Recombinant Rabbit Chloride intracellular channel protein 1 (CLIC1)

What is Chloride Intracellular Channel Protein 1 (CLIC1) and what are its primary functions?

CLIC1 is a 241 amino acid protein belonging to the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. It is highly conserved in Metazoa, suggesting an evolutionarily important role . CLIC1 exists in dual forms: as a soluble cytoplasmic protein and as an integral membrane protein with a single putative transmembrane region .

Functionally, CLIC1 serves as a chloride channel that contributes to various cellular processes including:

• Regulation of cyclic AMP-activated chloride currents in epithelial cells

• Modulation of phagosome acidification in macrophages

• Promotion of cancer progression, metastasis, and angiogenesis in tumor cells

• Facilitation of communication between tumor cells and endothelial cells via exosome-mediated activity

How is CLIC1 protein expression and localization regulated in different cell types?

CLIC1 demonstrates remarkable versatility in its subcellular localization, which varies depending on cell type and functional status. Research has shown that CLIC1:

• Is ubiquitously expressed across many tissues and organs

• Shows high expression levels in macrophages and human bronchial epithelial cells

• Can be localized to the nucleus, cytoplasm, or membrane depending on cellular conditions

• Translocates to specific cellular compartments during functional activation, such as to the phagosomal membrane during phagocytosis in macrophages

• May relocate from cytosol to plasma membrane under pathological conditions, as seen in microglia exposed to amyloid β-peptide in Alzheimer's disease models

The dynamic localization pattern of CLIC1 is critically linked to its diverse functions and appears to be regulated by redox status and pH changes in the cellular environment .

What are the optimal methods for expressing and purifying recombinant rabbit CLIC1 protein?

For optimal expression and purification of recombinant rabbit CLIC1:

• Expression System Selection:

  • Bacterial expression (E. coli) systems are commonly used for CLIC1 due to its relatively small size (27 kDa) and lack of post-translational modifications

  • Use pET vector systems with N-terminal His-tag for efficient purification

  • BL21(DE3) E. coli strains generally yield good expression levels

• Induction and Culture Conditions:

  • Induce expression at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG

  • Lower induction temperature (16-20°C) overnight often improves solubility

  • Supplementing growth media with 5-10% glycerol can enhance protein stability

• Purification Protocol:

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors

  • Purify using nickel affinity chromatography followed by size exclusion chromatography

  • Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) in buffers to maintain protein in reduced state

  • For functional studies, consider ion exchange chromatography as a final polishing step

Western blotting analysis can confirm purification using antibodies specific to CLIC1, such as those targeting residues surrounding Glu234 of human CLIC1 protein, which cross-react with rabbit CLIC1 .

How can researchers effectively measure CLIC1 channel activity in experimental models?

Several methodologies can be employed to assess CLIC1 channel activity:

• Planar Lipid Bilayer Electrophysiology:

  • Incorporate purified CLIC1 protein into artificial lipid bilayers

  • Record single-channel currents using patch-clamp techniques

  • Use symmetrical or asymmetrical chloride solutions to determine ion selectivity

  • Monitor channel activity under different pH conditions (optimal around pH 5.5-6.0) and redox states

• Cellular Electrophysiology:

  • Whole-cell patch-clamp recording in cells expressing CLIC1

  • Measure cyclic AMP-activated chloride currents before and after CLIC1 inhibition

  • Use specific chloride channel blockers (e.g., IAA-94, DIDS) as controls

• Functional Assays:

  • Assess phagosome acidification using pH-sensitive fluorophores like Oregon Green in macrophages

  • Measure reactive oxygen species production in phagocytic cells with and without CLIC1 expression

  • Monitor changes in organelle pH using ratiometric imaging techniques

• Membrane Translocation Assessment:

  • Use fractionation techniques to separate cytosolic and membrane proteins

  • Quantify CLIC1 distribution between fractions via western blotting

  • Visualize translocation using confocal microscopy with fluorescently-tagged CLIC1

How does CLIC1 contribute to tumor progression and what evidence supports its role as a potential cancer therapeutic target?

CLIC1 has emerged as an important player in cancer biology with multiple lines of evidence supporting its role in tumor progression:

• Expression Pattern in Cancer:

  • Overexpressed in multiple tumor types including renal cell carcinoma

  • Higher expression often correlates with advanced disease stages and poorer prognosis

• Mechanistic Contributions to Cancer:

  • Promotes cancer cell proliferation, migration, and invasion

  • Facilitates tumor angiogenesis by mediating communication between tumor cells and endothelial cells

  • Contributes to tumor growth via exosome-mediated functions

  • May influence tumor microenvironment by affecting immune cell function

• Experimental Evidence as Therapeutic Target:

  • Anti-CLIC1 antibodies suppress tumor growth in human renal cell carcinoma (RCC) xenograft models

  • Treatment with anti-CLIC1 antibodies induces tumor cell necrosis

  • Anti-CLIC1 therapy causes rapid regression of tumor vasculature, though not complete elimination

  • Both rabbit cornea and chick embryo chorioallantoic membrane (CAM) models demonstrated significant anti-tumor effects of CLIC1 targeting

These findings collectively support CLIC1 as a promising therapeutic target, particularly for highly vascularized tumors like clear cell renal cell carcinoma (cc-RCC).

What is the current understanding of CLIC1's role in inflammatory and neurodegenerative conditions?

CLIC1 has significant implications in both inflammatory and neurodegenerative pathologies:

• Inflammatory Conditions:

  • CLIC1 knockout mice show protection from development of serum transfer-induced K/BxN arthritis, indicating its role in inflammatory joint disease

  • CLIC1 is essential for proper macrophage function, particularly in phagosome acidification and reactive oxygen species production

  • Impaired phagosomal proteolytic capacity in CLIC1-deficient macrophages suggests its importance in processing inflammatory mediators

• Neurodegenerative Diseases:

  • In Alzheimer's disease models, CLIC1 expression is elevated in microglia upon exposure to amyloid β-peptide

  • CLIC1 translocates from cytosol to plasma membrane in microglia after amyloid β-peptide treatment

  • This translocation contributes to neurotoxicity through generation of superoxide anions

  • Inhibition of CLIC1 prevents neuronal apoptosis in neurons co-cultured with amyloid β-peptide treated microglia, identifying it as a potential therapeutic target for Alzheimer's disease

The dual role of CLIC1 in both inflammatory and neurodegenerative conditions highlights the interconnected nature of these pathological processes and suggests that CLIC1-targeted therapies might address multiple aspects of these diseases.

How do the redox-dependent conformational changes in CLIC1 affect its channel activity and biological function?

The redox-dependent activity of CLIC1 represents one of its most intriguing properties:

• Structural Transitions:

  • CLIC1 undergoes significant conformational changes in response to oxidizing conditions

  • These changes expose hydrophobic regions that facilitate membrane insertion

  • The glutathione S transferase fold domain contains critical cysteine residues that act as redox sensors

  • Under oxidizing conditions, intramolecular disulfide bonds form, stabilizing the membrane-inserted conformation

• Functional Consequences:

  • Oxidation increases the probability of CLIC1 membrane insertion and channel formation

  • Channel conductance properties differ between reduced and oxidized states

  • The pH sensitivity of CLIC1 is altered by its redox state, creating complex regulatory potential

  • These properties allow CLIC1 to respond to cellular stress conditions, particularly oxidative stress

• Biological Significance:

  • In phagocytes, the oxidative environment of the phagosome may trigger CLIC1 translocation and activation

  • In tumor microenvironments, hypoxia-induced redox changes may enhance CLIC1 membrane insertion and activity

  • Neurodegenerative conditions with oxidative stress components may pathologically activate CLIC1, contributing to disease progression

Understanding these redox-dependent mechanisms provides opportunities for targeted therapeutic approaches that specifically modulate CLIC1 activity under pathological conditions while preserving its physiological functions.

What is the relationship between CLIC1 and other chloride channels in maintaining cellular ion homeostasis?

CLIC1 operates within a complex network of chloride channels that collectively maintain cellular ion homeostasis:

• Comparative Expression Patterns:

  • In human bronchial epithelial cells (hBECs), CLIC family members are the most abundantly expressed chloride channel transcripts, with CLIC1 showing the highest expression levels

  • This suggests a dominant role for CLIC1 in chloride homeostasis in these cells

• Functional Complementarity:

  • CLIC1 modulates cyclic AMP-induced chloride currents, which are typically associated with CFTR (cystic fibrosis transmembrane conductance regulator)

  • This suggests potential compensatory or cooperative relationships between these channel types

  • In cystic fibrosis, where CFTR function is impaired, alterations are also observed in other chloride channels including Ca²⁺-activated Cl⁻ Channels, Outwardly rectified Cl⁻ Channels, and volume-activated Cl⁻ Channels

• Subcellular Specialization:

  • Unlike plasma membrane-localized chloride channels, CLIC1 can function in multiple subcellular compartments

  • This allows for compartment-specific regulation of chloride concentrations and electrical potential

  • The translocation ability of CLIC1 provides dynamic responsiveness to cellular needs

• Integrated Signaling:

  • CLIC1 activity appears to be integrated with other cellular signaling pathways, including reactive oxygen species production and phagosome acidification

  • This integration suggests CLIC1 functions as part of larger regulatory networks rather than as an isolated channel

What are the most significant challenges in generating functional recombinant CLIC1 for structural and functional studies?

Researchers face several challenges when producing functional recombinant CLIC1:

• Dual Conformational States:

  • CLIC1 exists in both soluble and membrane-inserted forms, making consistent preparation difficult

  • Solution: Carefully control redox conditions during purification; use non-denaturing detergents to stabilize membrane-inserted form when needed

• Functional Validation:

  • Confirming channel activity of recombinant protein is challenging due to spontaneous membrane insertion

  • Solution: Combine multiple approaches including planar lipid bilayer electrophysiology, liposome-based flux assays, and cell-based functional assays

• Post-translational Modifications:

  • Although minimal, potentially important post-translational modifications may be absent in bacterial expression systems

  • Solution: Compare protein from bacterial and eukaryotic expression systems; consider mammalian or insect cell expression for studies requiring native modifications

• Oxidation During Purification:

  • CLIC1 is sensitive to oxidation, which can alter its conformation and function

  • Solution: Maintain reducing conditions throughout purification; consider site-directed mutagenesis of critical cysteine residues for specific experiments

• Species-Specific Variations:

  • Subtle differences between rabbit CLIC1 and human CLIC1 may affect experimental outcomes

  • Solution: Perform comparative studies with both rabbit and human proteins when possible; carefully validate antibody cross-reactivity

How can researchers effectively design inhibitors or modulators specific to CLIC1 for experimental and therapeutic applications?

Developing specific CLIC1 modulators requires strategic approaches:

• Targeting Unique Structural Features:

  • Focus on regions that distinguish CLIC1 from other chloride channels and CLIC family members

  • The transition region between soluble and membrane-inserted forms offers a potential specific target

  • Use structure-based design informed by crystallographic data of the soluble form and modeling of the membrane form

• Exploiting Redox Sensitivity:

  • Design compounds that selectively interact with CLIC1's redox-sensing regions

  • Develop agents that prevent oxidation-induced conformational changes

  • Consider reversible oxidizing agents that specifically target CLIC1's critical cysteine residues

• Screening Strategies:

  • Implement high-throughput functional assays based on chloride flux or membrane insertion

  • Use cell-based screening with CLIC1-overexpressing lines to identify functional inhibitors

  • Develop competitive binding assays with known CLIC1 ligands

• Validation Methodology:

  • Confirm specificity by testing against multiple chloride channels and CLIC family members

  • Validate in both biochemical assays and cellular systems

  • Use CLIC1 knockout models as negative controls to confirm target engagement

• Application-Specific Optimization:

  • For research tools, prioritize specificity and reversibility

  • For potential therapeutic compounds, optimize pharmacokinetic properties and tissue penetration

  • Consider context-specific targeting, such as tumor-targeted delivery systems for anti-cancer applications

• Antibody-Based Approaches:

  • For specific experimental applications, develop and characterize function-blocking antibodies

  • Anti-CLIC1 antibodies have shown promise in tumor models and could serve as starting points for therapeutic development

What emerging technologies could advance our understanding of CLIC1 structure and function in native cellular environments?

Several cutting-edge technologies show promise for revealing new insights about CLIC1:

• Cryo-Electron Microscopy:

  • Could potentially capture CLIC1 in its membrane-inserted form, which has been challenging with traditional crystallography

  • May reveal oligomeric structures and interaction with membrane lipids

  • Could provide insights into conformational changes during soluble-to-membrane transition

• Advanced Live Cell Imaging:

  • Super-resolution microscopy techniques can track CLIC1 translocation in real-time

  • FRET-based sensors could monitor CLIC1 conformational changes in living cells

  • Correlative light and electron microscopy could connect functional states with ultrastructural localization

• Single-Cell Analysis:

  • Single-cell proteomics may reveal cell-specific CLIC1 expression patterns in heterogeneous tissues

  • Single-cell functional assays could uncover specialized roles in specific cell populations

  • Spatial transcriptomics could map CLIC1 expression in complex tissues like tumors or brain regions

• Genome Editing Technologies:

  • CRISPR-Cas9 knock-in of fluorescent tags at endogenous loci to study native expression levels

  • Base editing to introduce specific mutations for structure-function analysis

  • Inducible systems to control CLIC1 expression with temporal precision

• Artificial Intelligence Applications:

  • Machine learning approaches to predict CLIC1 interaction networks from large datasets

  • AI-assisted analysis of complex electrophysiological recordings

  • Computational modeling of CLIC1 dynamics in biological membranes

These technologies could collectively address key knowledge gaps regarding CLIC1's dynamic behavior in health and disease contexts.

What are the most promising translational research opportunities for CLIC1-targeted therapies?

Based on current knowledge, several translational pathways show particular promise:

• Oncology Applications:

  • Development of anti-CLIC1 antibodies or small molecule inhibitors for highly vascularized tumors like renal cell carcinoma

  • Combination approaches targeting both tumor cells and tumor vasculature via CLIC1 inhibition

  • Biomarker development based on CLIC1 expression patterns for patient stratification

• Neurodegenerative Disease Interventions:

  • CLIC1 inhibitors that specifically target microglial CLIC1 to reduce neurotoxicity in Alzheimer's disease

  • Development of central nervous system-penetrant CLIC1 modulators

  • Imaging agents that detect pathological CLIC1 expression or translocation as diagnostic tools

• Anti-Inflammatory Approaches:

  • Targeted modulation of CLIC1 in macrophages for conditions like rheumatoid arthritis

  • Selective manipulation of specific CLIC1 functions like phagosome acidification without affecting other cellular functions

  • Development of tissue-specific delivery systems to target inflammatory sites

• Respiratory Medicine:

  • Exploring CLIC1 modulators for bronchial epithelial cell function in conditions like asthma or chronic obstructive pulmonary disease

  • Potential complementary approaches for patients with cystic fibrosis by targeting non-CFTR chloride channels

• Diagnostic Applications:

  • Development of non-invasive methods to detect elevated CLIC1 expression as biomarkers for early cancer detection

  • Prognostic tools based on CLIC1 expression patterns or functional status

  • Companion diagnostics to identify patients most likely to respond to CLIC1-targeted therapies

The advancement of these translational opportunities depends on deeper mechanistic understanding of CLIC1 biology and continued development of specific modulatory tools.