LRRC8C Antibody

What is LRRC8C and why is it important for research?

LRRC8C, also known as AD158, functions as a non-essential but regulatory component of the volume-regulated anion channel (VRAC, also known as VSOAC). This channel plays a crucial role in maintaining constant cell volume in response to extracellular or intracellular permeability changes. The importance of LRRC8C extends beyond volume regulation, as recent research demonstrates its significant role in T cell function, immune responses, and development. Understanding LRRC8C is essential for investigating cellular homeostasis mechanisms, immune regulation, and potential therapeutic targets for related pathologies .

What applications can LRRC8C antibodies be used for?

LRRC8C antibodies, such as the 21601-1-AP polyclonal antibody, can be utilized in multiple experimental applications including:

The antibody demonstrates reactivity with human, mouse, and rat samples, making it versatile for cross-species studies. Published research confirms successful application in both Western Blot and immunofluorescence techniques .

What is the molecular profile of LRRC8C?

LRRC8C has the following molecular characteristics:

These properties are important to consider when planning experiments and interpreting results, particularly when confirming antibody specificity and target identification .

How should I optimize LRRC8C antibody use for immunohistochemistry?

For optimal immunohistochemistry results with LRRC8C antibody, tissue-specific antigen retrieval is crucial. For mouse heart tissue, evidence suggests using TE buffer (pH 9.0) for antigen retrieval. Alternatively, citrate buffer (pH 6.0) may be used, though comparative efficacy should be established for your specific tissue. Begin with a dilution range of 1:50-1:500, testing multiple concentrations to determine optimal signal-to-noise ratio for your specific tissue sample .

Validation controls should include known positive samples (e.g., mouse heart tissue) and negative controls (secondary antibody alone or isotype control). When possible, include LRRC8C knockout tissue as a definitive negative control, as researchers have used Lrrc8c-/- mice to validate antibody specificity in published studies .

How can I distinguish LRRC8C from other LRRC8 family members in my research?

Distinguishing LRRC8C from other family members (LRRC8A, B, D, and E) requires careful experimental design considering their structural similarities. For antibody-based detection, verify that your antibody was raised against unique epitopes of LRRC8C not conserved in other family members. The 21601-1-AP antibody targets LRRC8C specifically, using an LRRC8C fusion protein (Ag16213) as the immunogen .

For gene expression analysis, design primers spanning exon junctions unique to LRRC8C. Published research has utilized RT-qPCR and RNA-Seq targeting exon 4 of Lrrc8c (which encodes 94% of the LRRC8C protein) to confirm knockout in Lrrc8c-/- mice models . When analyzing protein complexes, consider that LRRC8C functions as part of heteromeric channels with the obligatory LRRC8A subunit, so co-immunoprecipitation approaches can help distinguish specific complexes containing LRRC8C.

How does LRRC8C function in T cell immune regulation?

LRRC8C plays a previously unrecognized inhibitory role in T cell function and adaptive immunity through a novel signaling pathway. Studies with Lrrc8c-/- mice have revealed that LRRC8C deficiency leads to:

• Increased T cell cycle progression and proliferation

• Enhanced T cell survival

• Elevated Ca2+ influx after TCR stimulation

• Increased cytokine production

• Exacerbated T cell-dependent immune responses

Mechanistically, LRRC8C mediates the transport of 2'3'cGAMP in T cells, which activates STING and subsequently p53 signaling. This LRRC8C-cGAMP-STING-p53 pathway functions as a novel inhibitory mechanism in T cells. When this pathway is disrupted in Lrrc8c-/- mice, the animals display enhanced T cell-mediated immunity with increased CNS infiltration of T cells in experimental autoimmune encephalomyelitis models and augmented T cell-dependent humoral immune responses to influenza infection .

What structural insights have been gained about LRRC8C in recent research?

Recent cryo-EM studies have provided significant structural insights into LRRC8C architecture and function. LRRC8C forms multimeric channels, typically hexameric or heptameric, with distinct domains:

• A transmembrane pore domain (PD)

• An extracellular subdomain (ESD)

• A cytoplasmic leucine-rich repeat domain (LRRD)

The boundary between the pore and cytoplasmic domain is particularly important for channel function. Disease-associated variants (V390L and a truncation) located at this boundary show increased structural flexibility compared to wild-type LRRC8C, suggesting destabilization of subunit interactions. This structural perturbation results in enhanced channel activation, even under isotonic conditions where wild-type channels remain closed .

These findings highlight the LRRD as an allosteric regulator of channel activity and demonstrate that LRRC8C plays a critical role in controlling VRAC gating beyond the obligatory LRRC8A subunit .

What are common challenges in detecting LRRC8C and how can they be addressed?

Researchers often encounter several challenges when detecting LRRC8C:

• Cross-reactivity with other LRRC8 family members: Use validated antibodies like 21601-1-AP that specifically target LRRC8C. Confirm specificity using knockout controls or siRNA knockdown samples.

• Heterogeneous expression levels: LRRC8C expression is upregulated in T cells stimulated with anti-CD3/CD28 and IL-2, and the Lrrc8c/Lrrc8a ratio increases with IL-2 or IL-15 treatment through STAT5 tetramerization . Consider these regulatory mechanisms when designing experiments and interpreting results.

• Detection in complex formations: Since LRRC8C functions in heteromeric complexes with LRRC8A, standard protein extraction methods may not preserve these interactions. Use appropriate detergents and extraction conditions that maintain protein-protein interactions when studying LRRC8C in its native complex.

• Storage and handling of antibodies: Store LRRC8C antibody at -20°C in the recommended buffer (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3). The antibody is stable for one year after shipment, and aliquoting is unnecessary for -20°C storage .

How can I validate LRRC8C functionality in my experimental system?

Validating LRRC8C functionality requires assessing its channel activity and physiological effects:

• Electrophysiological measurements: Record VRAC currents in response to hypotonic challenge. Lrrc8c-/- mice completely lack VRAC currents and regulatory volume decrease (RVD) upon osmotic swelling .

• Regulatory volume decrease (RVD) assays: Monitor cell volume recovery after hypotonic challenge using techniques like Coulter counter or fluorescent volume indicators. Compare wild-type cells with LRRC8C-depleted cells to assess the contribution of LRRC8C to RVD.

• Transport assays: Measure transport of specific substrates like 2'3'cGAMP, which is mediated by LRRC8C in T cells .

• Functional readouts: In T cells, assess proliferation, survival, Ca2+ influx, and cytokine production after TCR stimulation, as these parameters are enhanced in LRRC8C-deficient T cells .

• STING and p53 activation: Measure downstream signaling pathways regulated by LRRC8C, including STING activation and p53 expression and signaling .

What disease associations have been identified for LRRC8C variants?

Recent research has identified de novo pathogenic variants in LRRC8C that cause a previously unrecognized congenital syndrome. Two different human individuals with the same syndrome affecting blood vessels, brain, eyes, and bones were found to harbor de novo variants in the LRRC8C gene .

These variants were located in a region encoding the boundary between the pore and a cytoplasmic domain—a region depleted of sequence variations in control subjects. The two variants studied were:

• A conservative amino acid substitution (V390L)

• A truncation removing approximately 50% of the protein

Despite their different nature, both variants led to similar structural and functional consequences: increased flexibility of the protein structure and constitutive channel activation even under isotonic conditions. These findings established a dominant gain-of-function effect as the pathogenic mechanism .

The pleiotropic phenotype of this novel clinical entity indicates the fundamental roles of VRACs in different tissues and organs, opening new avenues for understanding LRRC8C function in development and disease .

How might LRRC8C antibodies contribute to therapeutic research?

LRRC8C antibodies hold significant potential for therapeutic research based on emerging understanding of LRRC8C's role in immune regulation and disease:

• Immune modulation: Given that LRRC8C suppresses T cell activation through the cGAMP-STING-p53 pathway, antibodies detecting this protein could facilitate screening for compounds that modulate this pathway. Such modulators might be valuable for treating autoimmune conditions (by enhancing LRRC8C function) or improving anti-tumor immunity (by inhibiting LRRC8C function) .

• Biomarker development: The recently identified role of LRRC8C variants in a congenital syndrome affecting multiple organ systems suggests LRRC8C antibodies could help identify biomarkers of disease progression or treatment response in affected individuals .

• Structural studies: Antibodies recognizing specific conformations of LRRC8C could help elucidate the structural basis of channel activation, contributing to structure-based drug design targeting VRACs.

• Cell-specific targeting: The differential expression of LRRC8C across tissues and cell types offers opportunities for targeted therapeutic approaches, with antibodies serving as research tools to validate such targeting strategies.