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

What is the basic molecular structure and function of LRRC8E?

LRRC8E is a protein that functions as a non-essential component of the volume-regulated anion channel (VRAC). Like other LRRC8 family members, the protein consists of a transmembrane pore domain and cytoplasmic leucine-rich repeat (LRR) domains. LRRC8E heteromerizes with LRRC8A (the obligatory subunit) to form functional VRAC channels that regulate cell volume by transporting chloride ions and various organic osmolytes across the plasma membrane .

The protein features multiple leucine-rich repeat motifs that are involved in protein-protein interactions. These structural features are critical for the assembly and function of VRAC heteromers. The structure consists of both transmembrane regions that form the ion-conducting pore and cytoplasmic domains that modulate channel activity .

How does LRRC8E differ from other members of the LRRC8 family?

Among the LRRC8 proteins (LRRC8A-E), LRRC8E has distinct functional and structural characteristics:

• Unlike LRRC8A, which is essential for VRAC function, LRRC8E serves as an optional subunit .

• LRRC8E shows different substrate selectivity compared to other family members. For example, LRRC8E-containing channels are more efficient at conducting negatively charged aspartate compared to LRRC8D-containing channels, which preferentially transport neutral compounds .

• Expression patterns differ between LRRC8 family members. In some tissues like the kidney, LRRC8E is predominantly found in the urothelial lining of the papilla and in acid-secreting α-cells and bicarbonate-secreting β-ICs, while other LRRC8 proteins show different tissue distributions .

What is the expression pattern of LRRC8E in rat tissues?

In rat tissues, LRRC8E shows a distinctive expression pattern:

In the kidney specifically, LRRC8E is found in basolateral membranes of intercalated cells but was not detected in principal cells (marked by AQP2) . This distinct expression pattern suggests specialized functions in specific cell types.

What are the recommended methods for producing recombinant rat LRRC8E protein?

For producing recombinant rat LRRC8E protein, the following methodological approach is recommended:

• Expression System Selection: HEK293 cells are preferred for mammalian expression as they produce properly folded and post-translationally modified LRRC8E proteins. For bacterial expression, E. coli systems using BL21(DE3) can be employed for truncated or partial domains .

• Construct Design:

  • For full-length protein: Include the entire coding sequence with a signal peptide

  • For structural studies: Consider using fusion constructs with tags like GST at N-terminus

  • For epitope mapping: Express fragments containing specific regions (250-500 aa) as done with human LRRC8E

• Purification Strategy:

  • Use affinity chromatography (His, GST, or Fc tags)

  • Follow with size exclusion chromatography to obtain homogeneous preparations

  • Consider adding stabilizing agents (glycerol 5-10%) to prevent aggregation

• Quality Control:

  • Verify proper folding using circular dichroism

  • Confirm oligomeric state by size exclusion chromatography and native PAGE

  • Test functionality in artificial liposomes or reconstituted systems

When producing recombinant fragments for antibody generation or binding studies, choose regions with high antigenicity and minimal similarity to other LRRC8 family members to ensure specificity.

How can researchers accurately quantify LRRC8E protein levels in tissue or cell samples?

Accurate quantification of LRRC8E requires careful methodological considerations:

• Immunoblotting with Calibration:

  • Use purified recombinant LRRC8E fragments as calibration standards

  • Create standard curves with 3-4 concentrations in the range of expected sample concentration

  • Include positive and negative controls (knockout tissues/cells are ideal negative controls)

  • Calculate absolute protein amounts using linear regression analysis of calibration curves

• Sample Preparation Optimization:

  • Use membrane protein extraction buffers containing 1% Triton X-100 or 0.5% DDM

  • Include protease inhibitors to prevent degradation

  • Normalize loading based on total protein (BCA or Bradford assay)

  • Ensure complete solubilization by incubating at 37°C for 30 minutes

• Cross-Validation Methods:

  • Combine immunoblotting with mass spectrometry-based quantification

  • Use selected reaction monitoring (SRM) for targeted quantification

  • Employ qPCR for mRNA levels as complementary data

As demonstrated in murine cells, the relative abundance of different LRRC8 subunits can vary significantly. For example, in C2C12 myoblasts, LRRC8E levels were approximately 5-fold lower than LRRC8B, C, and D .

How does the stoichiometry of LRRC8A/LRRC8E heteromers affect channel properties?

The stoichiometry of LRRC8A/LRRC8E heteromers significantly impacts channel function and properties:

Notably, sequential co-immunoprecipitation experiments have shown that a single VRAC complex can contain multiple different LRRC8 subunits simultaneously, suggesting complex stoichiometric arrangements rather than simple binary heteromers .

What is the mechanism by which LRRC8E-containing VRACs transport substrates differently than other LRRC8 combinations?

The substrate selectivity of LRRC8E-containing VRACs involves complex mechanisms:

• Structural Determinants:

  • The pore-lining regions of LRRC8E contain distinctive amino acid residues that create unique electrostatic environments

  • The cytoplasmic LRR domains influence pore conformation through allosteric mechanisms

  • Specific residues in transmembrane domains form substrate interaction sites

• Substrate Preference:
  Research has shown that LRRC8E-containing channels, when paired with LRRC8A:

  • Efficiently conduct negatively charged amino acids (aspartate, glutamate)

  • Show higher ATP permeability compared to other combinations

  • Form channels with distinct kinetics and voltage-dependence

• Experimental Evidence:
  Radiotracer efflux studies in astrocytes revealed that:

  • LRRC8A+LRRC8E efficiently transport D-aspartate

  • LRRC8A+LRRC8D preferentially transport neutral osmolytes like taurine and myo-inositol

• Biophysical Properties:
  Single channel recordings demonstrate that LRRC8A/E heteromers exhibit:

  • Higher open probability at positive potentials

  • Different rectification properties

  • Distinctive response to modulators like carbenoxolone

The functional differences likely arise from the unique three-dimensional arrangement of the channel pore and the influence of cytoplasmic domains on channel gating.

How can researchers effectively modulate LRRC8E-containing VRAC activity for functional studies?

Modulating LRRC8E-containing VRAC activity requires sophisticated approaches:

• Pharmacological Modulators:

  • Carbenoxolone acts from the extracellular side, binding to multiple sites and inhibiting LRRC8-mediated currents (IC₅₀ ≈ 20-30 μM)

  • Addition of 1 mM ATP blocks LRRC8A/LRRC8E-mediated currents by approximately 50%

  • DCPIB is a commonly used but non-specific VRAC inhibitor

• Genetic Approaches:

  • CRISPR-Cas9 knockout of LRRC8E combined with rescue experiments using wildtype or mutant constructs

  • RNAi-mediated knockdown using targeted siRNAs against non-conserved regions

  • Expression of dominant-negative truncated constructs

• Novel Tools: Synthetic Nanobodies:
  Recent advances have generated synthetic nanobodies (sybodies) targeting the LRR domain of LRRC8A that either inhibit or enhance channel activity. Similar approaches could be developed for LRRC8E-specific modulation .

• Reconstitution Systems:

  • Purified LRRC8A/LRRC8E channels can be reconstituted into artificial liposomes or lipid bilayers

  • The protein stoichiometry can be precisely controlled

  • Channel activity can be measured using fluorescent indicators or electrophysiological methods

When designing functional studies, it's important to consider that the precise regulation of VRAC activation involves not only subunit composition but also post-translational modifications and interaction with regulatory proteins.

What is known about the potential involvement of LRRC8E in disease states?

While less studied than other LRRC8 family members, LRRC8E has shown associations with certain disease conditions:

• Neuropsychiatric Disorders:

  • One study found that the LRRC8E gene was nominally associated with panic disorder

  • The role of LRRC8E in neurotransmitter transport suggests potential involvement in neurodevelopmental disorders

• Cancer Biology:

  • LRRC8A/LRRC8D are required for cisplatin uptake into cancer cells

  • By comparison, LRRC8E's role in drug transport is still being investigated

  • Altered expression of LRRC8E might affect cancer cell volume regulation and response to osmotic stress

• Genetic Disorders:

  • Recent identification of disease-causing variants in the related LRRC8C gene suggests possible mechanisms by which LRRC8E dysfunction might contribute to pathology

  • The LRRC8C variants caused constitutive channel activity even under isotonic conditions, suggesting similar gain-of-function mutations could occur in LRRC8E

• Renal Physiology:

  • LRRC8E's specific expression in certain renal cell types suggests a specialized role in kidney function

  • Disruption might contribute to defects in acid-base balance given its expression in acid/bicarbonate-secreting cells

The study of LRRC8E in disease contexts is emerging, and more research is needed to fully establish its pathophysiological roles.

How can recombinant LRRC8E be used for developing diagnostic or therapeutic approaches?

Recombinant LRRC8E has several potential applications in diagnostic and therapeutic development:

• Diagnostic Applications:

  • Development of specific antibodies for immunohistochemistry to detect altered LRRC8E expression patterns in tissues

  • Creation of reporter systems to monitor VRAC activity in patient-derived cells

  • Production of protein standards for quantitative immunoassays

• Therapeutic Target Identification:

  • Screening platforms using purified recombinant LRRC8E or LRRC8A/E heteromers

  • Development of subunit-specific modulators to selectively target certain VRAC compositions

  • Structure-based drug design utilizing the unique features of LRRC8E

• Antibody Development Strategy:
  Similar to the approach used for LRRC8 proteins in diagnostic lateral flow assays, recombinant LRRC8E could be used to:

  • Generate highly specific monoclonal antibodies

  • Develop diagnostic tests for conditions with altered LRRC8E expression

  • Create binding proteins that modulate channel activity

• Allosteric Modulators:
  Building on successful approaches with other LRRC8 proteins, researchers could develop:

  • Synthetic nanobodies (sybodies) targeting specific epitopes of LRRC8E

  • Small molecule modulators that bind to the LRR domain

  • Peptide-based inhibitors derived from interaction interfaces

While these applications are promising, they require further validation of LRRC8E's role in specific disease states and better understanding of its structural-functional relationships.

What are the current gaps in our understanding of LRRC8E function?

Despite significant advances, several knowledge gaps remain in LRRC8E research:

• Subunit-Specific Functions:

  • The unique contributions of LRRC8E to VRAC function compared to other LRRC8 proteins remain incompletely understood

  • Whether LRRC8E has VRAC-independent functions is unknown

  • The significance of heteromers containing multiple different LRRC8 subunits (beyond binary LRRC8A/E combinations) requires further investigation

• Regulatory Mechanisms:

  • How cell volume sensing leads to activation of LRRC8E-containing channels remains elusive

  • Potential phosphorylation sites and other post-translational modifications that regulate LRRC8E function are poorly characterized

  • Interaction partners beyond other LRRC8 family members are largely unknown

• Physiological Roles:

  • The functional significance of tissue-specific expression patterns of LRRC8E needs clarification

  • The role of LRRC8E in normal development and aging requires investigation

  • Whether LRRC8E participates in additional cellular processes beyond volume regulation is uncertain

• Structural Details:

  • High-resolution structures of LRRC8E-containing heteromers are currently lacking

  • The conformational changes during channel activation remain to be determined

  • The precise substrate binding sites and permeation pathway need definition

Addressing these gaps will require integrative approaches combining structural biology, electrophysiology, cell biology, and in vivo studies.

What novel techniques are emerging for studying LRRC8E interactions and functions?

Cutting-edge techniques are advancing our ability to study LRRC8E:

• Advanced Structural Biology Approaches:

  • Cryo-electron microscopy to resolve heteromeric channel structures at high resolution

  • Single-particle analysis to capture different conformational states

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics and interaction interfaces

• Functional Genomics:

  • CRISPR-based screening to identify regulators and interaction partners

  • Base editing for introducing specific mutations without disrupting the reading frame

  • Optical biosensors to monitor VRAC activity in real-time

• Advanced Imaging:

  • Super-resolution microscopy to visualize channel distribution and dynamics

  • FRET-based sensors to detect conformational changes

  • Correlative light and electron microscopy to link function with ultrastructure

• Synthetic Biology:

  • Engineered cells with defined LRRC8 compositions

  • Artificial intelligence-guided protein design to create LRRC8E variants with altered functions

  • Development of optogenetic tools to control VRAC activity with light

• In vivo Models:

  • Conditional knockout mouse models with tissue-specific LRRC8E deletion

  • Gene-edited rat models to study LRRC8E function in native contexts

  • Patient-derived induced pluripotent stem cells to investigate disease-relevant phenotypes

These emerging technologies promise to provide deeper insights into the molecular mechanisms of LRRC8E function and its role in health and disease.