Recombinant Rabbit Chloride intracellular channel protein 6 (CLIC6)

What is the basic structure of CLIC6 and how does it differ from other CLIC family members?

CLIC6 belongs to the family of chloride intracellular channel proteins that exist in both soluble and transmembrane forms. Crystallographic studies at 1.8 Å resolution reveal that CLIC6 adopts a monomeric arrangement with high structural conservation to other CLICs . The protein can be subdivided into a thioredoxin (TRX) domain (residues 363-442) and an α-helical domain (residues 456-592), connected by an intervening linker region .

The TRX domain adopts a thioredoxin fold consisting of four anti-parallel β strands (β1–β4) flanked by two α helices (α1 and α2), while the α-helical domain contains seven intertwined helices (α3–α9) . Unlike other CLICs, CLIC6 exhibits high selectivity for chloride ions over other anions, which is a distinguishing feature .

How do researchers confirm successful expression of recombinant CLIC6?

When expressing recombinant CLIC6, successful protein production can be confirmed through:

• Western blotting: Using specific anti-CLIC6 antibodies at a dilution of 1:500-1:3000 . Multiple commercially available antibodies can detect CLIC6 across various species, including rabbit CLIC6.

• Subcellular localization: Immunofluorescence microscopy using wheat germ agglutinin as a plasma membrane marker. Upon ectopic expression in cell lines like HEK-293, CLIC6 localizes near the plasma membrane .

• Functional verification: Patch-clamp electrophysiology to detect IAA-94-sensitive chloride currents. In whole-cell configuration, CLIC6 exhibits characteristic voltage-dependent activation with fast gating that closes at negative membrane voltages and opens upon depolarization .

What are the biophysical characteristics of CLIC6 ion channels?

CLIC6 forms functional ion channels with the following biophysical properties:

• Conductance: Approximately 3 pS

• Voltage dependence: Shows voltage-dependent activation with V₁/₂ = 14.062 mV

• Gating kinetics: Fast gating that closes at negative membrane voltages and opens upon depolarization to positive voltages

• Rectification: Displays rectification at positive holding potentials, a unique feature not observed in other CLIC family members

• Single-channel behavior: Exhibits distinct substates in addition to a large current, with a substate at 50% level of the main opening

• Inhibition: Sensitive to IAA-94 (10 μM), which blocks channel activity by approximately 48-53%

How does CLIC6 ion selectivity compare to other anions and cations?

CLIC6 demonstrates high selectivity for chloride ions over other ions. Experimental data shows:

The selectivity sequence is established as: Cl⁻ >> Br⁻ = F⁻. This high selectivity for chloride ions distinguishes CLIC6 from other CLIC proteins, which typically form poorly selective ion channels .

How do pH and redox conditions regulate CLIC6 activity?

CLIC6 function is regulated by both pH and redox conditions:

pH Regulation:

• CLIC6 activity is significantly enhanced at acidic pH (6.2) compared to physiological pH (7.2)

• The histidine residue at position 648 (H648) in the C-terminus is critical for pH sensitivity

• Mutation of H648 to alanine (H648A) abolishes pH sensitivity and reduces IAA-94 sensitivity

Redox Regulation:

• Reducing conditions (DTT treatment) significantly decrease CLIC6 activity

• The cysteine residue at position 487 (C487) in the N-terminus functions as a redox sensor

• Mutation of C487 to alanine (C487A) significantly reduces channel activity and eliminates sensitivity to DTT

• Oxidative conditions induce an increase in CLIC6 hydrophobicity and mild oligomerization, enhanced by membrane mimetics

What is the tissue distribution pattern of CLIC6 and how can researchers detect tissue-specific expression?

CLIC6 shows differential expression across tissues:

For detection of tissue-specific expression, researchers can use:

• qRT-PCR with beta-actin as an internal control

• Western blotting with specific antibodies at dilutions of 1:500-1:3000

• Immunohistochemistry using purified antibodies against internal epitopes of CLIC6

• Electrophysiological recording of IAA-94-sensitive currents

What methods can researchers use to manipulate CLIC6 expression in experimental models?

Researchers have successfully manipulated CLIC6 expression using:

• Ectopic Expression:

  • Transient transfection in HEK-293 cells for functional studies

  • Co-expression with fluorescent markers (GFP) for identification of transfected cells

• Gene Silencing:

  • Lentivirus-mediated shRNA with RFP reporter for CLIC6 knockdown

  • Verification of knockdown efficiency by qPCR (48 hours post-transduction)

• Mutational Analysis:

  • Site-directed mutagenesis of key residues:

    • H648A to study pH sensitivity

    • C487A to investigate redox regulation

    • Q383A to examine structural flexibility

• Recombinant Protein Administration:

  • Use of varying levels of CLIC6 recombinant protein for treatment in disease models

How is CLIC6 implicated in cancer development and what experimental approaches can be used to study this relationship?

CLIC6 has been implicated in multiple cancer types with both pro-tumorigenic and anti-tumorigenic effects:

Cancer Associations:

• Implicated in breast, ovarian, lung, gastric, and pancreatic cancers

• Demonstrates anti-tumor effects in hepatocellular carcinoma (HCC)

• Shows altered expression profile in breast cancer

Experimental Approaches:

• In vivo models:

  • Subcutaneous xenograft model of HCC with CLIC6 recombinant protein treatment

  • Measurement of tumor size and weight after 21 days of treatment

  • Histopathological assessment using hematoxylin-eosin staining

• Apoptosis Assessment:

  • Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining to evaluate apoptosis in tumor tissue cells

  • Analysis of apoptosis-related proteins including cleaved caspase-3 and Bax/Bcl-2 ratio

• Immunological Analysis:

  • Quantification of cytokine mRNA levels (IL-6, IL-1β, IFN-γ, TNF-α, IL-17A) in liver or tumor tissues

  • Assessment of immune cell balance by analyzing transcription factors (Foxp3, GATA3, T-bet, ROR-γt)

• Signal Pathway Investigation:

  • Analysis of JAK1/STAT pathway involvement in CLIC6-mediated inhibition of HCC

  • Examination of JNK phosphorylation status

What are the cancer-associated mutations in CLIC6 and how might they affect protein function?

Analysis of cancer databases reveals:

• 93 cancer-associated mutations annotated in the longest splice variant of CLIC6

• 58% classified as substitution missense mutations in highly conserved positions

• Mutational clusters in helix α3 (residues 456-461) and the amino terminal half of helix α8 (residues 571-574)

• A551T mutation (corresponding to A461 in mouse CLIC6) associated with both familial goiter and colon adenocarcinoma

• Several mutations in the putative transmembrane region (R384, L385, L389) and β1-α1 loop (G373, G377)

• G467E mutation (G377 in mouse CLIC6) found in two malignant melanoma cases

For functional characterization of these mutations, researchers can:

• Generate site-directed mutants in recombinant expression systems

• Assess structural impacts using crystallography or SAXS analysis

• Evaluate channel function using patch-clamp electrophysiology

• Examine membrane association and protein stability

• Investigate protein-protein interactions, particularly with dopamine receptors

How does the structural flexibility of CLIC6 contribute to its function and how can it be experimentally assessed?

CLIC6 demonstrates significant structural plasticity:

• Comparison of relative chain conformations yields r.m.s.d of 1.7 Å for all 226 Cα atoms

• Separate overlay of TRX or α-helical domains shows better alignments (r.m.s.d of 0.6 Å and 0.5 Å, respectively)

• Inter-domain relative angle can change by 6.1° (D431-L451-V465, Cα)

• Small-angle X-ray scattering (SAXS) analysis reveals multiple elongated conformations in solution

This flexibility is functionally significant as it likely enables CLIC6's metamorphic transition between soluble and membrane-bound forms. The inter-domain interface plays a critical role in stabilizing the crystal structure conformation, with a single inter-domain hydrogen bond between Q383 in the putative transmembrane region and T577 in the C-terminal end of helix α8 .

Experimental Assessment Methods:

• SAXS analysis with ensemble optimization method (EOM) to reveal the range of conformations in solution

• Perturbation of the inter-domain interface through site-directed mutagenesis (e.g., Q383A) followed by structural analysis

• Gel filtration chromatography to assess changes in migration properties reflecting conformational changes

• GNOM-derived PDDF analysis to determine maximum dimension (Dmax) changes

• Molecular envelope calculation and comparison with crystal structure using CRYSOL

What techniques can resolve the structural transitions between soluble and membrane-bound CLIC6?

The metamorphic transition of CLIC6 between soluble and membrane-bound forms represents a fundamental research challenge. Researchers can employ:

• Oxidative conditions to induce increases in CLIC6 hydrophobicity and mild oligomerization

• Membrane mimetics to enhance membrane association and structural transitions

• Fluorescence-based techniques to monitor changes in hydrophobicity and membrane insertion

• Hydrogen-deuterium exchange mass spectrometry to identify regions undergoing conformational changes

• Electron paramagnetic resonance spectroscopy with site-directed spin labeling to track movement of specific domains

• Cryo-electron microscopy to potentially resolve membrane-inserted oligomeric structures

• Computational approaches including molecular dynamics simulations to model the transition process

Experimental Tools and Resources

Based on published research, the following electrophysiological approaches are recommended:

Whole-cell patch-clamp:

• Use NMDG-Cl solutions (135 mM NMDG-Cl in bath solution, 130 mM NMDG-Cl in pipette)

• Apply voltage steps ranging from -100 to +100 mV followed by a step to -40 mV for tail currents

• Include 10 μM IAA-94 as a specific blocker to identify CLIC6-mediated currents

• Compare currents before and after IAA-94 application to quantify CLIC6 contribution

Single-channel recordings:

• Cell-attached configuration with 130 mM chloride in pipette and 4.2 mM in cytoplasm

• Record at both +100 mV and -100 mV for 100 seconds

• Look for multiple conductance states, including a substate at 50% of main opening

• Calculate open probability (Po) before and after IAA-94 application

Automated patch-clamp:

• SyncroPatch 384i system enables high-throughput recordings with consistent parameters

• Monitor seal resistance, capacitance, and series resistance in real-time

• Use step protocols to obtain current-voltage relationships

• Validate findings from automated systems with traditional manual patch-clamp

How does CLIC6 transition between soluble and membrane-bound forms and what factors influence this process?

The metamorphic nature of CLIC6 allows transitions between soluble and membrane-inserted forms:

• Redox regulation appears critical in this process:

  • Oxidative conditions induce increased hydrophobicity

  • Cysteine residue C487 functions as a redox sensor

  • Reducing agents like DTT affect the inserted channel's activity

• pH modulation affects CLIC6 conformation:

  • Acidic pH enhances CLIC6 activity

  • Histidine residue H648 mediates pH-dependent conformational changes

• Structural flexibility is an inherent property that facilitates transition:

  • Inter-domain interface is maintained by a single hydrogen bond (Q383-T577)

  • Perturbation of this bond results in significant changes to CLIC6 structure

  • EOM analysis reveals multiple elongated conformations in solution

• Membrane association is enhanced by:

  • Presence of membrane mimetics under oxidative conditions

  • Putative transmembrane region in the TRX domain

Future research directions should focus on:

• Identifying lipid compositions that promote membrane insertion

• Determining the oligomeric state of membrane-inserted CLIC6

• Resolving the structure of membrane-bound CLIC6

• Developing reagents that can selectively target specific conformational states

What is the relationship between CLIC6 and dopamine receptor signaling and how can it be experimentally investigated?

CLIC6 has been shown to interact with dopamine D3 receptors , though this interaction requires further characterization:

• Co-localization studies have demonstrated CLIC6 expression in brain regions with dopamine receptors

• Functional interactions show mixed results:

  • Cotransfection of CLIC6 with dopamine D3-receptors in CHO cell lines failed to present CLIC6-mediated Cl⁻ fluxes

  • The physiological significance of the CLIC6-dopamine receptor interaction remains unclear

Experimental approaches to investigate this relationship include:

• Co-immunoprecipitation to confirm physical interaction between CLIC6 and dopamine receptors

• FRET/BRET assays to study proximity and potential conformational changes

• Electrophysiology in neuronal systems expressing both proteins

• Calcium imaging to assess dopamine-induced signaling with and without CLIC6

• Radioligand binding to determine if CLIC6 affects dopamine receptor pharmacology

• Behavioral studies in animal models with CLIC6 manipulation in dopaminergic pathways

This research direction is particularly relevant for understanding CLIC6's role in neuropsychiatric conditions involving dopaminergic signaling.