Recombinant Mouse Leucine-rich repeat-containing protein 55 (Lrrc55)

What is Lrrc55 and where is it primarily expressed in mice?

Lrrc55 (Leucine-rich repeat-containing protein 55) is a paralog of Lrrc26 that functions as a γ-subunit of BK channels. Under normal physiological conditions, Lrrc55 shows high expression in neurons with negligible expression in pancreatic islets of non-pregnant mice . In situ hybridization studies have revealed that Lrrc55 mRNA is abundantly expressed in specific brain regions, particularly in the granule cell and molecular cell layers of the cerebellum and throughout the pons .

Interestingly, Lrrc55 expression increases dramatically (>60-fold) in pancreatic islets during pregnancy, with expression levels in pregnant islets significantly exceeding those in the hypothalamus, which normally has relatively high Lrrc55 expression . This upregulation appears to be unique to islets and is not observed in other tissues during pregnancy, suggesting tissue-specific regulatory mechanisms controlling Lrrc55 expression .

How does Lrrc55 expression change during pregnancy and metabolic stress?

Lrrc55 undergoes remarkable expression changes during pregnancy in pancreatic islets. The expression increases by more than 60-fold, peaking around day 15 of gestation in mice, and then declining toward pre-pregnancy levels postpartum . This upregulation is dependent on prolactin receptor (PrlR) signaling, as the increase is blunted in PrlR+/- mice .

Beyond pregnancy, Lrrc55 expression also increases under metabolic stress conditions:

The upregulation in metabolic stress conditions suggests that Lrrc55 may serve as part of an adaptive response to cellular stress in pancreatic β-cells .

What experimental methods are used to detect and manipulate Lrrc55 expression?

Several methodological approaches can be employed to study Lrrc55:

For detection:

• In situ hybridization using DIG-labeled RNA probes: This technique involves synthesizing N-terminal DNA fragments of mouse LRRC55 gene, cloning into vectors (like pBbluescriptSK), and generating antisense RNA probes using T7 transcriptase .

• Quantitative PCR (qPCR): For measuring relative mRNA expression levels in different tissues or under varying conditions.

For manipulation:

• Adenoviral overexpression: Researchers have successfully overexpressed Lrrc55 in mouse islets and INS-1-832/13 cells using adenovirus vectors to study its functional effects .

• siRNA-mediated knockdown: Small interfering RNAs (siRNAs) targeting Lrrc55 can degrade mRNA post-transcription to prevent translation. Ribo-modified siRNAs offer increased stability, improved specificity, and reduced immunogenicity .

For functional analysis:

• TUNEL assays and caspase-3 activation measurements to assess apoptosis in Lrrc55-overexpressing cells.

• Western blotting to evaluate expression of ER stress pathway components.

• Calcium imaging to investigate intracellular calcium homeostasis .

How is Lrrc55 related to BK channels and their function?

Lrrc55 has been identified as a regulatory γ-subunit of large-conductance calcium-activated potassium (BK) channels. Specifically, it has been classified as the γ4 subunit within the LRRC family of BK channel regulatory proteins . When co-expressed with BK α-subunits in heterologous systems, Lrrc55 produces a modest negative shift (approximately -50 mV) in voltage-dependent BK channel activation at low calcium concentrations (0 Ca²⁺), but notably shows no significant shift at elevated calcium levels .

This differs from other family members like Lrrc26 (γ1) and Lrrc52 (γ2), which produce more substantial shifts in BK gating. The functional hierarchy of these subunits based on their ability to shift BK gating leftward is as follows:

BK channels are expressed in both rodent and human β-cells where they participate in membrane repolarization, regulate calcium entry, influence action potential amplitude, and modulate glucose-stimulated insulin secretion .

What is the molecular mechanism by which Lrrc55 protects β-cells from apoptosis?

Lrrc55 exerts protective effects against β-cell apoptosis through multiple interconnected mechanisms:

• Attenuation of ER stress pathways: Overexpression of Lrrc55 leads to downregulation of key ER stress markers induced by glucolipotoxicity or thapsigargin treatment. This includes reduced expression of IRE-1α and CHOP, critical components of the unfolded protein response (UPR) that can trigger apoptosis when chronically activated .

• Modulation of apoptotic signals: Lrrc55 decreases the Bax/Bcl-2 ratio and reduces caspase-9 activation, shifting the balance away from pro-apoptotic signaling toward pro-survival pathways .

• Calcium homeostasis maintenance: Lrrc55 attenuates palmitate-induced depletion of intracellular calcium stores. Optimal calcium concentration in the ER lumen is essential for proper protein folding and chaperone function .

The protective effects are quantifiable in experimental systems:

These findings suggest that Lrrc55 is upregulated during physiological (pregnancy) and pathological (diabetes) states to protect β-cells from stress-induced apoptosis, maintaining functional β-cell mass .

How does Lrrc55 affect insulin biosynthesis and secretion in pancreatic β-cells?

Research indicates that Lrrc55 has nuanced effects on insulin biosynthesis and secretion:

• Insulin content and gene expression: Overexpression of Lrrc55 in β-cells leads to increased insulin content and prevents palmitate-induced reduction in insulin expression. Interestingly, this effect appears independent of major insulin transcription factors, as no significant changes in Pdx-1 or MafA expression were observed .

• Glucose-stimulated insulin secretion (GSIS): Lrrc55 has minimal direct effect on GSIS, suggesting its primary function is not to regulate acute insulin release but rather to maintain β-cell health and insulin production capacity .

• Indirect effects via BK channels: As a γ-subunit of BK channels, Lrrc55 may indirectly modulate calcium dynamics and membrane potential, which are crucial for insulin secretion. BK channels participate in membrane repolarization and regulate calcium entry in β-cells .

The protective effect on insulin biosynthesis may be related to Lrrc55's ability to maintain proper ER function and calcium homeostasis, as these are essential for proper insulin folding and processing. During pregnancy, when insulin demand increases dramatically, upregulation of Lrrc55 may help sustain the enhanced insulin synthesis capacity while protecting β-cells from ER stress-induced dysfunction .

What is the relationship between Lrrc55 expression and diabetic conditions?

Lrrc55 expression demonstrates significant associations with diabetic and pre-diabetic conditions:

• Upregulation in diabetic models: Lrrc55 is upregulated by more than 10-fold in islets of obese db/db mice, a well-established model of metabolic syndrome and diabetes .

• Response to glucolipotoxicity: When mouse islets are exposed to palmitate (a saturated fatty acid) in high glucose conditions (mimicking diabetogenic environment), Lrrc55 expression increases approximately 15-fold within 24 hours .

• ER stress induction: Thapsigargin, a SERCA inhibitor that induces ER stress (a common feature in diabetic β-cells), significantly increases Lrrc55 expression within hours of exposure .

• Genetic models of β-cell dysfunction: Lrrc55 is one of the most upregulated genes in transgenic mice with β-cell-specific deletion of TNF receptor 2 (βTRAF2) or βTRAF3, which are models of glucose intolerance and impaired β-cell function .

These observations suggest that Lrrc55 upregulation represents an adaptive response to cellular stress in β-cells during diabetic conditions. The protein may be part of a compensatory mechanism that attempts to protect β-cells from stress-induced apoptosis when faced with increased metabolic demands and glucolipotoxicity .

How can Lrrc55-targeted interventions be applied in diabetes research?

Based on current understanding of Lrrc55's protective functions in β-cells, several research applications can be considered:

• Therapeutic target exploration: As Lrrc55 protects β-cells from glucolipotoxicity-induced apoptosis, it represents a potential therapeutic target for preventing β-cell failure in diabetes. Research could focus on developing compounds that enhance Lrrc55 expression or activity in β-cells .

• Biomarker development: Changes in Lrrc55 expression might serve as a biomarker for β-cell stress and adaptation in diabetes progression.

• Mechanistic studies of β-cell failure: Using genetic manipulation of Lrrc55 (via siRNA knockdown or adenoviral overexpression ) could help elucidate the molecular mechanisms of β-cell adaptation and failure in diabetes.

• Pregnancy-related diabetes research: Given its dramatic upregulation during pregnancy, Lrrc55 may play a role in gestational diabetes pathophysiology when its normal upregulation is impaired .

• ER stress modulation: The ability of Lrrc55 to attenuate ER stress makes it a useful experimental tool for dissecting the UPR pathways in β-cells under various stressors .

Methodologically, researchers can utilize adenoviral vectors for overexpression studies or Ribo-modified siRNAs for knockdown experiments . For in vivo applications, tissue-specific genetic manipulation using Cre-lox systems could provide insights into Lrrc55's role in specific physiological contexts.

How does Lrrc55 function compare between neuronal and pancreatic islet contexts?

Lrrc55 shows interesting tissue-specific expression patterns and potentially different functional roles between neuronal and pancreatic contexts:

Neuronal expression and function:

• Constitutively expressed in neurons, particularly in the cerebellum (granule cell and molecular cell layers) and pons

• Likely functions as a γ-subunit of neuronal BK channels, modulating their voltage dependence

• May participate in neuronal excitability regulation and action potential shaping

Pancreatic islet expression and function:

• Negligible expression in non-pregnant conditions

• Dramatically upregulated (>60-fold) during pregnancy in a prolactin receptor-dependent manner

• Also upregulated in response to metabolic stress and in diabetic conditions

• Protects β-cells from ER stress and apoptosis

• Helps maintain insulin biosynthesis under stress conditions

The dramatic difference in baseline expression between these tissues suggests tissue-specific regulatory mechanisms controlling Lrrc55 gene expression. The pregnancy-specific upregulation in islets points to hormonal regulation that is absent in neuronal tissue .

From a functional perspective, while Lrrc55 in neurons likely primarily serves to modulate BK channel activity, its role in pancreatic islets appears more complex, involving ER stress regulation and calcium homeostasis beyond simple channel modulation .

What are the critical gaps in current Lrrc55 research?

Despite significant advances in understanding Lrrc55's role in pancreatic islets and neurons, several important knowledge gaps remain:

• Regulatory mechanisms: The precise molecular pathways controlling Lrrc55 upregulation during pregnancy and in response to stress remain incompletely characterized.

• Interaction partners: Beyond BK channels, the full range of Lrrc55's molecular interactions in β-cells is unknown.

• Structure-function relationship: The specific protein domains responsible for Lrrc55's protective effects against ER stress have not been fully mapped.

• Genetic variation: The impact of Lrrc55 genetic variants on β-cell function and diabetes susceptibility has not been explored.

• Translational relevance: While Lrrc55 is upregulated in mouse models of diabetes, its expression patterns in human diabetic islets remain to be characterized .

What experimental approaches should be prioritized for advancing Lrrc55 research?

Future research on Lrrc55 would benefit from several methodological approaches:

• Conditional knockout models: Development of β-cell-specific and inducible Lrrc55 knockout mice would help define its necessity in pregnancy adaptation and stress protection.

• Protein interaction studies: Comprehensive proteomic analysis to identify Lrrc55 binding partners in β-cells beyond BK channels.

• Structure-function analysis: Creation of domain-specific mutants to map regions responsible for ER stress protection versus BK channel modulation.

• Calcium dynamics investigation: Advanced calcium imaging techniques to clarify how Lrrc55 maintains calcium homeostasis in stressed β-cells.

• Translational studies: Analysis of LRRC55 expression and function in human islets under normal, pregnant, and diabetic conditions to validate findings from mouse models .