Recombinant Mouse Transmembrane protein 150C (Tmem150c)

What is Mouse Transmembrane protein 150C (Tmem150c)?

Tmem150c is a transmembrane protein consisting of 249 amino acids with six predicted transmembrane domains. It has been investigated as a potential mechanosensitive protein in mouse sensory neurons. The full amino acid sequence is: MDGKKCSVWMFLPLVFTLFTSAGLWIVYFIAVEDDKILPLNSAARKSGAKHAPYISFAGDDPPASCVFSQVMNMAAFLALVVAVLRFIQLKPKVLNPWLNISGLAALCLASFGMTLLGNFQLTNDEEIHNVGTSLTFGFGTLTCWIQAALTLKVNIKNEGRRAGIPRVILSAVITLCVVLYFILMAQDIHMYAARVQWGLVMCFLAYFGTLAVEFRHYRYEIVCSEYQENFLSFSESLSEASEYQTDQV . This protein has been studied in the context of its potential role in mechanosensation, although recent evidence challenges this proposed function .

What are the alternative names and identifiers for Tmem150c?

Tmem150c is also known as Tentonin 3 (Ttn3). In research databases, it may be identified by its UniProt ID Q8C8S3 for the mouse variant . When reviewing literature and ordering research materials, it's important to search using both nomenclatures to ensure comprehensive coverage of relevant studies.

What is the predicted structure and topology of Tmem150c?

Tmem150c is predicted to have six transmembrane domains with intracellular N- and C-termini. While the crystal structure has not been definitively resolved, structural prediction algorithms suggest a membrane-spanning topology typical of transmembrane proteins involved in sensory functions. Understanding this structural arrangement is crucial for designing experiments related to protein function, particularly when investigating potential mechanosensitive properties or interactions with known mechanosensitive channels like Piezo proteins .

What expression systems are recommended for producing recombinant Tmem150c?

Recombinant Tmem150c can be effectively expressed in E. coli expression systems, particularly when fused to an N-terminal His tag for purification purposes . For researchers investigating protein function rather than structure, mammalian expression systems like N2a cells (especially N2a-P1KO cells with CRISPR/Cas9-mediated deletion of endogenous Piezo1) are recommended for functional studies . The choice of expression system should be guided by the specific experimental requirements:

What are the optimal storage and reconstitution conditions for recombinant Tmem150c?

Recombinant Tmem150c protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt. For working aliquots, storage at 4°C for up to one week is recommended. Repeated freeze-thaw cycles should be avoided to maintain protein integrity. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol (5-50% final concentration) is recommended before aliquoting for long-term storage at -20°C/-80°C . These storage parameters are critical for maintaining protein stability and function in subsequent experiments.

How can Tmem150c knockout mouse models be generated and validated?

Two approaches for generating Tmem150c knockout mice have been documented in the literature:

• CRISPR/Cas9-mediated deletion: This method involves designing guide RNAs targeting the Tmem150c/Ttn3 locus. Verification of knockout can be performed using PCR amplification of the wild-type allele (using primers Tmem150c WT Rev1: 5′-TACCTGATGTATGGAGCATGCTTC-3′ and Tmem150c WT Fw1: 5′-TACTTTATAGCCGTGGAAGATGAC-3′) and the mutant allele (using primers MEMT 1 Fw: 5′-CTCAATAACAGCCACAAGGAAAG-3′ and MEMT 1 Rev: 5′-ACTGGCAGGGTTGTGTAAG-3′) .

• Gene trap approach: The international knockout mouse project (KOMP) has generated Tmem150c mice using an ES cell clone with a LacZ cassette insertion. Genotyping can be performed by amplifying the wild-type band, neomycin cassette, and LacZ cassette using specific primer sets .

Complete validation of knockout models should include immunoblotting using antibodies against the C-terminal part of TMEM150C (e.g., ABN2266; Millipore) to confirm absence of protein expression .

What is the controversial role of Tmem150c in mechanosensation?

• Expression of TMEM150C in neuroblastoma cells lacking endogenous Piezo1 failed to generate mechanosensitive currents in response to indentation, membrane stretch, or substrate deflection.

• Detailed physiological evaluation of Tmem150c knockout mice revealed no quantitative alterations in the properties of cutaneous sensory fibers.

• Locomotion analysis in Tmem150c knockout mice showed no indication of altered gait compared to wild-type controls, contradicting claims about its necessity for proprioception .

These conflicting findings highlight the importance of rigorous experimental controls and multiple methodological approaches when investigating novel mechanosensitive proteins.

What electrophysiological approaches are recommended for studying Tmem150c function?

To properly investigate Tmem150c's potential mechanosensitive properties, multiple complementary electrophysiological approaches should be employed:

• Cell indentation: Using a fire-polished glass pipette (tip size 2–3 μm) manipulated by a piezo-driven micromanipulator. The probe should be positioned at approximately 60° near the cell body, with movements executed in the in/out axis of the device. Movement velocity should be set at approximately 3.5 μm/ms .

• Membrane stretch: This approach applies negative pressure to cell-attached patches to assess channel activation under membrane tension.

• Substrate deflection: Using pillar arrays to provide precise mechanical stimuli to cells while recording electrical responses.

When implementing these techniques, it is crucial to use appropriate control cells (such as cells expressing known mechanosensitive channels like Piezo1/2) and to account for potential confounding factors like endogenous mechanosensitive channels .

How does Tmem150c potentially interact with Piezo ion channels?

While TMEM150C was initially proposed to function as an independent mechanosensitive channel, subsequent studies suggested it might instead modulate the kinetics of PIEZO2 channel activation . Researchers investigating this potential modulatory role should consider:

• Co-expression studies with Piezo2 in heterologous systems, comparing kinetic parameters of mechanically-activated currents with and without Tmem150c expression.

• Co-immunoprecipitation experiments to assess physical interactions between Tmem150c and Piezo proteins.

• Domain mapping studies to identify regions of Tmem150c potentially involved in Piezo channel modulation.

It's worth noting that despite these theoretical interactions, a detailed physiological evaluation of Tmem150c knockout mice did not reveal alterations in sensory neuron mechanosensitivity, challenging the functional relevance of any potential Piezo-Tmem150c interactions in vivo .

How can contradictory findings about Tmem150c's mechanosensitive properties be reconciled?

The contradictory findings regarding Tmem150c's role in mechanosensation may be reconciled through several methodological considerations:

• Expression system artifacts: Early studies reporting mechanosensitive properties of Tmem150c in HEK293 cells were later attributed to endogenous PIEZO1 channels in these cells . Researchers should use Piezo1-knockout cell lines for mechanosensitivity assays.

• Incomplete gene ablation: Some knockout mouse models, particularly the KOMP-generated line, may not achieve complete ablation of Tmem150c expression in sensory neurons . Verification of complete protein loss through immunoblotting is essential.

• Redundancy and compensation: Even with complete knockout, functional redundancy among sensory mechanotransduction proteins may mask phenotypes. Conditional and inducible knockout approaches may help mitigate developmental compensation.

• Context-dependency: Tmem150c might function in specific cellular contexts or require specific interacting partners that were absent in some experimental paradigms.

What are key considerations for interpreting developmental expression patterns of Tmem150c?

Studies have shown that Tmem150c gene expression is developmentally regulated in sensory neurons, with induction coinciding with the appearance of mechanosensitivity when sensory axons reach their peripheral targets . When interpreting these developmental patterns, researchers should consider:

• Temporal correlation versus causal relationship: While Tmem150c expression coincides with the acquisition of mechanosensitivity, knockout studies suggest this correlation may not indicate a causal role in mechanotransduction.

• Region-specific expression: Analysis of expression patterns should include multiple tissue types and neuronal subtypes to identify potential specialized functions.

• Comparative analysis: Examining expression patterns across different species may provide evolutionary insights into Tmem150c function.

• Alternative functions: The developmental regulation of Tmem150c may reflect roles unrelated to mechanosensation, such as neuronal maturation or synapse formation.

What are emerging directions for future Tmem150c research?

Despite the evidence challenging Tmem150c's role in cutaneous mechanosensation, several promising research directions remain:

• Investigation of potential roles in other sensory modalities or non-sensory cellular functions.

• Exploration of potential redundancy with other Tmem150 family members (Tmem150a, Tmem150b).

• Application of proteomics approaches to identify Tmem150c-interacting proteins that might provide clues to its true biological function.

• Single-cell transcriptomics to precisely define the neuronal subtypes expressing Tmem150c and correlate expression with specific functional properties.

• Conditional and inducible knockout approaches to bypass potential developmental compensation and examine acute effects of Tmem150c loss.

These approaches may help resolve the current controversies and uncover the biological significance of this evolutionarily conserved protein.

What transfection methods are most effective for Tmem150c expression studies?

For Tmem150c expression studies in cell lines, lipid-based transfection using reagents like FuGeneHD has proven effective. A recommended protocol involves mixing 1 μg of Tmem150c DNA plasmid with 3 μl of FuGeneHD and 300 μl of OptiMem, incubating for 15 minutes at room temperature, then adding the mixture to cells. Successfully transfected cells can be identified by co-expressed fluorescent markers at least 24 hours post-transfection . For neuronal cultures, which may be more difficult to transfect, alternative methods like nucleofection or viral transduction may yield higher efficiency.

How should researchers validate antibodies for Tmem150c detection?

Validation of antibodies for Tmem150c detection should include:

• Western blotting using tissues from both wild-type and Tmem150c knockout mice to confirm antibody specificity.

• Testing in multiple tissue types known to express Tmem150c at different levels.

• Comparison of results obtained with antibodies targeting different epitopes (e.g., N-terminal versus C-terminal domains).

• Verification of expected molecular weight (accounting for any post-translational modifications).

Published studies have successfully used rabbit antibodies against the C-terminal part of TMEM150C (ABN2266; Millipore) for immunoblotting . Proper validation ensures reliable detection and avoids misinterpretation of expression patterns.

What histological techniques are useful for studying Tmem150c expression patterns?

For researchers investigating Tmem150c expression patterns in tissues, several approaches have been documented:

• X-gal staining in LacZ reporter mice (like the KOMP-generated line), which can be counterstained with nuclear fast-red for better visualization .

• Immunohistochemistry using validated anti-Tmem150c antibodies.

• In situ hybridization to detect Tmem150c mRNA transcripts.

• Single-cell RNA sequencing for high-resolution analysis of expression in specific cell populations.

Each approach has strengths and limitations, and combining multiple techniques provides the most comprehensive view of expression patterns. Appropriate controls, including tissues from knockout mice, should always be included to ensure specificity.

What is the current consensus on Tmem150c function in sensory physiology?

Based on the most recent evidence, the scientific consensus is shifting away from the hypothesis that TMEM150C forms a mechanosensitive ion channel or plays a critical role in sensory mechanotransduction. Detailed physiological analysis of Tmem150c knockout mice revealed no alterations in the properties of cutaneous sensory fibers or locomotor function . While earlier studies suggested potential roles in mechanosensation or modulation of Piezo channels, these findings may have been confounded by methodological issues including incomplete gene ablation and the presence of endogenous mechanosensitive channels in expression systems.

What standards should be applied when evaluating new mechanosensitive channel candidates?

The contradictory findings regarding Tmem150c highlight the importance of rigorous standards when evaluating potential mechanosensitive channels. Researchers should:

• Utilize multiple mechanical stimulation paradigms (indentation, stretch, substrate deflection).

• Employ appropriate null background expression systems (e.g., cells lacking endogenous mechanosensitive channels).

• Generate and thoroughly validate genetic knockout models, confirming complete protein loss.

• Perform detailed in vivo phenotypic analysis using appropriate physiological assays.

• Seek convergent evidence from multiple experimental approaches before making definitive claims about mechanosensitive properties.

These standards will help prevent premature or incorrect attribution of mechanosensitive properties to proteins that may serve other cellular functions.