Recombinant Human Leucine-rich repeat-containing protein 38 (LRRC38)

What is the structure and function of LRRC38?

LRRC38 is a leucine-rich repeat-containing protein with a size of approximately 35 kDa. It has a predicted extracellular LRR domain structure and single transmembrane topology similar to other members of the LRRC family. The protein contains six LRR units in the middle with two cysteine-rich regions called LRRNT and LRRCT flanking on the N- and C-terminal sides, respectively . Functionally, LRRC38 modifies the BK channel's voltage dependence of activation toward more negative voltages, producing a modest but reproducible shift of V₁/₂ by -19 ± 1 mV in the absence of intracellular calcium . Due to this modulatory effect on BK channels, LRRC38 has been designated as a member of the BK channel γ-subunit family, specifically as γ4 .

To study LRRC38 structure-function relationships, researchers should consider using site-directed mutagenesis of key residues within the LRR domain, followed by patch-clamp recordings to assess functional changes in BK channel modulation. Structural studies may incorporate techniques such as X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure and interaction interface with BK channels.

What is the tissue expression profile of LRRC38?

LRRC38 exhibits a distinct tissue-specific expression pattern, which is crucial for understanding its physiological roles. Quantitative real-time PCR (qPCR) analysis has revealed that LRRC38 is mainly expressed in skeletal muscle, adrenal gland, and thymus . Low-level expression has also been detected in brain and cerebellum . This expression profile differs from other LRR family members, such as LRRC26 (highly expressed in salivary gland, prostate, and trachea) and LRRC55 (specific to the nervous system) .

For researchers aiming to characterize LRRC38 expression, qPCR methodology should employ specific primers such as those previously reported (though specific LRRC38 primer sequences are not provided in the search results). Sample preparation should include RNA extraction using commercial kits (e.g., TaKaRa MiniBEST Universal RNA Extraction Kit), followed by cDNA generation and real-time PCR with appropriate internal controls such as GAPDH . Western blot analysis can provide protein-level verification using specific antibodies against LRRC38, such as those available from commercial sources .

How does LRRC38 influence BK channel electrophysiological properties?

LRRC38 modifies BK channel gating by shifting the voltage dependence of activation toward more negative voltages by approximately -19 ± 1 mV in the virtual absence of intracellular calcium . This effect, while smaller than that of other LRRC family members (LRRC26, LRRC52, and LRRC55), is still significant and reproducible . The functional consequence of this modulation is that BK channels can activate at less depolarized membrane potentials in the presence of LRRC38.

To study this modulatory effect, researchers should employ patch-clamp electrophysiology techniques, preferably using the whole-cell configuration to record macroscopic currents. Expression systems such as HEK-293 cells are suitable for co-expression of BK α-subunits with LRRC38 . A cotranslational expression method using fusion constructs can ensure reproducible measurement of the electrophysiological properties . Voltage protocols should examine a wide range of membrane potentials (typically -100 mV to +300 mV) and various intracellular calcium concentrations to fully characterize the influence of LRRC38 on BK channel gating parameters.

What experimental approaches are available for manipulating LRRC38 expression?

Several experimental approaches are available for both overexpression and knockout of LRRC38. For overexpression studies, cotranslational expression methods using fusion constructs have been successfully employed to ensure reliable expression and measurement of LRRC38 effects . This approach involves creating fusion constructs where the BK α-subunit is linked to LRRC38, followed by cleavage at an internal signal peptide site.

For gene silencing or knockout studies, CRISPR/Cas9 technology provides a powerful approach. CRISPR/Cas9 KO Plasmids specific for LRRC38 are commercially available (e.g., sc-433936 for mouse LRRC38) . These plasmids contain guide RNA (gRNA) sequences that target specific regions of the LRRC38 gene for cleavage and subsequent knockout . When implementing CRISPR/Cas9 knockout, researchers should include appropriate controls and validation methods such as sequencing, Western blotting, or functional assays to confirm successful gene editing.

What are the molecular mechanisms underlying LRRC38 modulation of BK channels?

The molecular mechanisms by which LRRC38 modulates BK channels involve complex protein-protein interactions that affect channel gating. The LRR domain in LRRC38 likely provides a structural framework for protein-protein interactions, typically through the concave β-sheet side . Unlike other BK channel auxiliary subunits (β-subunits), LRRC38 and other γ-subunits have a distinct structure and modulatory mechanism .

To investigate these molecular interactions, researchers should consider employing co-immunoprecipitation studies to confirm physical association between LRRC38 and BK α-subunits. Mutational analyses targeting specific residues in both LRRC38 and BK channels can help identify crucial interaction sites. Advanced techniques such as FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) can be used to study the dynamics of these interactions in living cells.

For detailed mechanistic studies, researchers might apply single-channel patch-clamp recordings to determine whether LRRC38 affects open probability, conductance, or kinetic properties of BK channels. Comparison with allosteric gating models can provide insights into how LRRC38 influences the energy landscape of channel gating.

How can researchers optimize the production of functional recombinant LRRC38?

Producing functional recombinant LRRC38 presents several challenges due to its transmembrane nature and need for proper folding of the LRR domain. Successful production strategies typically involve mammalian expression systems rather than bacterial systems to ensure appropriate post-translational modifications and protein folding.

For recombinant expression, researchers should consider using HEK-293 cells or similar mammalian cell lines with appropriate expression vectors containing strong promoters (e.g., CMV) . The inclusion of N-terminal signal sequences is critical, as these function as fully cleavable internal signal peptides . When designing expression constructs, careful consideration should be given to the inclusion of epitope tags (e.g., His, FLAG, or HA tags) for purification and detection purposes, preferably positioned to avoid interference with protein function.

Validation of recombinant LRRC38 functionality should include electrophysiological studies to confirm its ability to modulate BK channels, comparing the magnitude of voltage shift with previously reported values (-19 ± 1 mV) . Western blot analysis using specific antibodies can confirm proper expression and processing of the recombinant protein .

What approaches are most effective for studying LRRC38 in native tissues versus heterologous expression systems?

Studying LRRC38 in native tissues versus heterologous expression systems requires different methodological approaches, each with distinct advantages and limitations. In native tissues where LRRC38 is endogenously expressed (skeletal muscle, adrenal gland, and thymus) , researchers must contend with the complexity of multiple channel types and regulatory mechanisms.

For native tissue studies, researchers should first confirm LRRC38 expression using qPCR and immunohistochemistry or Western blotting . Functional studies can employ patch-clamp electrophysiology on isolated cells or tissue slices, combined with pharmacological tools to isolate BK currents (e.g., paxilline or iberiotoxin as BK channel blockers). Comparison between tissues with different LRRC38 expression levels can provide insights into its physiological role.

In heterologous expression systems, such as HEK-293 cells, researchers have greater control over protein expression levels and can study LRRC38 in isolation or in combination with specific BK channel variants . The cotranslational expression method using fusion constructs of BKα and LRRC38 has proven effective for ensuring reliable expression and functional assessment . This approach allows for systematic mutagenesis studies and detailed biophysical characterization without the confounding variables present in native tissues.

How does LRRC38 compare functionally with other members of the LRRC family that modulate BK channels?

LRRC38 belongs to a family of BK channel γ-subunits that includes LRRC26, LRRC52, and LRRC55, all of which modulate BK channel gating but with varying degrees of effect . Among these, LRRC38 (designated as γ4) produces the smallest shift in voltage dependence (-19 ± 1 mV), while LRRC26 (γ1) produces the largest (-140 mV), followed by LRRC52 (γ2, -101 ± 4 mV) and LRRC55 (γ3, -51 ± 2 mV) .

To compare these subunits effectively, researchers should employ consistent experimental conditions when expressing each subunit with BK channels. Patch-clamp electrophysiology should examine a wide range of voltage and calcium conditions, as the modulatory effects of some γ-subunits (e.g., LRRC52 and LRRC55) show calcium dependence . Constructing voltage-activation curves (G-V curves) and measuring V₁/₂ values across different calcium concentrations can provide comprehensive functional comparisons.

Additionally, researchers should consider the possibility of co-expression of multiple γ-subunits in native tissues. Co-immunoprecipitation and immunohistochemistry studies can help determine whether different γ-subunits associate with BK channels in the same cells or whether they show mutually exclusive expression patterns.

What is known about LRRC38's potential role in disease contexts?

While the search results don't specifically address LRRC38's role in disease contexts, other LRRC family members have been implicated in various pathological conditions. For example, LRRC3B has been associated with cancer progression and immune responses in non-small cell lung cancer and breast cancer .

To investigate potential disease associations of LRRC38, researchers should consider analyzing changes in LRRC38 expression in disease states affecting tissues where it is normally expressed, such as muscle disorders, adrenal pathologies, or immune dysfunction. This can involve comparing LRRC38 expression levels between healthy and diseased tissues using qPCR, Western blotting, or immunohistochemistry .

Functional studies can examine whether altered LRRC38 expression affects BK channel activity in disease models, potentially contributing to cellular dysfunction. CRISPR/Cas9-mediated knockout of LRRC38 in relevant cell lines or animal models can help elucidate its physiological and pathophysiological roles . Researchers might also explore potential genetic associations by examining LRRC38 polymorphisms or mutations in patient populations with relevant disorders.

What are the best practices for validating LRRC38 antibodies for research applications?

Validating antibodies for LRRC38 research is crucial for obtaining reliable results. A comprehensive validation approach should include multiple complementary techniques to confirm antibody specificity and sensitivity.

First, researchers should test the antibody in Western blot analysis using both overexpression systems (positive control) and LRRC38 knockout samples (negative control) . Expected molecular weight for LRRC38 is approximately 35 kDa . The use of appropriate loading controls and blocking reagents (e.g., Quickblock blocking buffer) is essential for reducing background and non-specific binding .

Immunocytochemistry or immunohistochemistry validation should examine staining patterns in tissues known to express LRRC38 (skeletal muscle, adrenal gland, thymus) versus tissues with minimal expression . Antibody specificity can be further confirmed using siRNA knockdown or CRISPR/Cas9 knockout of LRRC38, which should result in reduced or absent staining .

For optimal results in Western blotting, researchers should follow protocols similar to those described in the literature, which typically involve protein extraction using RIPA buffer with protease inhibitors, SDS-PAGE separation, transfer to PVDF membranes, and detection using enhanced chemiluminescence substrates .

How can researchers effectively design CRISPR/Cas9 experiments for LRRC38 functional studies?

Designing effective CRISPR/Cas9 experiments for LRRC38 functional studies requires careful consideration of target sites, delivery methods, and validation strategies. Commercial CRISPR/Cas9 KO plasmids for LRRC38 are available (e.g., sc-433936 for mouse LRRC38) and utilize guide RNA sequences derived from the Genome-scale CRISPR Knock-Out (GeCKO) v2 library .

When designing custom CRISPR/Cas9 systems for LRRC38, researchers should select guide RNA sequences targeting early exons or critical functional domains to maximize the likelihood of generating loss-of-function mutations. Multiple guide RNAs should be designed and tested to identify the most efficient targeting strategy. For validation, researchers should sequence the targeted region to confirm mutations and assess LRRC38 expression at both mRNA (using qPCR) and protein (using Western blot) levels .

Functional validation is essential and should include electrophysiological studies to confirm the loss of LRRC38-mediated modulation of BK channels. This typically involves patch-clamp recordings comparing BK channel properties in wild-type versus LRRC38 knockout cells, with expected results showing a positive shift in voltage dependence of activation in knockout cells .

What electrophysiological protocols are optimal for characterizing LRRC38 effects on BK channels?

Optimal electrophysiological protocols for characterizing LRRC38 effects on BK channels should be designed to comprehensively assess voltage and calcium dependence of channel activation. Whole-cell patch-clamp recordings in expression systems such as HEK-293 cells provide the most reliable approach .

For voltage protocols, researchers should apply voltage steps from holding potentials of -80 to -100 mV to test potentials ranging from -100 to +300 mV, with increments of 10-20 mV. Duration of voltage steps should be sufficient to reach steady-state activation (typically 50-200 ms). To assess calcium dependence, experiments should be performed with various intracellular calcium concentrations, ranging from virtually calcium-free (<10 nM) to saturating levels (>100 μM) .

Data analysis should include construction of conductance-voltage (G-V) curves and determination of voltage of half-maximal activation (V₁/₂) and slope factor (k). Comparison of these parameters between BK channels expressed alone and with LRRC38 will quantify the modulatory effect, with expected results showing a negative shift in V₁/₂ by approximately -19 ± 1 mV in the absence of calcium . Additional protocols might include measuring activation and deactivation kinetics and examining potential effects on single-channel properties using inside-out patch recordings.

How can researchers investigate potential interactions between LRRC38 and other ion channel regulatory proteins?

Investigating interactions between LRRC38 and other ion channel regulatory proteins requires a multifaceted approach combining biochemical, functional, and imaging techniques. Co-immunoprecipitation (co-IP) experiments can provide direct evidence of physical interactions between LRRC38 and potential binding partners .

For co-IP studies, researchers should generate lysates from cells expressing tagged versions of LRRC38 and the protein of interest, followed by immunoprecipitation with antibodies against the tag or the protein directly. Western blot analysis can then detect co-precipitated proteins . Controls should include immunoprecipitation with non-specific IgG and samples lacking expression of one of the proteins.

Functional studies using patch-clamp electrophysiology can assess whether co-expression of LRRC38 with other regulatory proteins produces additive, synergistic, or antagonistic effects on BK channel properties . This approach can reveal functional interactions even in the absence of direct physical binding. Advanced imaging techniques such as FRET or BRET can visualize protein-protein interactions in living cells, providing insights into the dynamics and subcellular localization of these interactions.

What are the emerging technologies that could advance LRRC38 research?

Several emerging technologies hold promise for advancing LRRC38 research. Cryo-electron microscopy (cryo-EM) could reveal the structural basis of LRRC38 interaction with BK channels at near-atomic resolution, providing insights into the mechanism of channel modulation. Recent advances in cryo-EM have enabled visualization of membrane protein complexes in unprecedented detail.

CRISPR-based technologies beyond gene knockout, such as CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), offer opportunities for precise modulation of LRRC38 expression . These approaches could help elucidate the consequences of varying LRRC38 expression levels in different cellular contexts.

Single-cell transcriptomics and proteomics could provide comprehensive data on LRRC38 expression patterns at unprecedented resolution, potentially revealing cell-type-specific expression profiles not detectable in bulk tissue analyses . This approach could identify novel cellular contexts where LRRC38 plays important physiological roles.

Optogenetic and chemogenetic tools could be developed to enable precise temporal control of LRRC38 function, allowing researchers to investigate the acute consequences of LRRC38 modulation in complex physiological systems. Such tools would be particularly valuable for studying LRRC38's role in excitable tissues like skeletal muscle and adrenal gland .

What are the knowledge gaps in understanding LRRC38's physiological roles?

Despite the characterization of LRRC38's molecular properties and expression patterns, significant knowledge gaps remain regarding its physiological roles. The functional significance of LRRC38 expression in skeletal muscle, adrenal gland, and thymus remains largely unexplored . Future research should investigate how LRRC38-mediated modulation of BK channels influences muscle contractility, hormone secretion, and immune function in these tissues.

The regulatory mechanisms controlling LRRC38 expression under different physiological and pathological conditions are poorly understood. Studies examining transcriptional, post-transcriptional, and post-translational regulation of LRRC38 could provide insights into how its expression and function are dynamically regulated.

The potential interplay between LRRC38 and other ion channel regulatory proteins in native tissues remains to be elucidated. Given the modest effect of LRRC38 on BK channel gating compared to other γ-subunits, it may function cooperatively with other regulatory mechanisms to fine-tune channel activity in specific cellular contexts .

The evolutionary conservation and divergence of LRRC38 function across species could provide insights into its fundamental biological roles. Comparative studies examining LRRC38 orthologs in different species could reveal evolutionarily conserved functions and species-specific adaptations.