Recombinant Rat Metal transporter CNNM4 (Cnnm4)

Basic Research Questions

• What is the domain structure of rat CNNM4 and how does it compare to human CNNM4?

  Rat CNNM4 shares significant structural homology with human CNNM4, consisting of four major domains:

  • An extracellular domain

  • A DUF21 transmembrane domain

  • A Bateman module (containing two CBS domains, CBS1 and CBS2)

  • A cyclic nucleotide binding-like domain (cNMP)

  The Bateman module forms two main clefts, S1 and S2, where S2 accommodates nucleotide binding. The cNMP domain, despite its name, cannot bind cyclic nucleotides but plays a crucial role in dimerization . Crystal structure analysis has shown that the Bateman module of CNNM4 forms homodimers that undergo conformational changes from a twisted to a flat disk shape upon MgATP binding .

  Domain	Residues (Human CNNM4)	Function
  Bateman module	359-511	MgATP binding, dimerization
  cNMP domain	545-730	Dimerization, conformational restriction
  DUF21 transmembrane	N-terminal region	Membrane anchoring, ion transport
  C-terminal tail	731-775	Unknown/regulatory

• How does CNNM4 function in magnesium transport at the molecular level?

  CNNM4 functions as a magnesium extrusion mediator through an electroneutral Na⁺/Mg²⁺ exchange mechanism. The protein:

  1. Localizes to the basolateral membrane of epithelial cells

  2. Exchanges intracellular Mg²⁺ with extracellular Na⁺

  3. Requires ATP and Mg²⁺ binding at separate sites in the Bateman module

  Imaging analyses with Magnesium Green reveal that CNNM4-expressing cells show rapid decrease in intracellular Mg²⁺ when transferred from high Mg²⁺ to Mg²⁺-free solution, compared to minimal changes in control cells . The mechanism involves positive cooperativity between ATP and Mg²⁺ binding, as the Bateman module has unusually acidic surface in the S2 nucleotide-binding site, explaining the low nucleotide affinity that increases with Mg²⁺ co-binding for charge compensation .

  Importantly, electrophysiological analyses show that CNNM4 expression induces no significant electronic currents, supporting its role as an electroneutral exchanger rather than an ion channel .

• What phenotypes are observed in CNNM4-knockout mice, and what do they tell us about CNNM4 function?

  CNNM4-knockout mice exhibit two primary phenotypes that provide insight into CNNM4 function:

  1. Hypomagnesemia: Due to intestinal malabsorption of magnesium, confirming CNNM4's essential role in transcellular Mg²⁺ transport across intestinal epithelia .

  2. Defective amelogenesis: Consistent with human Jalili syndrome, where CNNM4 mutations cause amelogenesis imperfecta with cone-rod dystrophy .

  Additionally, CNNM4 Adipoq-cKO mice (adipocyte-specific knockout) show decreased Mg²⁺ levels in interstitial fluid from subcutaneous white adipose tissue (scWAT) during cold exposure and increased body weight gain under high-fat diet conditions .

  These findings collectively establish CNNM4 as crucial for:

  • Intestinal Mg²⁺ absorption

  • Tooth enamel formation

  • Metabolic processes related to adipose tissue function

Experimental Design and Methodology

• What expression systems are optimal for producing functional recombinant rat CNNM4, and what purification strategies yield the highest purity?

  Based on the available commercial products and research methodologies, several expression systems have been used successfully:

  Expression System	Application	Advantages	Challenges
  E. coli	Basic structural studies	Cost-effective, high yield	May lack post-translational modifications
  Yeast	Functional studies	Better folding than E. coli	Moderate yield
  Baculovirus	Functional and structural studies	Preserves most mammalian PTMs	More complex system
  Mammalian cells (HEK293)	Transport studies, interaction assays	Native-like folding and modifications	Lower yield, higher cost
  Cell-free expression	Rapid production for screening	Avoids toxicity issues	Limited post-translational modifications

  For purification, most commercial recombinant rat CNNM4 proteins utilize His-tagging strategies, with purity typically reaching ≥85% as determined by SDS-PAGE . For structural studies requiring higher purity, researchers have employed a "divide and conquer" strategy, creating separate constructs for individual domains:

  • CNNM4ᴮᴬᵀ (residues 359–511)

  • CNNM4ᶜᴺᴹᴾ⁻ᶜᵗᵃⁱˡ (residues 545–775)

  • CNNM4ᶜᴺᴹᴾ (residues 545–730)

  • CNNM4ᴮᴬᵀ⁻ᶜᴺᴹᴾ⁻ᶜᵗᵃⁱˡ (residues 356–775)

  • CNNM4ᴮᴬᵀ⁻ᶜᴺᴹᴾ (residues 356–730)

  This approach facilitates crystallization by eliminating unstructured regions that might impede the process.

• What techniques are most effective for measuring CNNM4-mediated Mg²⁺ transport activities in vitro?

  Several complementary techniques have proven effective for measuring CNNM4-mediated Mg²⁺ transport:

  1. Inductively Coupled Plasma Emission Spectroscopy (ICP-ES)

     • Quantifies changes in intracellular elemental composition

     • Shows increased sodium and decreased magnesium in CNNM4-expressing cells

     • Advantage: Provides absolute quantification of multiple elements simultaneously

  2. Fluorescent Mg²⁺ Indicators (e.g., Magnesium Green)

     • Real-time monitoring of intracellular Mg²⁺ levels

     • Protocol: Preload cells with 40 mM Mg²⁺, then transfer to Mg²⁺-free solution

     • CNNM4-expressing cells show rapid decrease in fluorescence intensity

     • Advantage: Provides temporal dynamics of transport

  3. Interstitial Fluid Mg²⁺ Measurement

     • Direct measurement of Mg²⁺ in tissue interstitial fluid

     • Demonstrated decreased levels in CNNM4 Adipoq-cKO mice and increased levels with CNNM4 overexpression

     • Advantage: Provides in vivo relevance

  4. Electrophysiological Analyses

     • Whole-cell patch clamp to detect potential currents

     • Shows no significant electronic currents with CNNM4 expression, supporting electroneutral exchange mechanism

     • Advantage: Distinguishes between channel and transporter/exchanger mechanisms

• How can genetic mutations in CNNM4 be introduced and evaluated for their impact on protein function?

  The evaluation of CNNM4 mutations involves several key methodologies:

  1. Site-Directed Mutagenesis

     • Generate specific mutations using PCR-based techniques

     • Example: The pathogenic variants p.(Gly492Cys), p.(Gly492Asp), and p.(Thr495Ile) were created by site-directed mutagenesis of CNNM4 in pCMV-Tag4A vector

  2. Protein Stability and Expression Analysis

     • Western blot: Quantify protein levels at different time points post-transfection

     • RT-qPCR: Assess mRNA stability

     • For example, p.(Gly492Cys) and p.(Gly492Asp) showed decreased protein levels at 48-hours post-transfection by 10.5-fold and 5.8-fold respectively compared to WT

  3. Half-life Determination

     • mRNA stability: Treat with actinomycin D and measure decay rates

     • Protein stability: Treat with cycloheximide to block new synthesis

     • Example findings: The half-life of p.(Gly492Cys), p.(Gly492Asp), and p.(Thr495Ile) CNNM4 proteins was 0.61h, 1.18h, and 12.11h respectively, compared to WT that was too stable to fit the decay equation

     CNNM4 Variant	mRNA Half-life	Protein Half-life
     Wild type	>24h	Too stable to measure
     p.(Gly492Cys)	1.1h	0.61h
     p.(Gly492Asp)	2.6h	1.18h
     p.(Thr495Ile)	14h	12.11h

  4. Localization Studies

     • Immunofluorescence microscopy to determine cellular localization

     • Co-localization with F-actin to assess plasma membrane targeting

     • Findings: Mutant proteins showed normal localization to the plasma membrane despite functional defects

  5. Mg²⁺ Transport Assays

     • Fluorescence-based assays to measure transport activity

     • Assess impact of mutations on transport kinetics

     • Findings: Mutant proteins exhibited reduced Mg²⁺ extrusion activity despite proper localization

Advanced Research Questions

• What are the known interaction partners of CNNM4 and how do these interactions affect its function?

  Several key interaction partners of CNNM4 have been identified:

  1. Phosphatases of Regenerating Liver (PRLs)

     • PRL-1 binds to the Bateman module of CNNM4

     • This interaction abrogates CNNM4's Mg²⁺-efflux capacity

     • Results in increased intracellular Mg²⁺ concentration

     • Promotes tumor growth in cancer contexts

     • SAXS analysis confirms one CNNM4 BAT-cNMP-Ctail dimer binds to two separate PRL-1 molecules

  2. IQCB1

     • Physical interaction demonstrated by co-immunoprecipitation studies

     • May be relevant to CNNM4's role in retinal function

     • Mutations in IQCB1 cause another form of retinal dystrophy (Senior-Loken syndrome)

  3. CREB (cAMP Response Element-Binding protein)

     • Transcriptional regulator that binds to the CRE region of CNNM4 promoter

     • Binding increases upon β-adrenergic stimulation (CL316243)

     • Verified through ChIP experiments and luciferase reporter assays

     • Mediates cold-induced upregulation of CNNM4 in adipocytes

  These interactions reveal CNNM4 as part of complex regulatory networks involving:

  • Cancer progression (PRL interaction)

  • Retinal function (IQCB1 interaction)

  • Thermogenic response (CREB-mediated regulation)

  Research approaches to study these interactions include co-immunoprecipitation, SAXS analysis, ChIP assays, and reporter gene assays.

• How do ATP and Mg²⁺ binding cooperatively regulate CNNM4 function, and what structural changes are involved?

  ATP and Mg²⁺ binding to CNNM4 involves a sophisticated cooperative mechanism:

  1. Binding Sites and Cooperativity

     • ATP and Mg²⁺ bind at non-overlapping sites within the Bateman module

     • The S2 cleft of the Bateman module accommodates ATP

     • The S2 site has an unusually acidic surface, rarely seen in typical ATP binding sites

     • Electrostatic repulsion between negatively charged residues and ATP's polyphosphate chain explains low nucleotide affinity

     • Mg²⁺ co-binding provides charge compensation, enhancing ATP affinity

  2. Structural Transitions

     • ATP and Mg²⁺ binding triggers a conformational change in the Bateman module

     • The module transforms from a twisted conformation to a flat disk-shaped dimer

     • The cNMP domain dimer forms a rigid cleft that restricts this Mg²⁺-induced sliding

     • Specifically, the CBS1 motifs of the Bateman module insert into the cavity formed by the cNMP dimer

  3. Functional Consequences

     • This structural reorganization is crucial for Mg²⁺ transport activity

     • Removal of the cNMP domain inhibits Mg²⁺ transport by CNNM proteins

     • The F631A mutation in the cNMP domain (strand β5) induces dissociation of CNNM4 cNMP dimers

  This mechanism is inverse to bacterial Mg²⁺ channels like MgtE, which require prior ATP binding to enhance Mg²⁺ affinity and promote channel closure at high Mg²⁺ concentrations. This difference reflects CNNM4's role in Mg²⁺ efflux rather than influx .

• What evidence supports CNNM4's role in disease pathogenesis, and how can recombinant CNNM4 be used to study these mechanisms?

  CNNM4 has been implicated in several pathological conditions:

  1. Jalili Syndrome

     • Caused by mutations in CNNM4

     • Characterized by cone-rod dystrophy and amelogenesis imperfecta

     • 24 different mutations documented to cause this condition

     • CNNM4-knockout mice exhibit both retinal and dental phenotypes similar to human patients

     Pathogenic mechanisms:

     • Decreased Mg²⁺ extrusion causing hypomagnesemia

     • Oxidative stress and retinal cell apoptosis

     • The p.R605X mutation increases apoptosis in cell models (shown by Annexin-APC/PI staining)

  2. Cancer Progression

     • CNNM4 interaction with PRLs promotes tumor growth

     • PRL binding inhibits CNNM4's Mg²⁺ efflux activity

     • Results in increased intracellular Mg²⁺ that favors tumor growth

  3. Metabolic Regulation

     • CNNM4 overexpression in scWAT ameliorates high-fat diet-induced weight gain

     • Improves insulin sensitivity and glucose homeostasis

     • Increases oxygen consumption and energy expenditure

     Parameter	Control mice	CNNM4-overexpressing mice
     Body weight gain on HFD	Higher	Reduced
     Thermogenic gene expression	Lower	Increased
     UCP1 protein levels	Lower	Increased
     VO₂ and energy expenditure	Lower	Promoted
     Insulin sensitivity	Reduced	Improved

  Research approaches using recombinant CNNM4:

  • Structure-function studies to understand how mutations affect protein stability and activity

  • Development of cell-based assays to screen for compounds that might rescue mutant protein function

  • In vitro studies of CNNM4-PRL interactions as potential cancer therapeutic targets

  • Local administration of recombinant CNNM4 or Mg²⁺ in animal models as potential therapeutic strategies

Practical Research Applications

• What are the critical quality control parameters for recombinant rat CNNM4, and how can researchers verify protein activity?

  Quality control of recombinant rat CNNM4 involves multiple parameters:

  1. Purity Assessment

     • SDS-PAGE under reducing and non-reducing conditions

     • Standard threshold: ≥85% purity as determined by SDS-PAGE

     • Coomassie Blue staining to visualize protein bands

     • Expected molecular weight: 87 kDa (calculated), observed at 90-100 kDa

  2. Protein Identity Confirmation

     • Western blot using specific anti-CNNM4 antibodies

     • Mass spectrometry for peptide mapping

     • Validated antibodies are available with specificities for different species (human, rat, mouse)

  3. Functional Verification

     • Mg²⁺ transport assays using fluorescent indicators

     • ATP binding assays (e.g., NMR titration experiments)

     • Cell-based assays showing proper localization to the plasma membrane

     • Dimerization assays (e.g., SEC-MALS, SAXS)

  4. Stability Assessment

     • Size-exclusion chromatography to detect aggregation

     • Thermal shift assays to determine stability

     • Storage at -20°C with 50% glycerol to maintain stability

  Activity verification methods:

  • Fluorescence-based Mg²⁺ efflux assays (preload cells with 40 mM Mg²⁺, then measure efflux)

  • Co-immunoprecipitation with known binding partners (e.g., PRL-1)

  • ATP binding assays showing cooperative binding with Mg²⁺

• How can researchers effectively design experiments to study the role of CNNM4 in tissue-specific contexts?

  Designing tissue-specific CNNM4 experiments requires careful consideration of several factors:

  1. Selection of Appropriate Model Systems

     • Tissue-specific knockout mice (e.g., CNNM4 Adipoq-cKO for adipose tissue)

     • Tissue-specific overexpression using AAV vectors (e.g., AAV-CNNM4 in scWAT)

     • Primary cells from relevant tissues (intestinal epithelia, ameloblasts, adipocytes)

     • Polarized cell lines for epithelial studies (e.g., MDCK cells)

  2. Verification of Tissue-Specific Targeting

     • Immunofluorescence co-staining with cell-type markers:

       • Perilipin (adipocytes)

       • TH (sympathetic neurons)

       • CD31 (vessels)

       • CD45 (immune cells)

     • qPCR analysis of CNNM4 expression across multiple tissues

  3. Functional Readouts for Different Tissues

     • Intestine: Mg²⁺ absorption measurements, transepithelial transport assays

     • Teeth: Amelogenesis assessment, enamel mineralization analysis

     • Retina: Electrophysiology, photoreceptor survival assays, apoptosis measurements

     • Adipose tissue: Thermogenic gene expression, UCP1 protein levels, oxygen consumption

  4. Intervention Strategies

     • Local administration of MgCl₂ (shown to promote M2 macrophage polarization in adipose tissue)

     • Local injection of CNNM4-expressing viral vectors

     • Tissue-specific CNNM4 silencing using siRNA or shRNA

  5. Experimental Controls

     • Include multiple tissues to confirm specificity (e.g., AAV-CNNM4 in scWAT showed no expression in liver, muscle, BAT, or brain)

     • Use appropriate Cre-driver lines for tissue-specific knockouts

     • Include wild-type littermates as controls for knockout studies

• What approaches can be used to investigate the regulatory mechanisms controlling CNNM4 expression and activity?

  Multiple approaches can elucidate CNNM4 regulatory mechanisms:

  1. Transcriptional Regulation

     • ChIP assays to identify transcription factor binding

       • CREB binds to the CRE region of CNNM4 promoter

       • Binding increases upon β-adrenergic stimulation

     • Luciferase reporter assays with wild-type and mutated promoter regions

       • PKA agonist forskolin increases luciferase activity

       • Mutating CREB binding site (TGACGTCA to ACTGCAGT) abolishes this response

     • RNA stability analyses using actinomycin D

       • Wild-type CNNM4 mRNA is highly stable (half-life >24h)

       • Mutations can dramatically reduce stability (half-life as low as 1.1h)

  2. Post-translational Modifications

     • Mass spectrometry to identify phosphorylation, ubiquitination, or other modifications

     • Cycloheximide chase assays to determine protein half-life

       • Wild-type CNNM4 protein is highly stable

       • Mutations can reduce half-life to as little as 0.61h

     • Proteasome inhibitors to assess degradation pathways

  3. Protein-Protein Interactions

     • Co-immunoprecipitation to identify novel binding partners

     • SAXS analysis to determine structural changes upon binding

     • Yeast two-hybrid screening for comprehensive interaction mapping

     • Proximity labeling techniques (BioID, APEX) to identify membrane-proximal interactors

  4. Allosteric Regulation

     • NMR titration experiments to study ATP and Mg²⁺ binding

     • SAXS analysis to monitor structural changes

     • Mutagenesis of key residues in binding sites to disrupt regulation

  5. Environmental Factors

     • Cold exposure increases CNNM4 expression in adipocytes

     • β-adrenergic stimulation (CL316243) enhances CREB binding to CNNM4 promoter

     • Dietary Mg²⁺ restriction may affect CNNM4 expression and activity