Recombinant Mouse Leucine-rich repeat-containing protein 8E (Lrrc8e)

What is the structural composition of mouse LRRC8E protein?

Mouse LRRC8E is characterized by multiple leucine-rich repeat (LRR) motifs which are typically 20-29 amino acids long containing the conserved consensus sequence LxxLxLxxN/CxL, where L may be substituted by isoleucine, phenylalanine, or valine . The full-length mouse LRRC8E protein consists of 795 amino acid residues . The amino acid sequence contains various structural domains that facilitate its function as a subunit of volume-regulated anion channels (VRACs). The protein's structure likely includes transmembrane domains that allow for its incorporation into the plasma membrane as part of the heteromeric VRAC complex.

How does the leucine-rich repeat motif in LRRC8E contribute to its function?

The leucine-rich repeat (LRR) motifs in LRRC8E provide a structural framework for protein-protein interactions . These repeats create a versatile platform that enables LRRC8E to interact with other LRRC8 family proteins to form heteromeric volume-regulated anion channels (VRACs) . Research indicates that LRR is a generally useful protein-binding motif, with proteins containing this repeat typically involved in protein-protein interactions . The LRR domains in LRRC8E likely contribute to the specificity of these interactions, influencing which combinations of LRRC8 proteins can assemble together. Different compositions of LRRC8 subunits affect the range of specificity for VRACs , suggesting that the LRR regions play a role in determining channel properties such as ion selectivity and conductance.

What is the primary role of LRRC8E in cellular physiology?

LRRC8E functions as a subunit of the heteromeric protein volume-regulated anion channel (VRAC) . VRACs play crucial roles in regulating cell size by transporting chloride ions and various organic osmolytes such as taurine and glutamate across the plasma membrane . While LRRC8E is not as crucial to VRAC activity as LRRC8A and LRRC8D , it contributes to the channel's functional diversity. Research has found that while LRRC8A and LRRC8D are necessary for VRAC function, they are not sufficient for the full range of usual VRAC activity . This is where other LRRC8 proteins like LRRC8E become important, as the different composition of these subunits affects the specificity range of VRACs . Thus, LRRC8E appears to have a modulatory role in fine-tuning VRAC function in specific cellular contexts.

What are the optimal methods for detecting mouse LRRC8E in tissue samples?

Enzyme-linked immunosorbent assay (ELISA) is a validated method for detecting mouse LRRC8E in biological samples. Commercial ELISA kits are available with a reported test range of 0.156 ng/ml to 10 ng/ml . These kits are optimized for detecting native LRRC8E in tissue homogenates, cell lysates, and other biological fluids . For accurate results, sample concentrations should be diluted to the mid-range of the kit's detection capability .

The methodological approach for ELISA detection typically involves:

• Sample preparation: Proper homogenization of tissues or lysis of cells

• Standard curve generation: Using recombinant LRRC8E protein

• Sample addition: To microtiter wells with biotin-conjugated antibody specific to LRRC8E

• Detection: Using avidin conjugated to horseradish peroxidase and TMB substrate

• Measurement: Spectrophotometric reading at 450nm after reaction termination

For other detection methods, western blotting using LRRC8E-specific antibodies, immunohistochemistry for tissue localization studies, or RT-qPCR for mRNA expression analysis can be employed, though these would require careful optimization.

How can recombinant mouse LRRC8E be optimally expressed and purified?

While the search results don't provide a specific protocol for mouse LRRC8E, methodological approaches can be derived from related protein work:

• Expression system selection: E. coli BL21(DE3) with appropriate expression vectors (such as pET series) has been successfully used for other LRR-containing proteins .

• Construct design: Including a C-terminal 6-His tag facilitates purification via affinity chromatography . The full amino acid sequence (residues 1-795) should be considered for expression .

• Buffer optimization: Since LRRC8E is part of a membrane channel complex, specialized buffer conditions may be required. A Tris-based buffer with 50% glycerol has been reported as a storage buffer for recombinant proteins . For membrane proteins with hydrophobic regions, zwitterionic detergents like Empigen BB may be necessary during solubilization and purification to suppress aggregation .

• Storage considerations: Store at -20°C, or -80°C for extended storage. Repeated freezing and thawing should be avoided. Working aliquots can be stored at 4°C for up to one week .

• Quality control: Verify protein identity and purity using SDS-PAGE, mass spectrometry, and functional assays specific to VRAC activity.

What controls should be included when studying LRRC8E function?

When investigating LRRC8E function, the following controls are methodologically important:

• Positive controls: Systems with confirmed VRAC activity, such as cells with verified expression of LRRC8A, which is necessary for VRAC function .

• Negative controls: Systems lacking VRAC activity, such as LRRC8A knockout cells, since LRRC8A is essential for channel formation .

• Specificity controls: Manipulations of other LRRC8 family members to distinguish LRRC8E's specific contribution, particularly important given that different combinations of LRRC8 subunits affect VRAC specificity .

• Expression verification: Quantification of LRRC8E and other VRAC components to ensure observed effects are attributable to the intended experimental manipulation.

• Physiological relevance: Confirmation that experimental conditions reflect relevant in vivo contexts, particularly regarding osmotic challenges that activate VRAC function .

• Functional redundancy assessment: Evaluation of whether other LRRC8 proteins compensate for LRRC8E in its absence, helping to delineate unique versus overlapping functions.

How does LRRC8E contribute to VRAC function compared to other LRRC8 family members?

LRRC8E is one of several LRRC8 proteins (along with LRRC8A, LRRC8B, LRRC8C, and LRRC8D) that can serve as subunits of volume-regulated anion channels (VRACs) . The comparative roles of these proteins reveal a functional hierarchy:

LRRC8A appears to be the essential component, as studies on LRRC8A-deficient mice show severe developmental issues including increased prenatal and postnatal mortality and multiple tissue abnormalities . LRRC8A is detected at higher levels on the surface of thymocytes than on other immune cells and is required for T cell development .

LRRC8D is also necessary for VRAC function but not sufficient for the full range of VRAC activity .

To study the specific contribution of LRRC8E to VRAC function, comparative analyses of VRAC activity in systems with different combinations of LRRC8 subunits would be required, using genetic approaches to selectively express or suppress LRRC8E while controlling for the presence of other family members.

What is the relationship between LRRC8E expression and cellular responses to osmotic challenges?

As LRRC8E is a subunit of VRACs , which are critical for cell volume regulation during osmotic stress, its expression likely influences cellular osmotic responses. VRACs are crucial to the regulation of cell size by transporting chloride ions and various organic osmolytes, such as taurine or glutamate, across the plasma membrane .

A methodological approach to investigating this relationship would include:

• Manipulation of LRRC8E expression (overexpression, knockdown, or knockout)

• Measurement of cellular responses to hypotonic or hypertonic challenges, including:

  • Changes in cell volume

  • Ion fluxes (particularly chloride)

  • Transport of organic osmolytes

• Time-course experiments tracking cellular adaptation to osmotic challenges

• Comparative studies across different cell types with varying endogenous levels of LRRC8E

Since different compositions of LRRC8 subunits affect the specificity of VRACs , varying the expression of LRRC8E relative to other family members might reveal how this subunit influences the sensitivity or kinetics of volume regulation in response to osmotic challenges.

Are there tissue-specific variations in LRRC8E function that researchers should be aware of?

While the search results don't provide direct evidence for tissue-specific variations in LRRC8E function, there are indications that LRRC8 family proteins may have tissue-specific roles. For instance, LRRC8A is expressed at higher levels on the surface of thymocytes than on other immune cells and plays an essential role in T lymphocyte development .

The search results also mention that transmembrane LRRC proteins are expressed exclusively in the nervous system , suggesting potential tissue-specific functions. Additionally, Creative Biolabs notes that LRRC proteins have diverse physiological functions including antibacterial reaction, maintenance of normal cardiac function, regulation of trafficking of membrane receptors, and regulation of ion channel activity .

Methodologically, researchers investigating tissue-specific variations should:

• Perform comparative expression analyses across different tissues

• Investigate whether LRRC8E forms tissue-specific complexes with other LRRC8 family members

• Conduct functional studies in different cell types

• Consider developmental timing, as the importance of LRRC8E might vary during different stages of tissue development

• Examine whether tissue-specific regulatory mechanisms control LRRC8E expression or function

How do functional differences between LRRC8 family members impact experimental design?

The functional differences between LRRC8 family members necessitate several experimental design considerations:

• Hierarchical importance: LRRC8A is essential for VRAC function, while LRRC8E has a more modulatory role . Experiments should control for LRRC8A expression when studying other family members.

• Subunit combinations: Different combinations of LRRC8 subunits affect VRAC specificity . Experimental designs should account for the endogenous expression profile of all LRRC8 proteins in the cellular system being used.

• Phenotypic severity: Knockout studies may produce different phenotypic severities depending on the LRRC8 member targeted. LRRC8A knockout mice show severe developmental abnormalities including increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities , whereas the phenotypes associated with LRRC8E deficiency might be more subtle.

• Reconstitution requirements: When studying recombinant LRRC8 proteins, co-expression of multiple family members may be necessary to form physiologically relevant VRAC complexes.

• Functional assay selection: Assays should detect both core VRAC activities and specialized functions that might be particularly influenced by LRRC8E, such as transport of specific organic osmolytes .

What approaches can distinguish between different LRRC8 proteins in experimental settings?

To distinguish between different LRRC8 proteins, researchers can employ several methodological approaches:

• Immunological methods: Using specific antibodies validated for their target LRRC8 protein. Commercial ELISA kits for mouse LRRC8E demonstrate the viability of this approach .

• Genetic techniques: Knockout or knockdown of specific LRRC8 genes, as demonstrated with LRRC8A in the search results , allows for studying systems with controlled expression of individual family members.

• Mass spectrometry: Mass spectrometry sequencing, mentioned for identifying other LRR proteins , can distinguish between LRRC8 proteins based on their unique peptide signatures.

• Tagged recombinant proteins: Expression of tagged versions of different LRRC8 proteins facilitates their specific detection and purification. His-tagged recombinant proteins have been used successfully for other LRR-containing proteins .

• Functional discrimination: Assays that exploit known differences in how each LRRC8 protein contributes to VRAC properties may help distinguish their activities.

• Structure-guided probes: Comparative sequence analysis and structural modeling could guide the design of probes or assays that target unique regions of each LRRC8 protein.

How can researchers effectively validate LRRC8E-specific antibodies?

Thorough validation of LRRC8E-specific antibodies is crucial for reliable research outcomes. A methodological approach includes:

• Positive controls: Use samples with confirmed LRRC8E expression, such as recombinant LRRC8E protein or cells/tissues known to express LRRC8E at high levels.

• Negative controls: Employ samples lacking LRRC8E, such as LRRC8E knockout/knockdown cells or tissues. The approach used for creating LRRC8A knockout mice could be adapted for LRRC8E.

• Cross-reactivity testing: Ensure the antibody doesn't detect other LRRC8 family members (LRRC8A-D), which is particularly important given the structural similarities within this protein family .

• Multi-method validation: Test antibody performance across different applications such as Western blotting, immunoprecipitation, immunohistochemistry, and flow cytometry.

• Epitope mapping: Identify which region of LRRC8E the antibody recognizes to predict potential cross-reactivity and interpret results when studying variants.

• Independent verification: Use multiple antibodies targeting different epitopes of LRRC8E, or complementary non-antibody methods like mass spectrometry to confirm results.

• Titration experiments: Determine optimal antibody concentrations for specific applications to ensure maximum signal-to-noise ratio.

What experimental approaches are most effective for studying LRRC8E interactions with other VRAC subunits?

To study LRRC8E interactions with other VRAC subunits, researchers can employ several methodological approaches:

• Co-immunoprecipitation: To identify protein-protein interactions between LRRC8E and other VRAC subunits in their native state.

• Proximity ligation assays: To visualize and quantify interactions between LRRC8E and other LRRC8 proteins in situ.

• FRET/BRET analysis: Using fluorescently or bioluminescently tagged LRRC8 proteins to monitor their interactions in living cells.

• Crosslinking studies: To stabilize transient interactions between VRAC subunits for subsequent analysis.

• Yeast two-hybrid screening: To identify novel interaction partners of LRRC8E.

• Recombinant protein co-expression: To study the formation of different VRAC subunit combinations and their functional properties.

The leucine-rich repeat motifs in LRRC8E provide a structural framework for protein-protein interactions , making them likely mediators of subunit associations. Different compositions of LRRC8 subunits affect the range of specificity for VRACs , highlighting the importance of understanding these interaction patterns.

How might LRRC8E function be related to pathological conditions?

While the search results don't directly link LRRC8E to specific pathological conditions, they do provide context for potential disease associations:

The LRRC8 protein family is associated with agammaglobulinemia-5 , suggesting potential roles in immune disorders. Given LRRC8E's role in VRAC function , and the importance of cell volume regulation in various physiological processes, dysfunction of LRRC8E could potentially contribute to conditions involving disrupted cellular homeostasis.

A recent study found that the LRRC8E gene was nominally associated with panic disorder , hinting at possible neuropsychiatric implications. Since transmembrane LRRC proteins are expressed exclusively in the nervous system , LRRC8E dysfunction might contribute to neurological disorders.

VRACs are crucial to the regulation of cell size by transporting chloride ions and various organic osmolytes across the plasma membrane , suggesting that LRRC8E abnormalities could be relevant to conditions involving osmotic imbalances or cell volume dysregulation.

Methodologically, researchers investigating potential disease associations should consider:

• Genetic association studies in patient populations

• Expression analysis in diseased versus healthy tissues

• Functional studies in disease models

• Investigation of LRRC8E variants identified in patient populations