Recombinant Human Metal transporter CNNM4 (CNNM4)

What is CNNM4 and what is its primary function in cellular physiology?

CNNM4 (Cyclin and CBS domain divalent metal cation transport mediator 4) is a membrane protein that primarily functions as a magnesium (Mg2+) efflux transporter. It operates as a Na+/Mg2+ exchanger, stimulating Mg2+ efflux from cells to maintain proper intracellular magnesium homeostasis . The protein contains several evolutionarily conserved domains including the DUF21 transmembrane domain, a Bateman module (containing two CBS motifs), and a cyclic nucleotide binding-like domain (cNMP) . CNNM4 was the first identified member of the CNNM family with conclusively demonstrated Mg2+-transporting capabilities .

What is the structural organization of the CNNM4 protein?

CNNM4 has a modular architecture consisting of:

• A transmembrane DUF21 domain

• An H0 helix connecting the transmembrane domain to the intracellular region

• A Bateman module (containing two CBS motifs)

• A linker connecting the Bateman module to the cNMP domain

• A cyclic nucleotide binding-like domain (cNMP)

• A C-terminal tail

How do CNNM4 expression levels vary across different tissues?

CNNM4 is abundantly expressed in the brain and intestinal tract . Expression has also been documented in adipose tissue, where it plays a role in thermogenesis and macrophage polarization . Research has shown altered expression of CNNM4 in various disease states, including decreased expression in subcutaneous white adipose tissue (scWAT) from obese individuals and increased expression in certain cancer types, including ovarian cancer . Experimental approaches to assess CNNM4 expression typically include qRT-PCR, Western blot analysis, and immunohistochemistry, which have been used to compare expression levels between normal and diseased tissues .

What is the biochemical mechanism by which the PRL-CNNM4 interaction regulates magnesium transport and tumor progression?

The interaction between CNNM4 and phosphatases of regenerating liver (PRL) represents a critical mechanism regulating intracellular Mg2+ levels with significant implications for tumor progression.

Biochemical Mechanism:

• PRLs (PRL-1, PRL-2, and PRL-3) physically interact with CNNM4 in a redox-sensitive manner

• This interaction occurs through the evolutionarily conserved DUF21 and CBS domains of CNNM4

• PRL binding to CNNM4 inhibits its Mg2+ efflux function, leading to increased intracellular Mg2+ concentration

• Elevated intracellular Mg2+ levels affect energy metabolism through AMPK/mTOR signaling

• This metabolic reprogramming promotes tumor cell proliferation and progression

Experimental Evidence:
IP experiments confirmed that all PRL family proteins (PRL-1, PRL-2, PRL-3) interact with CNNM4, and Mg2+ imaging analyses with Magnesium Green demonstrated that PRL expression suppresses CNNM4-mediated Mg2+ efflux . Importantly, tumor formation studies showed that only cells expressing PRL-3-WT (wild type), but not PRL-3 mutants that cannot bind CNNM4, demonstrated increased tumor nodules, establishing a direct correlation between the ability to bind CNNM4 and promotion of tumor formation .

For researchers studying this interaction, methodological approaches include co-immunoprecipitation assays with specific antibodies against CNNM4 and PRL proteins, Mg2+ efflux measurement using fluorescent indicators like Magnesium Green, and in vivo tumorigenesis analysis with wild-type and mutant proteins to establish functional relationships.

How does the structure of CNNM4's cNMP domain differ from canonical cyclic nucleotide binding domains, and what are the functional implications?

Despite its name suggesting cyclic nucleotide binding capabilities, the CNNM4 cNMP domain exhibits critical structural differences from canonical cyclic nucleotide binding domains (CNBD):

Structural Differences:

• Unusually long loop (approximately 50 amino acids longer) connecting strands β6 and β7 in the phosphate binding cassette (PBC)

• Abrupt turn of the polypeptide chain at residues 601-603 that distorts the canonical strand β2 and redirects the Y603 side chain toward the interior of the cavity, blocking space needed to accommodate cyclic nucleotides

• Absence of a conserved buried arginine to interact with the exocyclic phosphate of cAMP/cGMP

• Absence of a glutamate to fix the orientation of ribose 2"-OH

Functional Implications:

• The cNMP domain cannot bind or be regulated by cyclic nucleotides (cAMP or cGMP) despite its structural similarity to CNBDs

• Instead, it forms compact homodimers that contribute significantly to the dimerization of the full-length protein

• The cNMP domain dimerization defines morphological limits for the twisted-to-flat conformational change triggered within the Bateman module by MgATP binding

• Removal of the cNMP domain inhibits Mg2+ transport function

For researchers investigating this domain, methodologies such as X-ray crystallography (as used to obtain the 3.7 Å resolution structure), isothermal titration calorimetry (ITC), NMR titration experiments, and small angle X-ray scattering (SAXS) are appropriate to characterize structural features and binding properties.

What are the most effective experimental approaches for studying CNNM4-mediated magnesium transport in different cell types?

Studying CNNM4-mediated magnesium transport requires a combination of specialized techniques:

Magnesium Imaging:

• Fluorescent indicators like Magnesium Green for real-time monitoring of intracellular Mg2+ changes

• Protocol: Cells transfected with CNNM4 constructs are loaded with Mg2+, followed by exposure to Mg2+-free solution to artificially stimulate Mg2+ efflux

Genetic Manipulation:

• Stable expression of CNNM4 wild-type and mutant constructs via retroviral or lentiviral systems

• CRISPR/Cas9-mediated knockout models (tissue-specific conditional knockouts have been generated, e.g., CNNM4 Adipoq-cKO mice)

• RNA interference using shRNA for CNNM4 knockdown

Biochemical Approaches:

• Measurement of Mg2+ levels in subcellular compartments, interstitial fluid, and plasma

• Detection of magnesium-dependent signaling pathways (e.g., AMPK/mTOR)

In Vivo Models:

• Tissue-specific conditional knockout models (e.g., CNNM4 Adipoq-cKO, CNNM4 Fabp4-cKO)

• Adeno-associated virus (AAV)-mediated overexpression of CNNM4 in specific tissues

• Local administration of Mg2+ solutions to mimic CNNM4 function

When designing experiments, researchers should consider cell type-specific differences in CNNM4 expression and function, as well as potential compensatory mechanisms by other magnesium transporters.

How do CNNM4 expression patterns correlate with clinical outcomes in ovarian cancer patients?

CNNM4 expression in ovarian cancer (OV) shows significant correlations with patient outcomes:

Expression Pattern Analysis:

• CNNM4 is significantly upregulated in OV tissues compared to normal ovarian tissues

• Higher expression is associated with advanced histological grades

Survival Analysis:
The following table shows the distribution of clinical characteristics based on CNNM4 expression levels in ovarian cancer patients:

Clinical Correlations:

Methodologically, researchers can assess these correlations through integrated analysis of TCGA and GTEx data, construction of survival curves using Kaplan-Meier methods, and use of Cox regression models to identify independent prognostic factors.

What is the role of CNNM4 in thermogenesis and M2 macrophage polarization in adipose tissue?

CNNM4 plays a critical role in adipose tissue metabolism and immune function:

Mechanistic Findings:

• CNNM4 in adipocytes promotes M2 macrophage polarization in adipose tissue

• CNNM4-dependent Mg2+ efflux from adipocytes appears to be essential for this process

• CNNM4 Adipoq-cKO mice (with adipocyte-specific CNNM4 deletion) show:

  • Decreased M2 macrophage polarization in subcutaneous white adipose tissue (scWAT) and brown adipose tissue (BAT)

  • Increased adiposity and decreased UCP1 protein levels

  • Reduced thermogenic gene expression in scWAT

  • Lower oxygen consumption and energy expenditure under cold exposure

  • Impaired insulin sensitivity and glucose homeostasis

Clinical Relevance:

• CNNM4 expression is decreased in scWAT from obese mice on high-fat diet (HFD) and obese humans

• Mg2+ levels are decreased in the interstitial fluid of scWAT from HFD-fed mice

• CNNM4 expression in human scWAT negatively correlates with BMI and blood glucose levels

• CNNM4 expression positively correlates with M2 macrophage markers (CD301, MRC1, ARG1, IL10) and negatively with M1 macrophage markers (RANTES, MCP1, TNFα)

Therapeutic Potential:

• AAV-mediated CNNM4 overexpression in scWAT ameliorates HFD-induced obesity

• Local MgCl2 administration in scWAT promotes M2 macrophage polarization, increases thermogenic gene expression, and improves metabolic parameters

For researchers studying this aspect of CNNM4 function, methodologies include generation of tissue-specific knockout models, assessment of macrophage polarization through flow cytometry and gene expression analysis, measurement of thermogenic capacity through indirect calorimetry, and evaluation of metabolic parameters through glucose tolerance and insulin sensitivity tests.

What are the implications of CNNM4 mutations in genetic disorders, and how can functional studies help elucidate pathogenic mechanisms?

CNNM4 mutations have been primarily associated with Jalili syndrome, a rare multisystem disorder characterized by:

Clinical Features:

• Cone-rod dystrophy with bull's eye maculopathy

• Photophobia and nystagmus

• Amelogenesis imperfecta (defective tooth enamel development)

Genetic Findings:

• Several mutations have been identified, including a novel homozygous CNNM4 p.Arg236Trp variant found in patients of Guatemalan ancestry

• Evidence suggests a founder effect for some mutations, though diverse alleles exist within populations

Phenotypic Variability:

• Even patients with identical genotypes (e.g., homozygous p.Arg236Trp) show phenotypic differences

• One patient had disease mostly limited to the posterior pole, while another had significant peripheral sector retinal atrophy and pigment deposition

• This suggests factors beyond genotype influence disease severity

Research Approaches:

• Genetic Testing: Inherited retinal dystrophy panels or whole exome/genome sequencing

• Variant Classification: Using ACMG criteria to evaluate pathogenicity

• Functional Studies:

  • Expression studies of wild-type and mutant CNNM4 proteins

  • Mg2+ transport assays to assess functional consequences of mutations

  • Animal models to recapitulate disease phenotypes

• Structure-Function Analysis: Mapping mutations onto the known structural domains of CNNM4 to predict impact

Additional research implicates CNNM4 in male fertility, as CNNM4-deficient mice commonly exhibit male sterility , suggesting broader reproductive implications of CNNM4 mutations that warrant further investigation.

What are the optimal approaches for recombinant expression and purification of CNNM4 protein for structural studies?

Based on successful structural studies of CNNM4, the following approaches are recommended:

Domain Selection Strategy:

• "Divide and conquer" approach using individual domains is more successful than full-length protein

• Researchers have successfully expressed and crystallized:

  • CNNM4BAT (residues 359-511): Bateman module with preceding α-helix H0

  • CNNM4cNMP (residues 545-730): cNMP domain

  • Larger constructs (CNNM4BAT-cNMP-Ctail, residues 356-775) for solution studies

Expression Systems:

• E. coli for individual domains (BL21(DE3) or similar strains)

• Mammalian expression systems (HEK293, COS7) for functional studies of full-length protein

Purification Protocol:

• Affinity chromatography (His-tag, GST-tag)

• Size exclusion chromatography for final purity and to assess oligomeric state

• For crystallization, removal of flexible regions (e.g., the C-terminal tail following the cNMP domain)

Critical Considerations:

• Large unstructured stretches can impede crystallization:

  • Linker connecting domains (residues 512-544)

  • C-terminal segment (residues 731-775)

  • Long loop in CBS2 motif (residues 480-486)

  • Internal loop in cNMP domain (residues 639-699)

• The cNMP domain contains an unusually long loop (~50 aa) that is typically disordered in crystal structures

Biophysical Characterization:

• SEC-MALS (size exclusion chromatography with multi-angle light scattering) to determine oligomeric state

• SAXS (small angle X-ray scattering) for low-resolution structural information

• NMR for ligand binding studies

This methodological approach has yielded crystal structures at resolutions of up to 3.7 Å for the CNNM4 cNMP domain.

How can CNNM4-dependent magnesium homeostasis be targeted therapeutically, and what assays are most appropriate for drug screening?

The emerging role of CNNM4 in multiple diseases suggests potential therapeutic approaches:

Therapeutic Strategies:

• Modulation of CNNM4 Expression:

  • AAV-mediated overexpression of CNNM4 in specific tissues (demonstrated in scWAT for obesity)

  • siRNA/shRNA approaches for conditions where CNNM4 is overexpressed (e.g., certain cancers)

• Targeting CNNM4-PRL Interaction:

  • Small molecules disrupting the protein-protein interaction could restore CNNM4-mediated Mg2+ efflux in cancers

  • The crystal structure of the CNNM4-PRL complex provides a basis for structure-based drug design

• Direct Modulation of CNNM4 Activity:

  • Compounds enhancing or inhibiting Mg2+ transport

  • Modulators of conformational changes in the Bateman module

• Local Magnesium Supplementation:

  • Direct MgCl2 administration has shown efficacy in obesity models

Drug Screening Assays:

• Primary Screens:

  • Fluorescence-based Mg2+ efflux assays using Magnesium Green in CNNM4-expressing cells

  • FRET-based assays for CNNM4-PRL interaction

  • Cell proliferation assays in CNNM4/PRL-dependent cancer cells

• Secondary Validation:

  • Mg2+ measurements in subcellular compartments and interstitial fluid

  • Effects on AMPK/mTOR signaling pathway components

  • Thermogenic gene expression in adipocytes

  • M2 macrophage polarization assays

• In Vivo Models:

  • Tumor xenograft models for cancer applications

  • Diet-induced obesity models for metabolic applications

  • CNNM4 knockout/transgenic models for targeted validation

Each therapeutic approach requires careful consideration of tissue specificity and potential effects on magnesium homeostasis in other tissues, as CNNM4 plays diverse roles throughout the body.

What are the best experimental systems for studying the crosstalk between CNNM4 and cellular energy metabolism?

CNNM4's role in regulating energy metabolism can be investigated using:

Cellular Models:

• Cancer Cell Lines:

  • SW480, HEK293, B16, and CHO cells have been successfully used

  • Stable cell lines expressing wild-type or mutant CNNM4 and/or PRL proteins

• Adipocyte Models:

  • Primary beige adipocytes from WT and CNNM4 Adipoq-cKO mice

  • 3T3-L1 cells with CNNM4 manipulation

• Macrophage Systems:

  • Bone marrow-derived macrophages (BMDMs) exposed to conditioned medium from CNNM4-expressing cells

  • Co-culture systems of adipocytes and macrophages

Key Metabolic Pathways to Assess:

• AMPK/mTOR Signaling:

  • Western blotting for phosphorylated AMPK, mTOR, S6K, 4E-BP1

  • Effect of rapamycin (mTOR inhibitor) on CNNM4/PRL-expressing cells

• Energy Metabolism:

  • Seahorse XF analysis for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Measurements of ATP production and energy charge

• Thermogenic Pathway:

  • Expression of UCP1 and other thermogenic genes

  • Mitochondrial content and morphology

  • Indirect calorimetry for whole-body energy expenditure

In Vivo Models:

• Tissue-Specific Knockout Models:

  • CNNM4 Adipoq-cKO (adipocyte-specific)

  • CNNM4 Fabp4-cKO (adipose tissue)

  • Liver-specific models for NAFLD studies

• Metabolic Challenge Paradigms:

  • Cold exposure for thermogenesis assessment

  • High-fat diet for obesity studies

  • Glucose and insulin tolerance tests

• Advanced Techniques:

  • Metabolic cages for comprehensive assessment of energy balance

  • Stable isotope tracers to track specific metabolic pathways

  • In vivo imaging of tissue-specific metabolism

These experimental systems allow for comprehensive analysis of how CNNM4-mediated magnesium transport influences cellular and whole-body energy metabolism under physiological and pathological conditions.

What is the potential role of CNNM4 in non-alcoholic fatty liver disease (NAFLD) and how might it be therapeutically targeted?

Recent research has identified CNNM4 as a key regulator of magnesium in the liver and a potential therapeutic target for non-alcoholic fatty liver disease (NAFLD) :

Pathophysiological Context:

• Non-alcoholic steatohepatitis (NASH), a form of fatty liver disease with inflammation and fibrosis, affects approximately 1.7 billion people worldwide

• Magnesium deficiency is prevalent, with 79% of U.S. adults not meeting recommended intake

• CNNM4 facilitates magnesium transport out of the liver and influences Mg2+ homeostasis

Research Findings:

• Higher expression of CNNM4 protein has been observed in both patients with non-alcoholic steatohepatitis and mouse models of the disease

• CNNM4 is responsible for magnesium imbalance that contributes to liver disease development

Experimental Approaches:

• Expression Analysis:

  • Quantification of CNNM4 expression in liver biopsies from NAFLD/NASH patients versus controls

  • Correlation of expression with disease severity markers

• Functional Studies:

  • Liver-specific CNNM4 knockout or overexpression models

  • Assessment of hepatic lipid accumulation, inflammation, and fibrosis

  • Measurement of liver and serum magnesium levels

• Therapeutic Strategies:

  • CNNM4 inhibition to prevent excessive magnesium efflux from hepatocytes

  • Magnesium supplementation to counteract efflux effects

  • Combination approaches targeting both CNNM4 and dietary magnesium

This emerging field requires further research to elucidate the precise mechanisms by which CNNM4-mediated magnesium transport influences NAFLD pathogenesis and to develop effective therapeutic interventions.

How does CNNM4 expression in single-cell populations contribute to tissue heterogeneity and function in normal and disease states?

Single-cell analysis of CNNM4 expression reveals important insights into tissue heterogeneity:

Single-Cell Expression Patterns:

• Analysis of dataset GSE184880 using the Seurat package identified 18 cell subtypes that were further categorized into nine groups: T cells, NK cells, monocytes, B cells, epithelial cells, fibroblasts, tissue stem cells, endothelial cells, and smooth muscle cells

• CNNM4 expression varies across these cell types, suggesting cell type-specific functions

Research Approaches:

• Single-Cell RNA Sequencing:

  • Identification of cell clusters using tSNE or UMAP algorithms

  • Annotation of cell types using reference databases (e.g., celldex package)

  • Analysis of CNNM4 expression across identified cell populations

• Spatial Transcriptomics:

  • Mapping CNNM4 expression in spatial context within tissues

  • Correlation with tissue microenvironments and functional zones

• Functional Validation:

  • Cell type-specific CNNM4 knockout models

  • Single-cell western blotting or CyTOF for protein-level validation

  • Functional assays in isolated cell populations

Disease Relevance:

• In cancer: Analysis of tumor heterogeneity and identification of CNNM4-expressing cells within the tumor microenvironment

• In metabolic diseases: Characterization of CNNM4 expression in adipose tissue macrophage populations and their polarization states

• In genetic disorders: Investigation of cell type-specific consequences of CNNM4 mutations

This single-cell approach provides a more nuanced understanding of CNNM4 function beyond bulk tissue analysis and may reveal cell type-specific therapeutic targets in various disease states.

What are the cooperative or antagonistic relationships between CNNM4 and other magnesium transporters in different cellular contexts?

Understanding the interplay between CNNM4 and other magnesium transporters is critical for comprehending cellular magnesium homeostasis:

Key Magnesium Transport Systems:

• CNNM Family (CNNM1-4): Function primarily in Mg2+ efflux

• TRPM Channels (TRPM6, TRPM7): Involved in Mg2+ influx

• MagT Family: Mediates Mg2+ influx

• Mrs2: Mitochondrial Mg2+ transporter

• SLC41 Family: Na+/Mg2+ exchangers

Research Approaches:

• Co-expression Analysis:

  • Quantification of multiple transporters in the same tissue/cell type

  • Correlation of expression patterns across tissues and disease states

  • Single-cell analysis to identify co-expressing cells

• Functional Interaction Studies:

  • Sequential or simultaneous knockdown/overexpression of multiple transporters

  • Mg2+ flux measurements under various conditions

  • Compensation mechanisms after acute or chronic loss of specific transporters

• Subcellular Localization:

  • Co-localization studies using confocal microscopy

  • Analysis of transporter distribution in polarized cells

  • Investigation of transporter redistribution upon cellular stimulation

• Protein-Protein Interactions:

  • Co-immunoprecipitation of CNNM4 with other transporters

  • Proximity ligation assays to detect close associations

  • FRET/BRET approaches for real-time interaction monitoring

Physiological and Pathological Contexts:

• Cancer: How do changes in multiple Mg2+ transporters contribute to altered magnesium homeostasis in tumor cells?

• Metabolic Disorders: Is there coordinated regulation of Mg2+ transporters in response to metabolic challenges?

• Genetic Compensation: Do other transporters compensate for CNNM4 mutations in Jalili syndrome patients?

This comprehensive approach will provide a systems-level understanding of magnesium homeostasis and identify potential points for therapeutic intervention across various pathological conditions.

How can researchers overcome the challenges of studying the transmembrane domains of CNNM4?

The transmembrane DUF21 domain of CNNM4 presents significant technical challenges for structural and functional studies:

Major Challenges:

• Protein Expression and Purification:

  • Hydrophobic nature complicates heterologous expression

  • Detergent requirements for extraction and stability

  • Potential toxicity to expression hosts

• Structural Determination:

  • Difficulty in obtaining well-diffracting crystals

  • Dynamic nature of transmembrane regions

  • Limited success with conventional crystallography

• Functional Assays:

  • Complexity of reconstituting Mg2+ transport in artificial systems

  • Need for appropriate lipid environments

  • Difficulty in isolating DUF21 function from other domains

Methodological Solutions:

• Expression Strategies:

  • Use of specialized expression systems (CFES, insect cells)

  • Fusion with stability-enhancing tags (GFP, MBP)

  • Cell-free expression systems in the presence of nanodiscs or liposomes

• Structural Approaches:

  • Cryo-electron microscopy for full-length protein

  • Integrative structural biology combining multiple techniques

  • Computational modeling validated by experimental constraints

• Functional Reconstitution:

  • Proteoliposome-based transport assays

  • Nanodiscs for stable membrane protein incorporation

  • Giant unilamellar vesicles (GUVs) for single-vesicle transport studies

• Hybrid Approaches:

  • Chimeric proteins with well-characterized transmembrane domains

  • Split-domain complementation assays

  • Cross-linking and mass spectrometry for structural insights