CLIC4 Human

What is the cellular distribution pattern of CLIC4 in human tissues?

CLIC4 shows preferential localization to mitochondrial-associated membranes (MAMs) rather than pure mitochondrial fractions. Quantitative colocalization studies demonstrate CLIC4 has higher association with mitochondria (41.2 ± 0.6%, n=40 cells) and ER-mitochondria contact site proteins, including mitofusin 2 (34 ± 1%, n=25 cells) and ACSL4 (31 ± 2%, n=25 cells). In isolated crude mitochondria, CLIC4 shows 58 ± 10% association with mitochondria, while in neonatal cardiomyocytes it shows 40 ± 4% (n=28) association with MitoTracker-loaded mitochondria .

How does CLIC4 differ structurally and functionally from other CLIC family members?

CLIC4 demonstrates significantly higher mitochondrial colocalization compared to its paralog CLIC1. Specifically, CLIC1 shows lower colocalization to crude mitochondria (39 ± 8%, P=0.03) and neonatal cardiomyocytes (29 ± 3%, P=0.03, n=28) in comparison to CLIC4. Unlike CLIC1, which is primarily localized in the endoplasmic reticulum, CLIC4's predominant localization to MAMs appears to give it a distinct role in modulating mitochondrial physiology and cellular response to injury .

What are the recommended techniques for studying CLIC4 protein localization in cells?

For precise CLIC4 localization studies, a multi-technique approach is recommended. The most reliable methodology involves:

• Immunofluorescence with colocalization analysis using confocal microscopy (with organelle-specific markers like MitoTracker for mitochondria)

• Subcellular fractionation using differential centrifugation

• Purification of mitochondria using 30% Percoll gradient centrifugation to separate pure mitochondria (M3 fraction) from MAM fractions

• Western blot analysis of the various fractions using organelle-specific markers (e.g., GRP78 for ER/MAM)

This combined approach has revealed CLIC4's negligible presence in ultrapure mitochondrial fractions but significant presence in MAMs, establishing it as a MAM-specific Cl⁻ channel .

What cloning strategies are effective for expressing recombinant CLIC4 constructs?

The recommended approach for generating CLIC4 constructs involves:

• Using the pCDNA3.1-clic4 vector encoding N-terminal FLAG as a template

• Implementing site-directed mutagenesis via the "Quick change" method with specific primers (see table below)

• Using DH5α competent cells for transformation

• Screening positive colonies by Sanger sequencing

For protein expression and purification, researchers have successfully used:

• Human CLIC4 gene cloned into a pET-28a vector with N-terminal hexahistidine tag and TEV cleavage site

• Expression in C43 E.coli strain using M9 minimal media

• Purification via affinity chromatography with Ni IMAC

How is CLIC4 expression regulated in cancer progression?

CLIC4 functions as a tumor suppressor involved in processes including growth arrest, differentiation, and apoptosis. Its expression is diminished in tumor parenchyma during progression in squamous cell carcinoma (SCC) and other neoplasms. Importantly, this downregulation is not driven by genomic alterations but instead by post-transcriptional mechanisms. Screening and functional assays have identified miR-142-3p as a key regulator of CLIC4. Expression analysis reveals that CLIC4 and miR-142-3p are inversely correlated in head and neck (HN) SCC and cervical SCC, particularly in advanced stage cancers .

What advanced techniques can differentiate CLIC4 expression in tumor cells versus stromal compartments?

For researchers studying the complex tumor microenvironment, a combination of advanced techniques is required:

• In situ hybridization to localize miR-142-3p expression within tissue sections

• Single-cell RNA-sequencing to identify cell-type specific expression patterns

• Immunohistochemical analysis with cell-type specific markers

These combined approaches have revealed that stromal immune cells, not tumor cells, are the predominant source of miR-142-3p in HNSCC. Furthermore, single-cell expression data demonstrated that CLIC4 is lower in tumor epithelial cells than in normal epithelium, providing crucial insights into the cell-type specific regulation of CLIC4 in cancer progression .

What sex-specific differences exist in CLIC4 expression following ethanol exposure?

Sex-specific differences in CLIC4 response to ethanol have been documented using qRT-PCR analysis of the medial prefrontal cortex (mPFC) in C57BL/6J mice. Following acute ethanol exposure, female mice show a rapid increase in Clic4 mRNA expression that is notably absent in male mice. This sex-specific response suggests potential differences in how male and female brains process acute ethanol exposure at the molecular level .

What cell types predominantly express CLIC4 in brain tissue and how can they be quantified?

Immunohistochemical and stereological techniques have revealed that CLIC4 protein expression in brain tissue follows this pattern of abundance:

• Oligodendrocytes (most abundant)

• Microglia

• Astrocytes

• Neurons (minimal expression)

For comprehensive analysis of CLIC4-expressing cell populations in brain tissue, researchers should employ:

• Dual immunohistochemical labeling with cell-type specific markers

• Stereological counting methods for quantification

• qRT-PCR for mRNA expression analysis before and after experimental interventions

This methodological approach has established that oligodendrocytes likely play a key role in mediating CLIC4's response to ethanol in the brain .

What is the functional significance of CLIC4 in cardiac mitochondrial physiology?

CLIC4 plays a critical cardioprotective role through its localization to mitochondrial-associated membranes (MAMs). Experimental evidence using CLIC4 null cardiomyocytes demonstrated increased apoptosis and mitochondrial dysfunction when subjected to hypoxia-reoxygenation injury compared to wild-type cardiomyocytes. This indicates CLIC4's essential role in maintaining mitochondrial function during cardiac stress conditions .

How does CLIC4 expression and distribution change in heart failure pathology?

Heart failure induces complex changes in CLIC4 expression patterns that require sophisticated analysis techniques:

These observations suggest that reduced internalization of soluble CLIC4 to organelle membranes like MAMs may contribute to decreased functional activity in the intracellular membrane fraction, potentially contributing to heart failure pathology .

What computational methods are most effective for identifying potential CLIC4 inhibitors?

The crystal structure availability of soluble CLIC4 allows for sophisticated structure-based drug discovery approaches. A comprehensive methodology includes:

• Utilizing multiple predictive algorithms to identify consensus allosteric sites:

  • SiteMap

  • DeepSite

  • FTMap

  • DogSiteScore

  • ProBIS

• Characterizing potential binding pockets based on:

  • Surface exposure

  • Hydrophobicity

  • Hydrophilicity

  • Druggability (measured by Halgren's and DogSite scores)

• Performing virtual screening with blind docking using AutoDock Vina integrated in high-performance computing clusters

• Filtering compounds based on binding energies, non-preferential binding to GSH sites, and pharmacological relevance

What experimental validation methods should be used to confirm CLIC4 inhibitor efficacy?

For rigorous validation of potential CLIC4 inhibitors identified through computational screening, researchers should implement:

• Recombinant protein expression in appropriate bacterial systems (e.g., C43 E.coli strain)

• Protein purification via affinity chromatography with Ni IMAC

• In vitro binding and functional assays to confirm inhibitor interaction and efficacy

• GPU-accelerated molecular dynamics simulations to investigate allosteric inhibitory mechanisms

This combined computational and experimental approach has successfully identified new molecules with improved specificity compared to traditional non-selective CLIC inhibitors like IAA94 and A9C .

How does CLIC4 contribute to immune cell function and signaling?

Chloride ion channels, including CLIC4, play critical roles in modulating immunological interactions. Research indicates that chloride flux may utilize distinct signaling pathways to execute specific functions in cellular context-dependent manners. The CLIC family located in various cellular organelles is involved in diverse physiological functions and pathological conditions, including immune response regulation .

What experimental models are most appropriate for studying CLIC4's role in inflammatory conditions?

While specific experimental models aren't detailed in the search results, emerging research suggests that studying CLIC4's role in immunomodulation requires:

• Cell-type specific conditional knockout models

• Flow cytometry analysis of immune cell populations

• Cytokine profiling in response to inflammatory stimuli

• Examination of CLIC4 expression in immune cell subsets under various inflammatory conditions

Understanding CLIC4's role in immune signaling pathways may provide insights into novel therapeutic approaches for inflammatory and immune-mediated diseases .