal a distinctive anion selectivity profile of Br⁻ ≈ Cl⁻ > I⁻ . This selectivity profile provides important insights for researchers designing functional assays or investigating the physiological roles of CLIC1 in various cellular contexts. The preference for certain anions over others suggests specific structural properties of the channel pore that can be targeted in experimental designs.

What expression systems are optimal for producing functional recombinant bovine CLIC1?

The most established method for producing functional recombinant CLIC1 involves bacterial expression systems, particularly E. coli. The recommended approach involves:

• Generating a glutathione S-transferase (GST) fusion construct of CLIC1

• Expressing the fusion protein in an E. coli strain optimized for protein expression

• Purifying via glutathione affinity chromatography

• Releasing CLIC1 from the fusion partner using thrombin digestion

• Performing additional purification steps such as size exclusion chromatography

This methodology consistently yields highly pure protein (>90% as determined by reducing SDS-PAGE) that retains functional activity when reconstituted into artificial membrane systems .

What purification strategies maximize yield and functional integrity of recombinant CLIC1?

A multi-step purification protocol is recommended to obtain high-purity, functionally active CLIC1:

• Initial capture using affinity chromatography (glutathione for GST-fusion proteins or Ni-NTA for His-tagged constructs)

• Proteolytic removal of fusion tags (if using cleavable tags)

• Ion exchange chromatography to remove impurities

• Size exclusion chromatography as a polishing step

Typical formulation buffer conditions include 20 mM Tris, 150 mM NaCl, pH 8.0, which maintains protein stability. For long-term storage, lyophilization is recommended, as lyophilized proteins remain stable for up to 12 months when stored at -20°C to -80°C. Reconstituted protein solutions can be stored at 4-8°C for 2-7 days, while aliquots of reconstituted samples remain stable at < -20°C for 3 months .

What factors regulate CLIC1 membrane insertion and channel formation?

CLIC1 membrane insertion and channel formation are regulated by multiple factors:

• pH Dependence: Channel activity varies with pH, with distinct functional properties observed at different pH values

• Redox Regulation: Membrane insertion appears to be redox-regulated, potentially occurring only under oxidizing conditions

• Lipid Composition: The phospholipid composition of the target membrane influences insertion efficiency and channel properties

When investigating these factors experimentally, researchers should systematically control environmental conditions while monitoring channel formation using electrophysiological techniques or fluorescence-based assays .

How can researchers effectively reconstitute CLIC1 into artificial membrane systems?

For functional reconstitution of CLIC1 into artificial membrane systems:

• Prepare phospholipid vesicles using a defined lipid composition (typically phosphatidylcholine/phosphatidylethanolamine mixtures)

• Solubilize purified CLIC1 in a non-denaturing detergent

• Incorporate protein into vesicles using detergent dialysis

• Confirm successful reconstitution using proteoliposome flotation assays

• Assess chloride permeability using a valinomycin-dependent chloride efflux assay

This methodology has demonstrated increased vesicular chloride permeability with CLIC1 compared to control vesicles, confirming functional reconstitution .

What electrophysiological approaches best characterize CLIC1 channel properties?

The planar lipid bilayer technique provides the most detailed characterization of CLIC1 single-channel properties. This methodology involves:

• Formation of a stable lipid bilayer across an aperture separating two chambers

• Addition of purified CLIC1 to one chamber

• Application of defined voltage protocols while recording current

• Analysis of single-channel conductance, open probability, and ion selectivity

Using this technique, researchers have established that CLIC1 forms a voltage-dependent, Cl⁻-selective channel with rectifying current-voltage relationships. The open probability of CLIC1 channels can be modulated by inhibitors such as indanyloxyacetic acid-94, with an apparent IC₅₀ of 86 μM at 50 mV .

How can CLIC1 inhibition be achieved and quantified in experimental settings?

CLIC1 activity can be pharmacologically inhibited by compounds such as indanyloxyacetic acid-94 (IAA-94). When evaluating inhibition:

• Use reconstituted vesicles with established CLIC1 activity

• Apply varying concentrations of inhibitor compounds

• Measure chloride permeability using the valinomycin-dependent chloride efflux assay

• Calculate IC₅₀ values from dose-response curves

Studies have shown that CLIC1-dependent chloride permeability is inhibited by IAA-94 with an apparent IC₅₀ of 8.6 μM in vesicle-based assays, while in planar bilayers, the apparent IC₅₀ is 86 μM at 50 mV . This difference highlights the importance of experimental context when evaluating inhibitor efficacy.

How does CLIC1 contribute to cellular processes like proliferation and migration?

CLIC1 plays crucial roles in multiple cellular processes beyond its channel function. Research methodologies to investigate these roles include:

• Gene silencing experiments using shRNA or siRNA against CLIC1

• Phenotypic assays measuring cell proliferation, migration, and morphogenesis

• Cell cycle analysis using flow cytometry

• Integrin expression profiling via flow cytometry or immunoblotting

Studies in endothelial cells demonstrate that reduced CLIC1 expression causes significant reductions in migration, cell growth, branching morphogenesis, capillary-like network formation, and capillary-like sprouting. CLIC1 also regulates the cell surface expression of integrins important for angiogenesis, including β1 and α3 subunits, as well as αVβ3 and αVβ5 .

What analytical approaches can distinguish between ion channel-dependent and independent functions of CLIC1?

Distinguishing between channel-dependent and independent functions requires:

• Mutagenesis studies targeting residues critical for channel formation while preserving protein structure

• Pharmacological approaches using specific channel blockers

• Correlation analyses between channel activity and cellular phenotypes

• Complementation experiments with channel-competent versus channel-incompetent mutants

These approaches help delineate whether observed cellular effects depend on CLIC1's ion transport capabilities or stem from other protein interactions and signaling functions .

How is CLIC1 dysregulation implicated in cancer progression?

Multi-omics analyses reveal important connections between CLIC1 and cancer biology, particularly in gliomas:

• Expression Analysis: CLIC1 shows aberrant overexpression in glioma versus normal tissues

• Prognostic Correlation: Elevated CLIC1 expression correlates with worse survival outcomes

• Clinical Associations: Higher expression is observed in:

  • More advanced stage tumors (grade III versus grade II)

  • Wild-type IDH samples

  • Unmethylated MGMT samples

• Functional Impact: Suppressing CLIC1 results in apoptosis and attenuated cell motility in glioma cells

What molecular mechanisms connect CLIC1 to cellular pathways in disease states?

Pathway analyses reveal complex interactions between CLIC1 and cellular signaling networks:

• Gene Set Enrichment Analysis identifies tumorigenic and anticancer immunity pathways enriched in CLIC1-upregulated tumors

• CLIC1 expression positively correlates with:

  • Cancer-immunity cycle

  • Stromal activation

  • DNA damage repair pathways

  • Cell cycle regulation

These connections suggest that CLIC1 functions at the intersection of multiple pathways critical for cancer development and progression, making it a potential therapeutic target with pleiotropic effects .

How should researchers address potential artifacts in CLIC1 channel reconstitution experiments?

When designing CLIC1 reconstitution experiments, researchers should implement several controls to distinguish genuine channel activity from artifacts:

• Include protein-free liposome controls subjected to identical reconstitution procedures

• Use heat-denatured CLIC1 as a negative control to confirm that native protein structure is required

• Incorporate scrambled peptide controls when testing specific domains

• Implement pharmacological validation using known CLIC1 inhibitors (e.g., IAA-94)

• Perform parallel experiments with structurally related but functionally distinct proteins

These controls are essential because spontaneous pore formation can occur in liposomes during detergent dialysis, potentially mimicking channel activity .

What comparative approaches can distinguish species-specific variations in CLIC1 function?

To investigate species-specific variations in CLIC1 function:

• Perform sequence alignments and evolutionary analyses to identify conserved versus variable regions

• Express recombinant CLIC1 from multiple species (human, bovine, rodent) under identical conditions

• Compare biochemical properties including:

  • Membrane insertion efficiency

  • Ion selectivity profiles

  • Response to regulatory factors (pH, redox)

  • Inhibitor sensitivity

• Conduct cross-species complementation experiments in cellular models

These comparative approaches can reveal both fundamental conserved functions and species-specific adaptations that may be relevant for translational research .

What emerging technologies might advance CLIC1 structural and functional studies?

Several cutting-edge approaches show promise for advancing CLIC1 research:

• Cryo-electron microscopy for determining membrane-inserted CLIC1 structures

• High-throughput electrophysiology platforms for comprehensive pharmacological screening

• Advanced molecular dynamics simulations to model conformational transitions between soluble and membrane-inserted states

• Genetically encoded chloride sensors for real-time monitoring of CLIC1 activity in live cells

• CRISPR-Cas9 gene editing for generating precise functional mutations in endogenous CLIC1

These approaches could resolve longstanding questions about the structural basis of CLIC1's dual soluble/membrane states and its physiological regulation .

How might CLIC1 research contribute to therapeutic development in cancer and other diseases?

The therapeutic potential of targeting CLIC1 is suggested by several lines of evidence:

• Differential Drug Sensitivity: High CLIC1 expression correlates with increased sensitivity to multiple anticancer drugs including camptothecin, cisplatin, doxorubicin, erlotinib, paclitaxel, and rapamycin

• Immunotherapy Connections: CLIC1 expression correlates with immune cell infiltration and immune checkpoint expression

• Targeted Inhibition: Suppressing CLIC1 induces apoptosis and reduces cell motility in cancer models

Future therapeutic development may focus on novel CLIC1 inhibitors with improved selectivity, antibody-drug conjugates targeting CLIC1-expressing cells, or combination approaches leveraging CLIC1's connections to multiple cellular pathways .