Recombinant Human Transmembrane protein 100 (TMEM100)

What is the molecular structure of TMEM100 and how is it characterized?

TMEM100 is a small intracellular transmembrane protein containing two transmembrane domains. It has a molecular weight of approximately 17 kDa for the native protein, while recombinant versions with tags (such as GST and His) may have predicted molecular weights around 42.2 kDa . The human TMEM100 gene is located on chromosome 17q22 and encodes a protein of 134 amino acids . TMEM100 is primarily localized in the endoplasmic reticulum (ER) membrane of cells, rather than the plasma membrane, suggesting potential roles in posttranslational protein modification or intracellular sorting within ER-associated structures .

What are the primary biological functions of TMEM100 identified to date?

TMEM100 serves multiple critical biological functions across different systems. In vascular development, it functions as a downstream effector in the BMP9/BMP10-ALK1 signaling pathway essential for arterial endothelium differentiation and vascular morphogenesis during embryonic development . Genetic knockout studies in mice have demonstrated that TMEM100 deficiency leads to embryonic lethality with severe vascular abnormalities, including impaired differentiation of arterial endothelium and defective vascular morphogenesis . In neuronal systems, TMEM100 acts as a regulatory adaptor protein that modulates the interaction between two pain-sensing ion channels, TRPA1 and TRPV1, in sensory neurons . By weakening the physical association between these channels, TMEM100 relieves TRPV1's inhibition of TRPA1, thereby affecting pain signal transduction .

How does TMEM100 expression vary across different tissues and developmental stages?

TMEM100 exhibits dynamic expression patterns that are tissue-specific and developmentally regulated. During embryonic development, TMEM100 is highly enriched in arterial endothelial cells, consistent with its crucial role in vascular development . Studies using knockout mouse models have shown that TMEM100 expression begins to be detectable around embryonic day 8.5 (E8.5) in vascular structures . In adult tissues, TMEM100 expression has been reported in the lung, prostate, and kidney in both humans and mice . It is also expressed in sensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG), where it participates in pain signaling pathways . The differential expression pattern suggests context-dependent regulatory mechanisms and tissue-specific functions of TMEM100 that remain to be fully characterized.

What expression systems are most effective for producing functional recombinant TMEM100?

While E. coli has been used successfully to produce recombinant TMEM100 for certain applications (as evidenced by commercial availability of E. coli-expressed human TMEM100 with N-terminal GST and C-terminal His tags ), researchers should consider several factors when choosing an expression system. Mammalian expression systems may be preferred for studies requiring post-translational modifications and proper membrane protein folding. When expressing in E. coli, solubility can be a challenge as indicated by the presence of 8M urea in storage buffers of commercially available recombinant TMEM100 . For functional studies, particularly those examining TMEM100's role in protein-protein interactions or signaling pathways, mammalian cell lines such as HEK293 may provide more physiologically relevant protein conformation. Regardless of the chosen system, optimization of expression conditions including temperature, induction time, and cell density is critical for maximizing yield and biological activity.

What purification challenges are specific to TMEM100 and how can they be addressed?

Purification of recombinant TMEM100 presents several challenges common to membrane proteins. The hydrophobic transmembrane domains can lead to aggregation and poor solubility during extraction and purification processes. Based on commercial production protocols, TMEM100 appears to require denaturing conditions (8M urea) for solubilization . For researchers purifying their own recombinant TMEM100, a combination of detergents suitable for membrane protein extraction (such as n-dodecyl β-D-maltoside or CHAPS) may be necessary for initial solubilization from expression systems. Affinity chromatography using the engineered tags (such as His or GST) provides the primary purification step, potentially followed by size exclusion chromatography to remove aggregates. Critical quality control steps should include SDS-PAGE with Coomassie staining to verify purity (aim for >80% purity) , Western blotting for identity confirmation, and potentially circular dichroism to assess secondary structure if the protein is to be used in structural or functional studies.

What are the optimal storage conditions for maintaining TMEM100 stability and activity?

For long-term storage of purified recombinant TMEM100, temperatures of -80°C are recommended to maintain stability . The commercial preparation indicates stability for 12 months under proper storage conditions . Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and aggregation. For researchers working with the protein in functional assays, it's advisable to prepare small single-use aliquots before freezing. The buffer composition significantly impacts stability - commercially available recombinant TMEM100 is stored in 50 mM Tris-HCl, pH 8.0, with 8M urea . For applications requiring native protein, researchers should consider buffer exchange to remove denaturing agents and potentially include glycerol (10-20%) as a cryoprotectant. Before using in cell culture applications, filtration is recommended, with the caveat that some protein loss may occur during this process .

How does TMEM100 function within the BMP9/BMP10-ALK1 signaling pathway?

TMEM100 operates as a critical downstream effector in the BMP9/BMP10-ALK1 signaling pathway essential for vascular development. BMP9 and BMP10 activate the endothelial-specific receptor ALK1 (also called Acvrl1), leading to increased expression of TMEM100 . Mechanistically, microarray analysis identified TMEM100 as a gene whose expression is markedly augmented by BMP9 and BMP10 stimulation . When the ALK1 receptor is genetically deleted, TMEM100 expression significantly decreases, confirming its position downstream of ALK1 signaling . TMEM100 subsequently influences the activity of Notch- and Akt-mediated signaling, which are essential for vascular development . In TMEM100-null mice, the activity of these signaling pathways is downregulated, leading to vascular defects that phenocopy those seen in ALK1-deficient animals . This evidence positions TMEM100 as an indispensable link between ALK1 receptor activation and the downstream signaling events that drive arterial endothelium differentiation and vascular morphogenesis.

What experimental models are most informative for studying TMEM100's role in vascular development?

Several experimental models have proven valuable for investigating TMEM100's function in vascular development:

• Genetic knockout mouse models: TMEM100-null mice exhibit embryonic lethality with severe vascular abnormalities, providing a powerful tool for studying its role in vascular development . These models show impaired differentiation of arterial endothelium and defects in vascular morphogenesis that mirror the phenotypes observed in ALK1-deficient mice .

• Conditional knockout approaches: Cre-mediated deletion of TMEM100 specifically in endothelial cells recapitulates the null phenotypes, confirming the endothelial-autonomous function of TMEM100 .

• Cell culture systems: In vitro models using human umbilical vein endothelial cells (HUVECs) or human arterial endothelial cells (HAECs) treated with BMP9/BMP10 can demonstrate TMEM100 induction and its effects on endothelial cell differentiation markers.

• Zebrafish models: While not explicitly mentioned in the provided search results, zebrafish models offer advantages for real-time visualization of vascular development and are amenable to genetic manipulation of TMEM100.

When designing experiments with these models, researchers should consider time-dependent analyses, as TMEM100 null embryos show no detectable phenotypes until E8.5, with the earliest signs of deficiency observed in the vasculature at E9.0-9.5 .

What molecular markers should be examined when studying TMEM100's impact on arterial differentiation?

When investigating TMEM100's role in arterial differentiation, researchers should examine a specific panel of molecular markers:

This pattern of marker expression indicates that TMEM100 deficiency specifically compromises arterial specification while preserving general endothelial identity, highlighting TMEM100's selective role in arterial differentiation .

What molecular mechanisms underlie TMEM100's regulation of TRPA1-TRPV1 interactions?

TMEM100 functions as a critical adaptor protein that modulates the physical and functional interaction between two key pain-sensing ion channels, TRPA1 and TRPV1, in sensory neurons . The mechanism involves a carefully balanced regulation of channel activity:

When TMEM100 is present, it weakens the physical association between TRPA1 and TRPV1, which results in disinhibition (or release from inhibition) of TRPA1 activity . This molecular uncoupling increases the single-channel open probability of the TRPA1-TRPV1 complex . In contrast, when TMEM100 is absent or inhibited, TRPV1 forms a tight complex with TRPA1 that significantly suppresses TRPA1 activity . This mechanistic insight explains why conditional knockout of TMEM100 in dorsal root ganglion neurons or application of TMEM100 inhibitors can attenuate certain forms of pain .

The TMEM100-mediated regulation appears to be particularly important in inflammatory and neuropathic pain conditions where TRPA1 activity contributes to hypersensitivity. Recent research has extended these findings to trigeminal ganglion-mediated temporomandibular disorder (TMD) pain, demonstrating that TMEM100 contributes to this condition through its regulatory effect on TRPA1 activity in the TRPA1-TRPV1 complex .

What experimental approaches are effective for studying TMEM100's role in nociception?

Several experimental approaches have proven valuable for investigating TMEM100's function in pain signaling:

• Genetic manipulation models: Conditional knockout (cKO) of TMEM100 in specific neuronal populations (e.g., DRG or TG neurons) allows for tissue-specific evaluation of its role in pain signaling .

• Calcium imaging in sensory neurons: Ex-vivo Ca²⁺-imaging of trigeminal ganglia or dorsal root ganglia explants from transgenic mice (e.g., Pirt-GCaMP3 mice) enables visualization of neuronal activity in response to TRPA1 agonists with or without TMEM100 inhibition .

• Pharmacological interventions: Application of specific TMEM100 inhibitors (e.g., T-100 Mut) provides a complementary approach to genetic manipulation for studying TMEM100 function .

• Behavioral pain models: Various pain models can be employed, including:

  • Complete Freund's adjuvant (CFA)-induced inflammatory pain

  • Temporomandibular disorder (TMD) pain models

  • Mechanical hyperalgesia assessment using von Frey filaments

  • Bite force testing for orofacial pain

• Molecular interaction studies: Co-immunoprecipitation or FRET-based approaches can directly assess how TMEM100 affects the physical interaction between TRPA1 and TRPV1.

When designing such experiments, researchers should include appropriate controls and consider the specific pain modality (inflammatory, neuropathic, etc.) most relevant to their research question.

How might targeting TMEM100 offer advantages over direct TRPA1 or TRPV1 inhibition for pain management research?

Targeting TMEM100 represents a novel strategy for pain management that may offer several advantages over direct inhibition of TRPA1 or TRPV1 channels:

• Reduced side effects: Clinical trials with direct TRPA1 and TRPV1 inhibitors have been hampered by significant side effects, particularly off-target thermoregulatory effects and blunting of normal noxious sensation . Since TMEM100 modulates the interaction between these channels rather than blocking them entirely, targeting TMEM100 may preserve physiological pain sensation while reducing pathological pain.

• Pathway specificity: TMEM100 appears to specifically regulate the TRPA1-TRPV1 interaction in pain conditions without affecting other physiological functions of these channels when they operate independently. This specificity could lead to more targeted pain relief.

• Localized treatment potential: Research has demonstrated that local administration of TMEM100 inhibitors (e.g., into the temporomandibular joint or masseter muscle) can effectively attenuate pain . This suggests that TMEM100-based therapies could be applied topically or locally to specific pain sites, further reducing systemic side effects.

• Novel mechanistic approach: By targeting a regulatory protein (TMEM100) rather than the ion channels themselves, this approach represents a fundamentally different strategy for pain management—regulating channel interactions rather than blocking channel function entirely .

Preclinical evidence supports this approach, as conditional knockout of TMEM100 in DRG neurons or subcutaneous injection of TMEM100 inhibitor effectively reduced mechanical hyperalgesia in inflammatory pain models . Similar efficacy has been demonstrated for TMD pain when TMEM100 inhibitor was locally administered .

What techniques can resolve the structural determinants of TMEM100 interactions with partner proteins?

Elucidating the structural basis of TMEM100's interactions with partner proteins requires a multi-faceted approach combining several advanced techniques:

• Cryo-electron microscopy (Cryo-EM): This technique has revolutionized membrane protein structural biology and would be particularly valuable for resolving the structure of TMEM100 in complex with interaction partners like TRPA1 and TRPV1. Cryo-EM can capture these proteins in their native-like membrane environment.

• Cross-linking mass spectrometry (XL-MS): This approach can identify specific contact points between TMEM100 and its binding partners by chemically cross-linking interacting regions followed by mass spectrometric analysis.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): HDX-MS can map protein-protein interaction interfaces by identifying regions that show altered hydrogen-deuterium exchange rates upon complex formation.

• Site-directed mutagenesis combined with functional assays: Systematic mutation of potential interaction domains of TMEM100, followed by co-immunoprecipitation and functional assays (e.g., calcium imaging or electrophysiology), can identify critical residues for protein-protein interactions and their functional consequences.

• Peptide array technology: Overlapping peptides spanning the TMEM100 sequence can be immobilized on arrays and probed with potential binding partners to map interaction domains with high resolution.

For the specific interaction with TRPA1-TRPV1, researchers should focus on the two transmembrane domains of TMEM100 and its intervening loop region, as these are likely involved in modulating the physical association between these channels .

How can single-cell approaches advance our understanding of TMEM100 expression heterogeneity?

Single-cell technologies offer powerful approaches to uncover the heterogeneity of TMEM100 expression across different cell populations and states:

• Single-cell RNA sequencing (scRNA-seq): This technique can reveal the cell-type-specific expression patterns of TMEM100 in complex tissues such as the vascular endothelium during development or in sensory ganglia. It can identify previously unrecognized cell populations that express TMEM100 and correlate its expression with other genes to infer functional relationships.

• Single-cell ATAC-seq: By profiling chromatin accessibility, this method can identify regulatory elements controlling TMEM100 expression in specific cell types and developmental stages.

• Spatial transcriptomics: These methods (e.g., Visium from 10x Genomics or MERFISH) preserve spatial information while quantifying gene expression, allowing researchers to map TMEM100 expression in the context of tissue architecture.

• CyTOF or spectral flow cytometry: Using antibodies against TMEM100 and other markers, these techniques can quantify protein-level expression across large numbers of single cells and identify correlations with cell state markers.

• Live-cell imaging with fluorescent reporters: CRISPR knock-in of fluorescent tags at the endogenous TMEM100 locus can enable real-time visualization of its expression dynamics in living cells or tissues.

The heterogeneity in TMEM100 expression may help explain the differential sensitivity of various cell populations to vascular developmental cues or pain stimuli, potentially identifying cellular subsets that could be targeted for therapeutic intervention.

What are the emerging clinical implications of TMEM100 research for vascular and pain disorders?

TMEM100 research has significant emerging clinical implications across multiple disease areas:

• Hereditary vascular disorders:

  • Mutations in ALK1 signaling components cause hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. TMEM100, as a downstream effector of this pathway, may represent an additional causative gene or genetic modifier in these conditions .

  • Screening for TMEM100 mutations or expression changes could improve genetic diagnosis and risk stratification for these disorders.

• Chronic pain conditions:

  • TMEM100's role in regulating TRPA1-TRPV1 interactions positions it as a promising target for novel analgesics .

  • Temporomandibular disorders (TMD), which affect 5-12% of the population and are often refractory to current treatments, may benefit from TMEM100-targeted therapies .

  • Local administration of TMEM100 inhibitors has shown efficacy in preclinical pain models, suggesting potential for topical or local treatments with reduced systemic side effects .

• Biomarker development:

  • Expression levels of TMEM100 could potentially serve as biomarkers for vascular development disorders or pain sensitivity in certain conditions.

  • Quantifying TMEM100 expression or activity might help stratify patients for clinical trials of targeted therapies.

• Drug development strategies:

  • Rather than directly inhibiting ion channels like TRPA1 or TRPV1, which has led to clinical trial failures due to side effects, modulating their interaction through TMEM100-targeted compounds represents a novel approach to pain management .

  • Development of small molecule inhibitors, peptide mimetics, or antibody-based therapies targeting TMEM100 could yield new classes of analgesics with improved side effect profiles.

The translational potential of TMEM100 research highlights the importance of further characterizing its molecular functions and developing selective modulators of its activity for potential clinical applications.

What controls are essential when performing functional studies with recombinant TMEM100?

When conducting functional studies with recombinant TMEM100, several critical controls should be incorporated:

• Expression verification controls:

  • Western blotting to confirm expression and expected molecular weight

  • Immunofluorescence to verify subcellular localization (primarily ER rather than plasma membrane)

  • Use of tagged versions (GST, His, or fluorescent proteins) for tracking expression

• Functional negative controls:

  • Inactive mutant versions of TMEM100 (e.g., T-100 Mut used in pain studies)

  • Buffer-only treatments matching recombinant protein buffer composition

  • Non-relevant recombinant proteins of similar size and preparation method

• Positive controls for downstream pathways:

  • Direct activators of BMP9/BMP10-ALK1 signaling when studying vascular development

  • Direct TRPA1 agonists like JT010 when studying pain pathways

• System-specific controls:

  • For calcium imaging: baseline measurements before stimulation and maximum calcium response controls

  • For in vivo studies: sham operations or vehicle injections

  • For gene expression analysis: housekeeping genes and known BMP9/BMP10 target genes

• Validation across systems:

  • Comparison of results between different expression systems (e.g., E. coli vs. mammalian)

  • Correlation between in vitro and in vivo findings

  • Verification using both gain-of-function and loss-of-function approaches

How can researchers effectively measure TMEM100-mediated modulation of signaling pathways?

Researchers can employ several complementary approaches to measure TMEM100-mediated modulation of signaling pathways:

• Phosphorylation-specific assays:

  • Western blotting with phospho-specific antibodies targeting key components of the Notch and Akt pathways that are regulated by TMEM100

  • Phosphoproteomics to identify comprehensive changes in phosphorylation cascades

  • ELISA-based phosphorylation detection kits for quantitative analysis

• Transcriptional readouts:

  • qRT-PCR analysis of target genes known to be regulated by BMP9/BMP10-ALK1 signaling

  • RNA-seq to capture genome-wide transcriptional changes dependent on TMEM100

  • Reporter gene assays (e.g., luciferase) driven by Notch- or Akt-responsive promoters

• Protein-protein interaction assays:

  • Co-immunoprecipitation to assess TMEM100's impact on protein complexes

  • Proximity ligation assays to visualize and quantify TRPA1-TRPV1 interactions in the presence/absence of TMEM100

  • FRET-based approaches to measure real-time changes in protein interactions

• Functional readouts:

  • Calcium imaging in sensory neurons to assess TMEM100's effect on TRPA1-TRPV1 activity

  • Patch-clamp electrophysiology to measure single-channel properties of the TRPA1-TRPV1 complex

  • In vascular contexts, endothelial cell migration, tube formation, or arterial marker expression assays

• In vivo pathway indicators:

  • Genetic crosses with reporter mice for Notch (e.g., Hes1-GFP) or Akt signaling

  • Immunohistochemistry for pathway components and arterial markers in tissue sections

  • Functional vascular assessment through techniques like India ink injection to visualize vascular morphology

By combining multiple measurement approaches, researchers can build a comprehensive picture of how TMEM100 influences specific signaling pathways in different biological contexts.

What are the key considerations for interpreting phenotypes in TMEM100 genetic models?

When interpreting phenotypes in TMEM100 genetic models, researchers should consider several important factors:

• Developmental timing effects:

  • TMEM100-null embryos show no detectable phenotypes until E8.5, with vascular defects becoming apparent at E9.0-9.5

  • This temporal progression indicates stage-specific requirements for TMEM100 function

  • Conditional knockout systems with temporal control (e.g., tamoxifen-inducible Cre) can help distinguish developmental versus homeostatic requirements

• Tissue-specific roles:

  • Cre-mediated deletion specifically in endothelial cells recapitulates the null phenotype, confirming cell-autonomous functions in these cells

  • Conditional deletion in sensory neurons reveals distinct functions in pain signaling

  • Potential roles in other tissues (lung, prostate, kidney) remain to be fully characterized

• Signaling context dependency:

  • TMEM100 functions downstream of BMP9/BMP10-ALK1 in vascular contexts

  • It regulates TRPA1-TRPV1 interactions in sensory neurons

  • These distinct signaling roles suggest context-dependent functions that should be carefully defined

• Compensatory mechanisms:

  • Consider potential upregulation of related proteins or alternative pathways

  • Acute versus chronic deletion models may reveal different phenotypes due to compensation

  • Combined deletions of TMEM100 with interacting partners may uncover redundant functions

• Genetic background effects:

  • The penetrance and expressivity of TMEM100-related phenotypes may vary with genetic background

  • Backcrossing to ensure uniform background or using mixed backgrounds with appropriate controls is important

• Relationship to human disease:

  • Connect mouse phenotypes to potential human conditions (e.g., vascular malformations, pain disorders)

  • Consider whether TMEM100 might be a modifier gene in disorders associated with ALK1 pathway components