Recombinant Mouse Transmembrane protein 100 (Tmem100)

What is the molecular structure of Tmem100?

Tmem100 is a 134-amino acid protein with two hypothetical transmembrane domains (located at amino acids 53-75 and 85-107). It was first identified as a transcript from the mouse genome (FLJ10970) and is well-conserved across vertebrates. Importantly, Tmem100 is not structurally related to any known protein family in any species . Several splice variants or alternative gene transcripts appear to exist, though the primary structure contains the characteristic dual transmembrane organization that defines its functional properties .

What is the tissue distribution pattern of Tmem100 in mice?

Tmem100 displays a specific distribution pattern in the gastrointestinal tract where it is restricted to enteric neurons and vascular tissue in the muscularis propria across all regions of the mouse digestive system. Tmem100 immunoreactivity co-localizes with the pan-neuronal marker protein gene product 9.5 (PGP9.5) but does not co-localize with the glial marker S100β or Kit (a marker of interstitial cells of Cajal) . Additionally, Tmem100 expression has been demonstrated in neuronal cell bodies and fibers in the mouse brain and dorsal root ganglia, indicating its importance across multiple neuronal populations .

How can Tmem100 mRNA expression be detected in experimental settings?

Reverse transcription PCR represents an effective approach for detecting Tmem100 mRNA in tissue samples. Using specific primers (forward: 5′-TGG ACT GCC TTT CTG TGA GCT TGC A-3′, reverse: 5′-GGT GAC CAC AAC TTC CCT CTT GGG G-3′) under optimized conditions (94°C for 3 min, followed by 35 cycles at 94°C for 30s, 62°C for 30s, 72°C for 30s, and 72°C for 2 min final extension), researchers can amplify Tmem100 from tissues such as the gastrointestinal muscularis propria . The PCR product can be visualized on a 2% agarose gel, and confirmation of identity through sequencing is recommended for validation purposes .

How does Tmem100 regulate the interaction between TRPA1 and TRPV1 channels?

Tmem100 functions as a regulatory adaptor protein that modulates the physical and functional interaction between TRPA1 and TRPV1 ion channels in sensory neurons. When present, Tmem100 weakens the TRPA1-TRPV1 physical association, resulting in disinhibition of TRPA1 activity . Conversely, in the absence of Tmem100, TRPV1 forms a tight complex with TRPA1 that significantly suppresses TRPA1 activity . This regulatory mechanism has important implications for nociception and pain modulation, as both channels are critical mediators of sensory signaling in pain pathways.

What experimental evidence demonstrates Tmem100's role in pain modulation?

Multiple lines of experimental evidence establish Tmem100's role in pain:

• Conditional knockout of Tmem100 in dorsal root ganglion neurons significantly reduces mechanical hyperalgesia in inflammatory pain models

• Subcutaneous injection of Tmem100 inhibitor blunts complete Freund's adjuvant (CFA)-induced pain responses in mice

• Mice lacking Tmem100 do not develop secondary mechanical hypersensitivity during knee joint inflammation

• AAV-mediated overexpression of Tmem100 in articular afferents is sufficient to induce mechanical hypersensitivity in remote skin regions even without inflammation

• The role of Tmem100 in inflammatory pain appears to be sex-independent, as female Tmem100KO mice exhibit the same pain phenotype as males with respect to primary and secondary hypersensitivity

What is the mechanism of Tmem100-mediated "un-silencing" of mechanically insensitive nociceptors?

Inflammation upregulates the expression of Tmem100 in silent nociceptors, which are sensory afferents that are normally insensitive to noxious mechanical stimuli but become sensitized during inflammation . Electrophysiological studies demonstrate that overexpression of Tmem100 is both necessary and sufficient to un-silence these nociceptors in mice . When expressed in HEK293 cells, Tmem100 does not produce mechanotransduction currents nor does it modulate PIEZO2-mediated currents, indicating that Tmem100 is neither a channel itself nor a direct modulator of PIEZO2. Instead, it appears to un-silence PIEZO2 specifically in the cellular context of mechanically insensitive afferents .

How can researchers generate and validate Tmem100 conditional knockout models?

Researchers can effectively generate conditional Tmem100 knockout models using the Cre-lox system. A successful approach involves Advillin-Cre ER::Tmem100 fl/fl mice, where Advillin-Cre mice express tamoxifen-inducible Cre recombinase specifically in approximately 98% of trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons . Deletion of Tmem100 can be induced via daily intraperitoneal injection of tamoxifen (75 mg/kg) for 5 days . Validation should include PCR genotyping and quantitative RT-PCR to confirm reduced Tmem100 expression in target tissues.

What methods are effective for measuring Tmem100's impact on neuronal activity?

Ex-vivo calcium imaging of neuronal explants represents an effective approach for visualizing how Tmem100 influences neuronal activity. Using Pirt-GCaMP3 mice that express the genetically-encoded Ca²⁺ indicator GCaMP3 in >96% of DRG/TG neurons, researchers can prepare tissue explants equilibrated in artificial cerebrospinal fluid (ACSF) bubbled with 95% O₂/5% CO₂ at room temperature . Confocal microscopy at 488-nm wavelength allows visualization of calcium signals in response to specific stimuli, such as the TRPA1 agonist JT010 (100 nM), with or without pretreatment with Tmem100 inhibitors like T-100 Mut (200 nM) .

What subcellular localization techniques are most suitable for Tmem100 visualization?

Total internal reflection fluorescence (TIRF) microscopy of cells transfected with TMEM100-GFP fusion constructs provides excellent visualization of Tmem100's subcellular localization. Researchers have successfully employed C-terminal GFP-tagged ORF clones of Tmem100 transfected into HEK293 cells using Lipofectamine 2000 . After 24 hours of expression, cells can be fixed in 4% paraformaldehyde and examined by TIRF microscopy to determine membrane localization patterns, consistent with Tmem100's predicted function as a transmembrane protein .

How does Tmem100 contribute to temporomandibular disorder (TMD) pain mechanisms?

Tmem100 plays a crucial role in trigeminal ganglion (TG)-mediated temporomandibular disorder (TMD) pain by regulating TRPA1 activity within the TRPA1-TRPV1 complex . In TMD pain models involving TMJ inflammation or masseter muscle injury, genetic knockout or pharmacological inhibition of TRPA1 and TRPV1 attenuates pain behaviors . Importantly, Tmem100 co-expresses with TRPA1 and TRPV1 in TG neurons, and this co-expression increases in TG neurons innervating the TMJ and masseter muscle during inflammation . The inhibition of Tmem100 through either conditional knockout or local injection of Tmem100 inhibitor reduces TMD pain, suggesting its potential as a therapeutic target .

What advantages does Tmem100 offer as a potential therapeutic target compared to direct TRPA1/TRPV1 inhibition?

Targeting Tmem100 presents distinct advantages over direct inhibition of TRPA1 or TRPV1 channels for pain management. Clinical trials targeting TRPA1 and TRPV1 have been delayed or discontinued due to off-target thermoregulatory effects and blunting of normal noxious sensation . In contrast, Tmem100 functions as a modulator of channel interactions rather than inhibiting the channels themselves. This regulatory approach may provide a more nuanced intervention in pain transmission pathways . Additionally, local administration of Tmem100 inhibitors into affected tissues (such as TMJ or masseter muscle) can attenuate pain while avoiding potential side effects associated with systemic treatments .

How can researchers quantify Tmem100-associated pain behaviors in experimental animals?

The literature describes several approaches for quantifying Tmem100-associated pain in animal models:

• Bite force measurements to assess TMD-related pain, where reduced bite force indicates increased pain

• Von Frey filament testing to measure mechanical sensitivity in both primary affected areas and secondary sites (for measuring pain spread beyond inflammation sites)

• Assessment of gait alterations and weight-bearing changes in joint inflammation models

• Grimace scale scoring to evaluate spontaneous pain behaviors

• Response to chemical stimuli that activate TRPA1 or TRPV1 channels to assess channel-specific pain behaviors

Statistical analysis typically employs two-tail t-test, one-way ANOVA, or two-way ANOVA followed by appropriate post-hoc tests, with power analysis based on previous relevant studies .

How does Tmem100 interact with BMP and TGFβ signaling pathways in development?

Tmem100 appears to be involved in bone morphogenetic protein (BMP) signaling pathways, which are part of the broader transforming growth factor β (TGFβ) family . In the enteric nervous system, Tmem100 co-localizes with BMP4 in human colon neurons, suggesting functional interaction . This association may reflect Tmem100's role in development and differentiation of cells through these signaling pathways. The BMP signaling pathway is known to be involved in the development of the enteric nervous system, and Tmem100's expression pattern suggests it may contribute to neuronal development and differentiation through modulation of these pathways .

What approaches can resolve contradictory data between different Tmem100 knockout models?

When addressing contradictory results between different Tmem100 knockout models, researchers should consider:

• Specificity of knockout strategies: Global versus conditional knockouts may yield different phenotypes due to developmental compensation

• Temporal factors: Inducible systems (like tamoxifen-inducible Cre) versus constitutive knockouts

• Tissue specificity: Different phenotypes may emerge depending on which tissue-specific promoter drives Cre expression

• Genetic background effects: The same knockout on different mouse strains may produce varying results

• Sex differences: Though Tmem100's role in pain appears sex-independent , other functions might show sexual dimorphism

• Molecular compensation: Comprehensive transcriptomic analysis to identify upregulated genes that might compensate for Tmem100 loss

How can single-cell approaches advance our understanding of Tmem100 function?

Single-cell technologies offer powerful approaches for elucidating Tmem100 function:

• Single-cell RNA sequencing of sensory ganglia can identify specific neuronal subpopulations expressing Tmem100 and co-expression patterns with interacting partners like TRPA1 and TRPV1

• Patch-clamp electrophysiology combined with single-cell transcriptomics can correlate Tmem100 expression levels with functional properties of individual neurons

• CRISPR-based approaches for selective manipulation of Tmem100 in defined cell populations

• Live-cell imaging of fluorescently tagged Tmem100 can reveal dynamic trafficking and localization at single-cell resolution

• Calcium imaging in identified Tmem100-expressing neurons can determine how Tmem100 levels correlate with responsiveness to specific stimuli

How is Tmem100 expression altered in cancer microenvironments?

While the search results provide limited information on Tmem100 in cancer, there is evidence that TMEM100 expression has been associated with clinical stage in lung adenocarcinomas . The broader context of tumor microenvironment contributions to cancer development, progression, and therapeutic response suggests that Tmem100 may play a role in these processes. Comprehensive analysis of tumor microenvironment-related genes could potentially identify Tmem100 as a prognostic marker, though more specific research is needed to establish its role in different cancer types .

What transcriptional regulators control Tmem100 expression during inflammation?

The search results indicate that inflammation upregulates Tmem100 expression in silent nociceptors , but do not specify the transcriptional mechanisms. Based on the involvement of Tmem100 in BMP signaling, it is possible that inflammation-induced alterations in BMP/TGFβ pathway components might influence Tmem100 expression. The data showing that nerve growth factor (NGF) treatment induces up-regulation of Tmem100 in cultured CHRNA3-EGFP+ mechanically insensitive afferents suggests that NGF-responsive transcription factors may regulate Tmem100 expression during inflammation. Future research should focus on identifying the specific transcription factors and regulatory elements controlling Tmem100 expression in different inflammatory conditions.